U.S. patent application number 13/359199 was filed with the patent office on 2012-05-17 for 186 human secreted proteins.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Daniel P. Bednarik, Laurie A. Brewer, Kenneth C. Carter, D. Roxanne Duan, Reinhard Ebner, Gregory A. Endress, Ping Feng, Ann M. Ferrie, Carrie L. Fischer, Kimberly A. Florence, John M. Greene, Jing-Shan Hu, Hla Kyaw, David W. LaFleur, Yi Li, Paul A. Moore, Jian Ni, Henrik S. Olsen, Craig A. Rosen, Steven M. Ruben, Yanggu Shi, Daniel R. Soppet, Paul E. Young, Guo-Liang Yu, Zhizhen Zeng.
Application Number | 20120122762 13/359199 |
Document ID | / |
Family ID | 36181214 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122762 |
Kind Code |
A1 |
Ruben; Steven M. ; et
al. |
May 17, 2012 |
186 Human Secreted Proteins
Abstract
The present invention relates to novel human secreted proteins
and isolated nucleic acids containing the coding regions of the
genes encoding such proteins. Also provided are vectors, host
cells, antibodies, and recombinant methods for producing human
secreted proteins. The invention further relates to diagnostic and
therapeutic methods useful for diagnosing and treating diseases,
disorders, and/or conditions related to these novel human secreted
proteins.
Inventors: |
Ruben; Steven M.;
(Brookeville, MD) ; Rosen; Craig A.; (Pasadena,
MD) ; Soppet; Daniel R.; (McLean, VA) ;
Carter; Kenneth C.; (Rockville, MD) ; Bednarik;
Daniel P.; (Highland, MD) ; Endress; Gregory A.;
(Florence, MA) ; Yu; Guo-Liang; (Hillsborough,
CA) ; Ni; Jian; (Germantown, MD) ; Feng;
Ping; (Germantown, MD) ; Young; Paul E.;
(Sudbury, MA) ; Greene; John M.; (Gaithersburg,
MD) ; Ferrie; Ann M.; (Painted Post, NY) ;
Duan; D. Roxanne; (Bethesda, MD) ; Hu; Jing-Shan;
(Mountain View, CA) ; Florence; Kimberly A.;
(Rockville, MD) ; Olsen; Henrik S.; (Gaithersburg,
MD) ; Fischer; Carrie L.; (Burke, VA) ; Ebner;
Reinhard; (Gaithersburg, MD) ; Brewer; Laurie A.;
(Eagan, MN) ; Moore; Paul A.; (North Bethesda,
MD) ; Shi; Yanggu; (Omaha, NE) ; LaFleur;
David W.; (Washington, DC) ; Li; Yi;
(Sunnyvale, CA) ; Zeng; Zhizhen; (Lansdale,
PA) ; Kyaw; Hla; (Boonsboro, MD) |
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
36181214 |
Appl. No.: |
13/359199 |
Filed: |
January 26, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11745580 |
May 8, 2007 |
8106165 |
|
|
13359199 |
|
|
|
|
11144947 |
Jun 6, 2005 |
|
|
|
11745580 |
|
|
|
|
10164861 |
Jun 10, 2002 |
|
|
|
11144947 |
|
|
|
|
09149476 |
Sep 8, 1998 |
6420526 |
|
|
10164861 |
|
|
|
|
PCT/US98/04493 |
Mar 6, 1998 |
|
|
|
09149476 |
|
|
|
|
09882171 |
Jun 18, 2001 |
|
|
|
11144947 |
|
|
|
|
09809391 |
Mar 16, 2001 |
|
|
|
09882171 |
|
|
|
|
60040162 |
Mar 7, 1997 |
|
|
|
60043576 |
Apr 11, 1997 |
|
|
|
60047601 |
May 23, 1997 |
|
|
|
60056845 |
Aug 22, 1997 |
|
|
|
60043580 |
Apr 11, 1997 |
|
|
|
60047599 |
May 23, 1997 |
|
|
|
60056664 |
Aug 22, 1997 |
|
|
|
60043314 |
Apr 11, 1997 |
|
|
|
60047632 |
May 23, 1997 |
|
|
|
60056892 |
Aug 22, 1997 |
|
|
|
60043568 |
Apr 11, 1997 |
|
|
|
60047595 |
May 23, 1997 |
|
|
|
60056632 |
Aug 22, 1997 |
|
|
|
60043578 |
Apr 11, 1997 |
|
|
|
60040333 |
Mar 7, 1997 |
|
|
|
60043670 |
Apr 11, 1997 |
|
|
|
60047596 |
May 23, 1997 |
|
|
|
60056864 |
Aug 22, 1997 |
|
|
|
60043674 |
Apr 11, 1997 |
|
|
|
60047612 |
May 23, 1997 |
|
|
|
60056631 |
Aug 22, 1997 |
|
|
|
60043569 |
Apr 11, 1997 |
|
|
|
60047588 |
May 23, 1997 |
|
|
|
60056876 |
Aug 22, 1997 |
|
|
|
60043671 |
Apr 11, 1997 |
|
|
|
60043311 |
Apr 11, 1997 |
|
|
|
60038621 |
Mar 7, 1997 |
|
|
|
60043672 |
Apr 11, 1997 |
|
|
|
60047613 |
May 23, 1997 |
|
|
|
60056636 |
Aug 22, 1997 |
|
|
|
60043669 |
Apr 11, 1997 |
|
|
|
60047582 |
May 23, 1997 |
|
|
|
60056910 |
Aug 22, 1997 |
|
|
|
60043315 |
Apr 11, 1997 |
|
|
|
60047598 |
May 23, 1997 |
|
|
|
60056874 |
Aug 22, 1997 |
|
|
|
60043312 |
Apr 11, 1997 |
|
|
|
60047585 |
May 23, 1997 |
|
|
|
60056881 |
Aug 22, 1997 |
|
|
|
60043313 |
Apr 11, 1997 |
|
|
|
60047586 |
May 23, 1997 |
|
|
|
60056909 |
Aug 22, 1997 |
|
|
|
60040161 |
Mar 7, 1997 |
|
|
|
60047587 |
May 23, 1997 |
|
|
|
60056879 |
Aug 22, 1997 |
|
|
|
60047500 |
May 23, 1997 |
|
|
|
60056880 |
Aug 22, 1997 |
|
|
|
60047584 |
May 23, 1997 |
|
|
|
60056894 |
Aug 22, 1997 |
|
|
|
60047492 |
May 23, 1997 |
|
|
|
60056911 |
Aug 22, 1997 |
|
|
|
60040626 |
Mar 7, 1997 |
|
|
|
60047503 |
May 23, 1997 |
|
|
|
60056903 |
Aug 22, 1997 |
|
|
|
60047501 |
May 23, 1997 |
|
|
|
60056637 |
Aug 22, 1997 |
|
|
|
60047590 |
May 23, 1997 |
|
|
|
60056875 |
Aug 22, 1997 |
|
|
|
60047581 |
May 23, 1997 |
|
|
|
60056882 |
Aug 22, 1997 |
|
|
|
60047592 |
May 23, 1997 |
|
|
|
60056888 |
Aug 22, 1997 |
|
|
|
60040334 |
Mar 7, 1997 |
|
|
|
60047618 |
May 23, 1997 |
|
|
|
60056872 |
Aug 22, 1997 |
|
|
|
60047617 |
May 23, 1997 |
|
|
|
60056662 |
Aug 22, 1997 |
|
|
|
60047589 |
May 23, 1997 |
|
|
|
60056862 |
Aug 22, 1997 |
|
|
|
60047594 |
May 23, 1997 |
|
|
|
60056884 |
Aug 22, 1997 |
|
|
|
60047583 |
May 23, 1997 |
|
|
|
60056878 |
Aug 22, 1997 |
|
|
|
60040336 |
Mar 7, 1997 |
|
|
|
60047502 |
May 23, 1997 |
|
|
|
60056893 |
Aug 22, 1997 |
|
|
|
60047633 |
May 23, 1997 |
|
|
|
60056630 |
Aug 22, 1997 |
|
|
|
60047593 |
May 23, 1997 |
|
|
|
60056887 |
Aug 22, 1997 |
|
|
|
60040163 |
Mar 7, 1997 |
|
|
|
60047597 |
May 23, 1997 |
|
|
|
60056889 |
Aug 22, 1997 |
|
|
|
60047615 |
May 23, 1997 |
|
|
|
60056877 |
Aug 22, 1997 |
|
|
|
60047600 |
May 23, 1997 |
|
|
|
60056886 |
Aug 22, 1997 |
|
|
|
60047614 |
May 23, 1997 |
|
|
|
60056908 |
Aug 22, 1997 |
|
|
|
60048964 |
Jun 6, 1997 |
|
|
|
60057650 |
Sep 5, 1997 |
|
|
|
60048974 |
Jun 6, 1997 |
|
|
|
60057761 |
Sep 5, 1997 |
|
|
|
60049610 |
Jun 13, 1997 |
|
|
|
60061060 |
Oct 2, 1997 |
|
|
|
60051926 |
Jul 8, 1997 |
|
|
|
60058785 |
Sep 12, 1997 |
|
|
|
60052874 |
Jul 16, 1997 |
|
|
|
60055724 |
Aug 18, 1997 |
|
|
|
60057669 |
Sep 5, 1997 |
|
|
|
60190068 |
Mar 17, 2000 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
435/252.33; 435/320.1; 435/325; 435/352; 435/358; 435/365; 435/366;
435/367; 435/369; 435/455; 435/471; 435/6.12; 435/69.1; 435/7.1;
435/7.92; 436/86; 530/350; 530/387.9; 536/23.5 |
Current CPC
Class: |
A61P 5/00 20180101; G01N
33/566 20130101; A61P 17/02 20180101; C07K 14/47 20130101; C07K
14/4748 20130101; A61K 38/00 20130101; G01N 2800/00 20130101; A61P
25/00 20180101; A61P 9/00 20180101; A61P 13/12 20180101; A61P 35/00
20180101; A61P 15/00 20180101; A61P 43/00 20180101; A61P 31/00
20180101; A61P 7/00 20180101; A61P 11/00 20180101; A61P 1/00
20180101 |
Class at
Publication: |
514/1.1 ;
536/23.5; 435/320.1; 530/350; 530/387.9; 435/7.92; 435/6.12;
435/69.1; 435/7.1; 436/86; 435/455; 435/471; 435/252.33; 435/358;
435/365; 435/352; 435/325; 435/367; 435/369; 435/366 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12N 15/63 20060101 C12N015/63; C07K 14/435 20060101
C07K014/435; C07K 16/18 20060101 C07K016/18; G01N 33/53 20060101
G01N033/53; C12Q 1/68 20060101 C12Q001/68; C12P 21/00 20060101
C12P021/00; G01N 33/68 20060101 G01N033/68; C12N 15/85 20060101
C12N015/85; C12N 15/70 20060101 C12N015/70; C12N 1/21 20060101
C12N001/21; C12N 5/10 20060101 C12N005/10; A61P 7/00 20060101
A61P007/00; A61P 35/00 20060101 A61P035/00; A61P 13/12 20060101
A61P013/12; A61P 9/00 20060101 A61P009/00; A61P 11/00 20060101
A61P011/00; A61P 17/02 20060101 A61P017/02; A61P 25/00 20060101
A61P025/00; A61P 5/00 20060101 A61P005/00; A61P 15/00 20060101
A61P015/00; A61P 31/00 20060101 A61P031/00; A61P 1/00 20060101
A61P001/00; C12N 15/12 20060101 C12N015/12 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a polynucleotide
fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA
sequence included in ATCC.TM. Deposit No:Z, which is hybridizable
to SEQ ID NO:X; (b) a polynucleotide encoding a polypeptide
fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the
cDNA sequence included in ATCC.TM. Deposit No:Z, which is
hybridizable to SEQ ID NO:X; (c) a polynucleotide encoding a
polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded
by the cDNA sequence included in ATCC.TM. Deposit No:Z, which is
hybridizable to SEQ ID NO:X; (d) a polynucleotide encoding a
polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded
by the cDNA sequence included in ATCC.TM. Deposit No:Z, which is
hybridizable to SEQ ID NO:X; (e) a polynucleotide encoding a
polypeptide of SEQ ID NO:Y or the cDNA sequence included in
ATCC.TM. Deposit No:Z, which is hybridizable to SEQ ID NO:X, having
biological activity; (f) a polynucleotide which is a variant of SEQ
ID NO:X; (g) a polynucleotide which is an allelic variant of SEQ ID
NO:X; (h) a polynucleotide which encodes a species homologue of the
SEQ ID NO:Y; and (i) a polynucleotide capable of hybridizing under
stringent conditions to any one of the polynucleotides specified in
(a)-(h), wherein said polynucleotide does not hybridize under
stringent conditions to a nucleic acid molecule having a nucleotide
sequence of only A residues or of only T residues.
2. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding a
secreted protein.
3. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding
the sequence identified as SEQ ID NO:Y or the polypeptide encoded
by the cDNA sequence included in ATCC.TM. Deposit No:Z, which is
hybridizable to SEQ ID NO:X.
4. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises the entire nucleotide sequence of
SEQ ID NO:X or the cDNA sequence included in ATCC.TM. Deposit No:Z,
which is hybridizable to SEQ ID NO:X.
5. The isolated nucleic acid molecule of claim 2, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
6. The isolated nucleic acid molecule of claim 3, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
7. A recombinant vector comprising the isolated nucleic acid
molecule of claim 1.
8. A method of making a recombinant host cell comprising the
isolated nucleic acid molecule of claim 1.
9. A recombinant host cell produced by the method of claim 8.
10. The recombinant host cell of claim 9 comprising vector
sequences.
11. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of: (a) a polypeptide fragment of SEQ ID NO:Y or the
encoded sequence included in ATCC.TM. Deposit No:Z; (b) a
polypeptide fragment of SEQ ID NO:Y or the encoded sequence
included in ATCC.TM. Deposit No:Z, having biological activity; (c)
a polypeptide domain of SEQ ID NO:Y or the encoded sequence
included in ATCC.TM. Deposit No:Z; (d) a polypeptide epitope of SEQ
ID NO:Y or the encoded sequence included in ATCC.TM. Deposit No:Z;
(e) a secreted form of SEQ ID NO:Y or the encoded sequence included
in ATCC.TM. Deposit No:Z; (f) a full length protein of SEQ ID NO:Y
or the encoded sequence included in ATCC.TM. Deposit No:Z; (g) a
variant of SEQ ID NO:Y; (h) an allelic variant of SEQ ID NO:Y; and
(i) a species homologue of the SEQ ID NO:Y.
12. The isolated polypeptide of claim 11, wherein the secreted form
or the full length protein comprises sequential amino acid
deletions from either the C-terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated
polypeptide of claim 11.
14. A recombinant host cell that expresses the isolated polypeptide
of claim 11.
15. A method of making an isolated polypeptide comprising: (a)
culturing the recombinant host cell of claim 14 under conditions
such that said polypeptide is expressed; and (b) recovering said
polypeptide.
16. The polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical
condition, comprising administering to a mammalian subject a
therapeutically effective amount of the polypeptide of claim
11.
18. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the
polynucleotide of claim 1; and (b) diagnosing a pathological
condition or a susceptibility to a pathological condition based on
the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the
polypeptide of claim 11 in a biological sample; and (b) diagnosing
a pathological condition or a susceptibility to a pathological
condition based on the presence or amount of expression of the
polypeptide.
20. A method for identifying a binding partner to the polypeptide
of claim 11 comprising: (a) contacting the polypeptide of claim 11
with a binding partner; and (b) determining whether the binding
partner effects an activity of the polypeptide.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 11/745,580, filed May 8, 2007, which is a continuation of U.S.
application Ser. No. 11/144,947, filed Jun. 6, 2005, now abandoned,
which is a continuation of U.S. application Ser. No. 09/882,171,
filed Jun. 18, 2001, now abandoned, which is a continuation
application of U.S. application Ser. No. 09/809,391, filed Mar. 16,
2001, now abandoned, which claims benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application No. 60/190,068, filed
Mar. 17, 2000; and U.S. application Ser. No. 11/144,947 is a
continuation-in-part of U.S. application Ser. No. 10/164,861, filed
Jun. 10, 2002, now abandoned, which is a divisional of U.S.
application Ser. No. 09/149,476, filed Sep. 8, 1998, (now issued
U.S. Pat. No. 6,420,526, issued on Jul. 16, 2002), which is a
continuation-in-part of PCT International Application No.
PCT/US98/04493, filed Mar. 6, 1998 which claims benefit under 35
U.S.C. .sctn.119(e) based on the following U.S. Provisional
Applications: 60/040,162; 60/040,333; 60/038,621; 60/040,626;
60/040,334; 60/040,336; 60/040,163; (each filed Mar. 7, 1997); and,
60/047,600; 60/047,615; 60/047,597; 60/047,502; 60/047,633;
60/047,583; 60/047,617; 60/047,618; 60/047,503; 60/047,592;
60/047,581; 60/047,584; 60/047,500; 60/047,587; 60/047,492;
60/047,598; 60/047,613; 60/047,582; 60/047,596; 60/047,612;
60/047,632; 60/047,601, (each filed May 23, 1997); and, 60/043,580;
60/043,568; 60/043,314; 60/043,569; 60/043,311; 60/043,671;
60/043,674; 60/043,669; 60/043,312; 60/043,313; 60/043,672;
60/043,315, (each filed Apr. 11, 1997); and, 60/048,974, (filed
Jun. 6, 1997); and, 60/056,886; 60/056,877; 60/056,889; 60/056,893;
60/056,630; 60/056,878; 60/056,662; 60/056,872; 60/056,882;
60/056,637; 60/056,903; 60/056,888; 60/056,879; 60/056,880;
60/056,894; 60/056,911; 60/056,636; 60/056,874; 60/056,910;
60/056,864; 60/056,631; 60/056,845, 60/056,892, (each filed Aug.
22, 1997); and 60/057,761, (filed Sep. 5, 1997); and, 60/047,595;
60/047,599; 60/047,588; 60/047,585; 60/047,586; 60/047,590;
60/047,594; 60/047,589; 60/047,593; 60/047,614, (each filed May 23,
1997); and, 60/043,578, 60/043,576 (each filed Apr. 11, 1997); and,
60/047,501, (filed May 23, 1997); and, 60/043,670, (filed Apr. 11,
1997); and, 60/056,632; 60/056,664; 60/056,876; 60/056,881;
60/056,909; 60/056,875; 60/056,862; 60/056,887; 60/056,908, (each
filed Aug. 22, 1997); and, 60/048,964, (filed Jun. 6, 1997); and,
60/057,650, (filed Sep. 5, 1997); and, 60/056,884, (filed Aug. 22,
1997); and, 60/057,669, (filed Sep. 5, 1997); and, 60/049,610,
(filed Jun. 13, 1997); and, 60/061,060, (filed Oct. 2, 1997); and,
60/051,926, (filed Jul. 8, 1997); and, 60/052,874, (filed Jul. 16,
1997); and, 60/058,785, (filed Sep. 12, 1997); and, 60/055,724,
(filed Aug. 18, 1997); and, 60/040,161, (filed Mar. 7, 1997). Each
of the above referenced patent applications are hereby incorporated
by reference herein.
REFERENCE TO SEQUENCE LISTING AS TEXT FILE
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text file. The text file contains a
Sequence Listing entitled "PZ002P2C3D1-FINALSequenceListing.txt"
(1,003,751 bytes, created on Jan. 26, 2012), which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to newly identified polynucleotides,
polypeptides encoded by these polynucleotides, antibodies that bind
these polypeptides, uses of such polynucleotides, polypeptides, and
antibodies, and their production.
BACKGROUND OF THE INVENTION
[0004] Unlike bacterium, which exist as a single compartment
surrounded by a membrane, human cells and other eucaryotes are
subdivided by membranes into many functionally distinct
compartments. Each membrane-bounded compartment, or organelle,
contains different proteins essential for the function of the
organelle. The cell uses "sorting signals," which are amino acid
motifs located within the protein, to target proteins to particular
cellular organelles.
[0005] One type of sorting signal, called a signal sequence, a
signal peptide, or a leader sequence, directs a class of proteins
to an organelle called the endoplasmic reticulum (ER). The ER
separates the membrane-bounded proteins from all other types of
proteins. Once localized to the ER, both groups of proteins can be
further directed to another organelle called the Golgi apparatus.
Here, the Golgi distributes the proteins to vesicles, including
secretory vesicles, the cell membrane, lysosomes, and the other
organelles.
[0006] Proteins targeted to the ER by a signal sequence can be
released into the extracellular space as a secreted protein. For
example, vesicles containing secreted proteins can fuse with the
cell membrane and release their contents into the extracellular
space--a process called exocytosis. Exocytosis can occur
constitutively or after receipt of a triggering signal. In the
latter case, the proteins are stored in secretory vesicles (or
secretory granules) until exocytosis is triggered. Similarly,
proteins residing on the cell membrane can also be secreted into
the extracellular space by proteolytic cleavage of a "linker"
holding the protein to the membrane.
[0007] Despite the great progress made in recent years, only a
small number of genes encoding human secreted proteins have been
identified. These secreted proteins include the commercially
valuable human insulin, interferon, Factor VIII, human growth
hormone, tissue plasminogen activator, and erythropoietin. Thus, in
light of the pervasive role of secreted proteins in human
physiology, a need exists for identifying and characterizing novel
human secreted proteins and the genes that encode them. This
knowledge will allow one to detect, to treat, and to prevent
medical diseases, disorders, and/or conditions by using secreted
proteins or the genes that encode them.
SUMMARY OF THE INVENTION
[0008] The present invention relates to novel polynucleotides and
the encoded polypeptides. Moreover, the present invention relates
to vectors, host cells, antibodies, and recombinant and synthetic
methods for producing the polypeptides and polynucleotides. Also
provided are diagnostic methods for detecting diseases, disorders,
and/or conditions related to the polypeptides and polynucleotides,
and therapeutic methods for treating such diseases, disorders,
and/or conditions. The invention further relates to screening
methods for identifying binding partners of the polypeptides.
DETAILED DESCRIPTION
Definitions
[0009] The following definitions are provided to facilitate
understanding of certain terms used throughout this
specification.
[0010] In the present invention, "isolated" refers to material
removed from its original environment (e.g., the natural
environment if it is naturally occurring), and thus is altered "by
the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of
matter, or could be contained within a cell, and still be
"isolated" because that vector, composition of matter, or
particular cell is not the original environment of the
polynucleotide. The term "isolated" does not refer to genomic or
cDNA libraries, whole cell total or mRNA preparations, genomic DNA
preparations (including those separated by electrophoresis and
transferred onto blots), sheared whole cell genomic DNA
preparations or other compositions where the art demonstrates no
distinguishing features of the polynucleotide/sequences of the
present invention.
[0011] In the present invention, a "secreted" protein refers to
those proteins capable of being directed to the ER, secretory
vesicles, or the extracellular space as a result of a signal
sequence, as well as those proteins released into the extracellular
space without necessarily containing a signal sequence. If the
secreted protein is released into the extracellular space, the
secreted protein can undergo extracellular processing to produce a
"mature" protein. Release into the extracellular space can occur by
many mechanisms, including exocytosis and proteolytic cleavage.
[0012] In specific embodiments, the polynucleotides of the
invention are at least 15, at least 30, at least 50, at least 100,
at least 125, at least 500, or at least 1000 continuous nucleotides
but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb,
10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a
further embodiment, polynucleotides of the invention comprise a
portion of the coding sequences, as disclosed herein, but do not
comprise all or a portion of any intron. In another embodiment, the
polynucleotides comprising coding sequences do not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of
interest in the genome). In other embodiments, the polynucleotides
of the invention do not contain the coding sequence of more than
1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic
flanking gene(s).
[0013] As used herein, a "polynucleotide" refers to a molecule
having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA
contained within the clone deposited with the ATCC.TM.. For
example, the polynucleotide can contain the nucleotide sequence of
the full length cDNA sequence, including the 5' and 3' untranslated
sequences, the coding region, with or without the signal sequence,
the secreted protein coding region, as well as fragments, epitopes,
domains, and variants of the nucleic acid sequence. Moreover, as
used herein, a "polypeptide" refers to a molecule having the
translated amino acid sequence generated from the polynucleotide as
broadly defined.
[0014] In the present invention, the full length sequence
identified as SEQ ID NO:X was often generated by overlapping
sequences contained in multiple clones (contig analysis). A
representative clone containing all or most of the sequence for SEQ
ID NO:X was deposited with the American Type Culture Collection
("ATCC.TM."). As shown in Table 1, each clone is identified by a
cDNA Clone ID (Identifier) and the ATCC.TM. Deposit Number. The
ATCC.TM. is located at 10801 University Boulevard, Manassas, Va.
20110-2209, USA. The ATCC.TM. deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of
the deposit of microorganisms for purposes of patent procedure.
[0015] A "polynucleotide" of the present invention also includes
those polynucleotides capable of hybridizing, under stringent
hybridization conditions, to sequences contained in SEQ ID NO:X,
the complement thereof, or the cDNA within the clone deposited with
the ATCC.TM.. "Stringent hybridization conditions" refers to an
overnight incubation at 42 degree C. in a solution comprising 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about 65
degree C.
[0016] Also contemplated are nucleic acid molecules that hybridize
to the polynucleotides of the present invention at lower stringency
hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, lower stringency
conditions include an overnight incubation at 37 degree C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M
NaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide,
100 ug/ml salmon sperm blocking DNA; followed by washes at 50
degree C. with 1.times.SSPE, 0.1% SDS. In addition, to achieve even
lower stringency, washes performed following stringent
hybridization can be done at higher salt concentrations (e.g.
5.times.SSC).
[0017] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0018] Of course, a polynucleotide which hybridizes only to polyA+
sequences (such as any 3' terminal polyA+ tract of a cDNA shown in
the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in the definition of
"polynucleotide," since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone generated using oligo dT as a primer).
[0019] The polynucleotide of the present invention can be composed
of any polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. For example,
polynucleotides can be composed of single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions. In addition,
the polynucleotide can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. A polynucleotide may
also contain one or more modified bases or DNA or RNA backbones
modified for stability or for other reasons. "Modified" bases
include, for example, tritylated bases and unusual bases such as
inosine. A variety of modifications can be made to DNA and RNA;
thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[0020] The polypeptide of the present invention can be composed of
amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
[0021] "SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ
ID NO:Y" refers to a polypeptide sequence, both sequences
identified by an integer specified in Table 1.
[0022] "A polypeptide having biological activity" refers to
polypeptides exhibiting activity similar, but not necessarily
identical to, an activity of a polypeptide of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency. In the case
where dose dependency does exist, it need not be identical to that
of the polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the polypeptide
of the present invention (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity, and most
preferably, not more than about three-fold less activity relative
to the polypeptide of the present invention.)
[0023] Polynucleotides and Polypeptides of the Invention
Features of Protein Encoded by Gene No: 1
[0024] This gene is expressed primarily in testes tumor and to a
lesser extent in fetal brain. Therefore, polynucleotides and
polypeptides of the invention are useful as reagents for
differential identification of the tissue(s) or cell type(s)
present in a biological sample and for diagnosis of diseases and
conditions which include, but are not limited to, cancer
particularly of the testes, and defects of the central nervous
system such as seizure and neurodegenerative disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly cancer of the
testes and central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g. testes and other reproductive
tissue, brain and other tissue of the nervous system, and blood
cells, and spleen, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0025] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
treatment/diagnosis of testicular cancer and the treatment of
central nervous system disorders since this gene is primarily
expressed in the testes tumor and developing brain. Alternatively,
expression within fetal tissue and other cellular sources marked by
proliferating cells suggests that this protein may play a role in
the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0026] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:11 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1005 of SEQ ID NO:11, b is an integer
of 15 to 1019, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:11, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 2
[0027] This gene is expressed primarily in cancer tissues, such as
breast cancer, Wilm's tumor, and to a lesser extent in fetal
tissues. Therefore, polynucleotides and polypeptides of the
invention are useful as reagents for differential identification of
the tissue(s) or cell type(s) present in a biological sample and
for diagnosis of diseases and conditions which include, but are not
limited to, tumors, particularly, those found in the breast, or
developmental abnormalities or disorders. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the glandular tissues,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.
mammary tissue, fetal tissue, developmental tissue, and cancerous
and wounded tissues) or bodily fluids (e.g. breast milk, amniotic
fluid, serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder. The tissue distribution in
breast cancer cells indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment/diagnosis
of cancers and/or tumors, particularly, those found in the breast
since expression is mainly in cancer/tumor tissues. Similarly,
expression within embryonic tissue and other cellular sources
marked by proliferating cells suggests that this protein may play a
role in the regulation of cellular division, and may show utility
in the diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0028] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 321 as residues: Pro-11 to Thr-18, Leu-43 to Pro-50,
Gly-64 to Leu-72, Leu-81 to Lys-86.
[0029] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:12 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 451 of SEQ ID NO:12, b is an integer
of 15 to 465, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:12, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 3
[0030] The gene encoding the disclosed cDNA is believed to reside
on chromosome 11. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
11. This gene is expressed primarily in CD34 depleted buffy coat,
and to a lesser extent in spleen, chronic lymphocytic leukemia.
Therefore, polynucleotides and polypeptides of the invention are
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions: blood disorders or leukemias, diseases of
the immune or hematopoietic system. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., blood cells, and
spleen, and cancerous and wounded tissues) or bodily fluids (e.g.
lymph, serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0031] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of a
variety of immune system disorders. Expression of this gene product
in spleen and leukemia cells suggests a role in the regulation of
the proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0032] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:13 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 460 of SEQ ID NO:13, b is an integer
of 15 to 474, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:13, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 4
[0033] This gene is expressed primarily in CD34 depleted buffy
coat. Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or hematopoietic
disorders, particularly lymphocytic diseases. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., blood cells, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0034] The tissue distribution in CD34 depleted buffy coat suggests
that the protein product of this clone would be useful for the
treatment and diagnosis of hematopoetic related disorders such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0035] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:14 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 300 of SEQ ID NO:14, b is an integer
of 15 to 314, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:14, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 5
[0036] This gene is expressed primarily in CD34 depleted buffy
coat. Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or hematopoietic
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., blood cells, immune, hematopoietic, and cancerous
and wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0037] The tissue distribution in CD34 depleted buffy coat suggests
that the protein product of this clone would be useful for the
treatment and diagnosis of hematopoetic related disorders such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0038] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 324 as residues: Pro-13 to Lys-21.
[0039] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:15 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 599 of SEQ ID NO:15, b is an integer
of 15 to 613, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:15, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 6
[0040] This gene is expressed primarily in CD34 depleted buffy
coat. Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or hematopoietic
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g. immune, hematopoietic, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred epitopes include those comprising a sequence
shown in SEQ ID NO. 318 as residues: Lys-31 to Lys-39.
[0041] The tissue distribution in CD34 depleted buffy coat suggests
that the protein product of this clone would be useful for the
treatment and diagnosis of hematopoetic related disorders such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0042] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 325 as residues: Lys-31 to Lys-39.
[0043] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:16 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 342 of SEQ ID NO:16, b is an integer
of 15 to 356, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:16, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 7
[0044] This gene is expressed primarily in CD34 depleted buffy
coat, and to a lesser extent in the pineal gland. Therefore,
polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of
diseases and conditions: immune, hematopoietic, neural, or
endocrine disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g. immune, blood
cells, endocrine, neural, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. The tissue
distribution in CD34 depleted buffy coat suggests that the protein
product of this clone would be useful for the treatment and
diagnosis of hematopoetic related disorders such as anemia,
pancytopenia, leukopenia, thrombocytopenia or leukemia since
stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types.
[0045] Alternatively, expression within the pineal gland suggests
that the protein product of this clone would be useful for the
detection, treatment, and/or prevention of various endocrine
disorders and cancers, particularly Addison's disease, Cushing's
Syndrome, and disorders and/or cancers of the pancrease (e.g.
diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g.,
hyper-, hypopituitarism), thyroid (e.g. hyper-, hypothyroidism),
parathyroid (e.g. hyper-, hypoparathyroidism), hypothalamus, and
testes. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0046] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:17 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 400 of SEQ ID NO:17, b is an integer
of 15 to 414, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:17, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 8
[0047] The translation product of this gene shares sequence
homology with an organic cation transporter which is thought to be
important in organic cation uptake in the kidney and liver. (See
Accession No. 2343059.) Preferred polypeptide fragments comprise
the amino acid sequence
ITIAIQMICLVNXELYPTFVRNXGVMVCSSLCDIGGIITPFIVFRLREVWQALPLILFAVLGLLAAGVTLLLP-
E TKGVALPETMKDAENLGRKAKPKENTIYLKVQTSEPSGT (SEQ ID NO: 629) or
TMKDAENLGRKAKPKENT (SEQ ID NO: 630) as well as N-terminal and
C-terminal deletions of these fragments. Also preferred are
polynucleotide fragments encoding these polypeptide fragments.
[0048] This gene is expressed primarily in liver.
[0049] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: hepatic and renal diseases
where drug elimination/cation exchange (organic cation uptake) in
the liver and kidney are problematic. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the hepatic or renal system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
kidney and liver, and cancerous and wounded tissues) or bodily
fluids (e.g. bile, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0050] The tissue distribution and homology to organic cation
transporter indicate that polynucleotides and polypeptides
corresponding to this gene are useful as a polyspecific transporter
that is important for drug elimination in the liver (and possibly
kidney) since expression is found in the liver. Similarly, the
tissue distribution in liver suggests that the protein product of
this clone would be useful for the detection and treatment of liver
disorders and cancers (e.g. hepatoblastoma, jaundice, hepatitis,
liver metabolic diseases and conditions that are attributable to
the differentiation of hepatocyte progenitor cells). In addition
the expression in fetus would suggest a useful role for the protein
product in developmental abnormalities, fetal deficiencies,
pre-natal disorders and various would-healing models and/or tissue
trauma. Moreover, the homology to the organic ion transporter may
suggest that this gene or gene product could be used in the
treatment and/or detection of kidney diseases including renal
failure, nephritus, renal tubular acidosis, proteinuria, pyuria,
edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush
syndrome, glomerulonephritis, hematuria, renal colic and kidney
stones, in addition to Wilms Tumor Disease, and congenital kidney
abnormalities such as horseshoe kidney, polycystic kidney, and
Falconi's syndrome. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0051] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 327 as residues: Asn-64 to Asn-74, Gln-81 to
Gly-87.
[0052] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:18 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 455 of SEQ ID NO:18, b is an integer
of 15 to 469, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:18, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 9
[0053] This gene is expressed primarily in eosinophils induced with
IL-5, and to a lesser extent in fetal liver and spleen. The gene
encoding the disclosed cDNA is believed to reside on chromosome 15.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 15.
[0054] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: diseases of the immune
system, particularly allergies or asthma, in addition to hepatic,
or developmental disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., blood cells,
liver, and spleen, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0055] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for
treating/diagnosis of diseases involving esosinphil reactions since
expression seems to be concentrated in eosinophils and other
tissues involved in immunity such as the liver and spleen.
Similarly, expression within embryonic tissue and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0056] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:19 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 536 of SEQ ID NO:19, b is an integer
of 15 to 550, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:19, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 10
[0057] This gene is expressed primarily in tissues of hematopoietic
lineage and to a lesser extent in Hodgkins lymphoma. Any frame
shifts in this sequence can easily be clarified using known
molecular biology techniques.
[0058] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
the diagnosis of diseases and conditions which include, but are not
limited to, immune deficiencies or dysfunctions. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., hematopoietic cells, lymphoid and reticuloendothelial
tissues, and cancerous tissues) or bodily fluids (e.g. lymph,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0059] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for
treatment/diagnosis for lymphomas or immune dysfunction or as a
therapeutic protein useful in immune modulation based on expression
in anergic T-cells and lymphomas. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0060] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 18 as residues: Gly-6 to Asp-7, Pro-20 to Ser-21,
Ser-23 to Cys-24, Arg-26 to Arg-26.
[0061] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:20 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 759 of SEQ ID NO:20, b is an integer
of 15 to 773, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:20, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 11
[0062] This gene is expressed primarily in neutrophils and to a
lesser extent in activated lymphoid cells.
[0063] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the cell
type present in a biological sample and for diagnosis of diseases
and conditions: immune or hematopoietic disorders, particularly
inflammatory conditions. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., blood cells and
lymphoid tissue, immune, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0064] The tissue distribution suggests that the protein product of
this clone would be useful for the treatment and diagnosis of
hematopoetic related disorders such as anemia, pancytopenia,
leukopenia, thrombocytopenia or leukemia since stromal cells are
important in the production of cells of hematopoietic lineages. The
uses include bone marrow cell ex vivo culture, bone marrow
transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis, therefore, it can be used in immune disorders such
as infection, inflammation, allergy, immunodeficiency etc. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0065] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 330 as residues: Glu-40 to Lys-46.
[0066] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:21 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 977 of SEQ ID NO:21, b is an integer
of 15 to 991, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:21, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 12
[0067] This gene is expressed primarily in brain and to a lesser
extent in activated T-cells. It is likely that the open reading
frame containing the predicted signal peptide continues in the 5'
direction. Preferred polypeptide fragments comprise the amino acid
sequence PRVRNSPEDLGLSLTGDSCKL (SEQ ID NO:631).
[0068] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or neurodegenerative
disorders including ischemic shock, alzheimers and cognitive
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., blood cells, brain, other
tissue of the nervous system, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0069] The tissue distribution in brain suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system.
[0070] Alternatively, the tissue distribution in activated T-cells
suggests that the protein product of this clone would be useful for
the diagnosis and treatment of a variety of immune system
disorders. Expression of this gene product in tonsils suggests a
role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g. by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the
natural gene product may be involved in immune functions. Therefore
it may be also used as an agent for immunological disorders
including arthritis, asthma, immunodeficiency diseases such as
AIDS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersentivities, such as T-cell mediated
cytotoxicity; immune reactions to transplanted organs and tissues,
such as host-versus-graft and graft-versus-host diseases, or
autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
scleroderma and tissues. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0071] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 331 as residues: Ser-5 to Glu-14, Ile-21 to Pro-35,
Ser-65 to Asp-81, Cys-89 to Val-96, Lys-136 to Ser-145, Ile-152 to
Met-169, Arg-189 to Lys-196.
[0072] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:22 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 639 of SEQ ID NO:22, b is an integer
of 15 to 653, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:22, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 13
[0073] This gene was also recently cloned by other groups, naming
this calcium-activated potassium channel gene, hKCa4. (See
Accession No. AF033021, see also, Accession No. 2584866.) This gene
is mapped to human chromosome 19q13.2. A second signal sequence
likely exists upstream from the predicted signal sequence as
described in Table 1. Preferred polypeptide fragments comprise:
QADDLQATVAALCVLRGGGPWAGSWLSPKTPGAMGGDLVLGLGALRRRKRLL (SEQ NO: 632);
or
EQEKSLAGWALVLAXXGIGLMVLHAEMLWFGGCSAVNATGHLSDTLWLIPITFLTIGYGDVVPGTMWGK
IVCLCTGVMGVCCTALLVAVVARKLEFNKAEKHVHNFMMDIQYTKEMKESAARVLQEAWMFYKHTRR
KESHAARXHQRXLLAAINAFRQVRLKHRKLREQVNSMVDISKMHMILYDLQQNLSSSHRALEKQIDTLAG
KLDALTELLSTALGPRQLPEPSQQSK (SEQ ID NO: 633), as well as N-terminal
and C-terminal deletions. Also preferred are polynulcleotide
fragments encoding these polypeptide fragments.
[0074] This gene is expressed primarily in breast lymph node and
T-cells, and to a lesser extent in placenta.
[0075] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: hematologic, immune,
reproductive, or developmental disorders. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune or reproductive
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., lymphoid tissue, blood cells and placenta, neural, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph,
amniotic fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred epitopes
include those comprising a sequence shown in SEQ ID NO. 325 as
residues: Arg-13 to Lys-23.
[0076] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of a
variety of immune system disorders. Expression of this gene product
in tonsils suggests a role in the regulation of the proliferation;
survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages, including blood stem cells. This gene
product may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g. by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types.
[0077] Alternatively, expression within placental tissue and other
cellular sources marked by proliferating cells suggests that this
protein may play a role in the regulation of cellular division, and
may show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0078] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 332 as residues: Arg-13 to Lys-23.
[0079] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:23 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1472 of SEQ ID NO:23, b is an integer
of 15 to 1486, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:23, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 14
[0080] The translation product of this gene was found to have
homology with the human PAPS synthase, which is believed to be
involved in modification of L-selectin ligands (See Accession No.
e1204135.) Preferred polypeptide fragments comprise the amino acid
sequence YQAHHVSRNKRGQVVGTRGGFRGCTVWLTGLSGAGK (SEQ ID NO: 634),
SPFLLPGEVPASRGGSGPSPFSFSLRIDVLPPPPPPSRVLRSLLPGPGSAQPASMSGIKKQKTENQQKSTNVV-
Y
QAHHVSRNKRGQVVGTRGGFRGCTVWLTGLSGAGKNNDKFCPGGVLVSHAIPVNSWMGTMSVMALTES
PRWLHGPQSMEGPDRLLQVPAEELSLWSRVSF (SEQ ID NO: 635), VLPWRSTCLPCHPC
(SEQ ID NO: 636), or FLDGDNVRHGLNRIPQMASWPPKHGRS (SEQ ID NO: 637).
Also preferred are the polynucleotide fragments encoding this
polypeptide fragment.
[0081] It has been discovered that this gene is expressed primarily
in benign prostate hyperplasia, human umbilical vein endothelial
cells and to a lesser extent in smooth muscle and human endometrial
stromal cells-treated with estradiol.
[0082] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: inflammation, ischemia, and
restenosis, based on endothelial cell and smooth muscle cell
expression, and prostate diseases such as benign prostate
hyperplasia or prostate cancer. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the prostate or vessels of the
circulatory system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., prostate, endothelial cells, smooth muscle, and
endometrium, and cancerous and wounded tissues) or bodily fluids
(e.g. seminal fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0083] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for
treating/diagnosing diseases or conditions where the endothelial
cell lining of the veins and arteries of underlying smooth muscle
are involved. Alternatively, expression within prostate tissue
suggests that the protein product of this clone may show utility in
the study, detection, treatment, or prevention of a variety of
reproductive disorders, particularly prostate cancer. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0084] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 333 as residues: Arg-21 to Asp-26, Lys-35 to Lys-44,
Glu-49 to Asn-58.
[0085] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:24 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2309 of SEQ ID NO:24, b is an integer
of 15 to 2323, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:24, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 15
[0086] This gene is expressed primarily in human 6 week embryo, and
to a lesser extent in placenta.
[0087] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: reproductive disorders,
particularly developmental anomalies or fetal deficiencies.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly
reproductive or developmental in nature, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., embryonic tissue, developing,
differentiating, placenta, and cancerous and wounded tissues) or
bodily fluids (e.g. amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0088] The tissue distribution within human embryonic, placental,
and other cellular sources marked by proliferating cells suggests
that this protein may play a role in the regulation of cellular
division, and may show utility in the diagnosis and treatment of
cancer and other proliferative disorders. Similarly, embryonic
development also involves decisions involving cell differentiation
and/or apoptosis in pattern formation. Thus this protein may also
be involved in apoptosis or tissue differentiation and could again
be useful in cancer therapy. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0089] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 334 as residues: Lys-50 to Glu-57.
[0090] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:25 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 669 of SEQ ID NO:25, b is an integer
of 15 to 683, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:25, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 16
[0091] This gene is expressed primarily in kidney, amygdala, and to
a lesser extent in fetal tissues. The gene encoding the disclosed
cDNA is believed to reside on chromosome 14. Accordingly,
polynucleotides related to this invention are useful as a marker in
linkage analysis for chromosome 14.
[0092] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) present in a biological sample and for diagnosis of
diseases and conditions: kidney diseases, neurological disorders
and developmental abnormalities. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s). For a number of disorders of the above tissues,
particularly of the renal system or developing fetal tissues,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
kidney, amygdala, and fetal tissues, and cancerous and wounded
tissues) or bodily fluids (e.g., amniotic fluid, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0093] The tissue distribution in kidney suggests that this gene or
gene product could be used in the treatment and/or detection of
kidney diseases including renal failure, nephritus, renal tubular
acidosis, proteinuria, pyuria, edema, pyelonephritis,
hydronephritis, nephrotic syndrome, crush syndrome,
glomerulonephritis, hematuria, renal colic and kidney stones, in
addition to Wilms Tumor Disease, and congenital kidney
abnormalities such as horseshoe kidney, polycystic kidney, and
Falconi's syndrome. Similarly, expression within neural tissue
suggests that that the protein product of this clone would be
useful for the detection/treatment of neurodegenerative disease
states, behavioural disorders, or inflamatory conditions such as
Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, sexually-linked disorders,
or disorders of the cardiovascular system. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0094] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:26 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2022 of SEQ ID NO:26, b is an integer
of 15 to 2036, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:26, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 17
[0095] This gene is expressed primarily in ovarian cancer.
[0096] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: reproductive disorders,
particularly solid tumors similar to ovarian cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
reproductive system. expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., ovarian and other reproductive tissue, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph,
amniotic fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0097] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
treatment of solid tumors of the reproductive system such as
ovarian cancer. Similarly, expression within cellular sources
marked by proliferating cells suggests that this protein may play a
role in the regulation of cellular division, and may show utility
in the diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0098] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 336 as residues: Ser-51 to Val-56.
[0099] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:27 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 703 of SEQ ID NO:27, b is an integer
of 15 to 717, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:27, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 18
[0100] This gene is expressed primarily in brain medulloblastoma.
Preferred polypeptide fragments comprise the amino acid sequence:
IRHEQHPNFSLEMHSKGSSLLLFLPQL ILILPVCAHLHEELNC (SEQ ID NO: 638) and
SFFISEEKGHLLLQAERHPWVAGALVGVSGGLTLTTCSGPTEKPATKNYFLKRLLQEMHIRAN
(SEQ ID NO: 639), as well as N-terminal and C-terminal deletions.
Also preferred are polynucleotide fragments encoding these
polypeptide fragments.
[0101] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural disorders,
particularly tumors of the CNS. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the Central nervous system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0102] The tissue distribution suggests that the protein product of
this clone would be useful for the detection/treatment of
neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Similarly,
expression within cellular sources marked by proliferating cells
suggests that this protein may play a role in the regulation of
cellular division, and may show utility in the diagnosis and
treatment of cancer and other proliferative disorders. Similarly,
embryonic development also involves decisions involving cell
differentiation and/or apoptosis in pattern formation. Thus this
protein may also be involved in apoptosis or tissue differentiation
and could again be useful in cancer therapy. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0103] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:28 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 481 of SEQ ID NO:28, b is an integer
of 15 to 495, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:28, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 19
[0104] This gene is expressed primarily in adipocytes.
[0105] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: obesity. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
adipose tissues expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., adipocytes and cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0106] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
metabolic disorders, particularly obesity by regulating the
function and number of adipocytes. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0107] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:29 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 542 of SEQ ID NO:29, b is an integer
of 15 to 556, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:29, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 20
[0108] This gene is expressed primarily in B cell lymphoma.
[0109] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, the immune system with an emphasis on B cell lymphoma.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the tumors of the immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., blood cells, and lymphoid
tissue, and cancerous and wounded tissues) or bodily fluids (e.g.
lymph, serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0110] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of a
variety of immune system disorders, particularly for diagnosis and
treatment of B cell derived tumors. Expression of this gene product
in B-cells suggests a role in the regulation of the proliferation;
survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages, including blood stem cells. This gene
product may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g. by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0111] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:30 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 420 of SEQ ID NO:30, b is an integer
of 15 to 434, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:30, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 21
[0112] It is likely that the open reading frame containing the
predicted signal peptide continues in the 5' direction. Therefore,
in specific embodiments, polypeptides of the invention comprise the
following amino acid sequence: TRPISQLRHYCEPYTTWCQETYSQTKPK (SEQ ID
NO: 640). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0113] This gene is expressed primarily in immune cells, and to a
lesser extent in fetal tissues
[0114] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or reproductive
disorders, particularly inflammatory or developmental disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., cells of the immune system, and fetal tissues, and cancerous
and wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0115] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of a
variety of immune system disorders. Expression of this gene product
in immune cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Similarly, expression within fetal tissue and
other cellular sources marked by proliferating cells suggests that
this protein may play a role in the regulation of cellular
division, and may show utility in the diagnosis and treatment of
cancer and other proliferative disorders. Similarly, embryonic
development also involves decisions involving cell differentiation
and/or apoptosis in pattern formation. Thus this protein may also
be involved in apoptosis or tissue differentiation and could again
be useful in cancer therapy. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0116] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 340 as residues: Asp-10 to Pro-19, Ser-74 to Tyr-79,
Glu-95 to Lys-110.
[0117] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:31 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 701 of SEQ ID NO:31, b is an integer
of 15 to 715, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:31, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 22
[0118] It is likely that the open reading frame containing the
predicted signal peptide continues in the 5' direction. Therefore,
in specific embodiments, polypeptides of the invention comprise the
following amino acid sequence:
SQCRRKGTFLYFLPQTLSPHTSCPCSAGRPLPPPVLDSTPSSPSN (SEQ ID NO: 641).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0119] It has been discovered that this gene is expressed primarily
in ovarian tumor.
[0120] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: reproductive or endocrine
disorders, particularly tumors of the ovary. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of tumors of
the reproductive organs. expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., ovarian and other reproductive tissue and
cancerous and wounded tissues) or bodily fluids (e.g. lymph,
amniotic fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0121] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis,
treatment, and/or prevention of a variety of reproductive or
endocrine disorders, particularly ovarian. Protein, as well as,
antibodies directed against the protein may show utility as a
tissue-specific marker and/or immunotherapy target for the above
listed tissues.
[0122] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 341 as residues: Leu-22 to Gln-27.
[0123] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:32 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 472 of SEQ ID NO:32, b is an integer
of 15 to 486, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:32, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 23
[0124] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
1.
[0125] It has been discovered that this gene is expressed primarily
in fetal tissues, and to a lesser extent in osteoclastoma cell
line.
[0126] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: osteoporosis or arthritis.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the skeletal expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., bone cells, fetal tissue, developmental, and cancerous
and wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0127] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
conditions of a variety of skeletal disorders, particularly
abnormal bone remodeling due to enhanced activity of osteoclasts.
This may be useful as a specific marker for malignancies derived
from osteoclasts or their precursors. Alternatively, expression
within fetal tissues and other cellular sources marked by
proliferating cells suggests that this protein may play a role in
the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0128] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:33 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 711 of SEQ ID NO:33, b is an integer
of 15 to 725, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:33, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 24
[0129] The translation product of this gene shares sequence
homology with a periplasmic ribonuclease which is thought to be
important in degrading extracellular polynucleotides. In specific
embodiments, polypeptides of the invention comprise the following
amino acid sequence:
HSMLPSDVSILYHMKTLLLLQDTERLKHALEMFPEHCTMPPAFIGSCRNQIGRSSVPAAPNVEKYSRSIPKE
PTPMTWTQESYNLRGLFPSVHCRAHATHHLACPDPRXATPCDNSR (SEQ ID NO: 642).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0130] It has been discovered that this gene is expressed primarily
in serum treated smooth muscle cells
[0131] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: vascular disease such as
restenosis, atherosclerosis, stroke, or aneurysm. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
vasculature expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., smooth muscle, vascular, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0132] The tissue distribution in smooth muscle and homology to
ribonucleases indicate that polynucleotides and polypeptides
corresponding to this gene are useful for treatment of pathological
conditions of smooth muscle associated with bacterial or viral
infiltration. Protein, as well as, antibodies directed against the
protein may show utility as a tissue-specific marker and/or
immunotherapy target for the above listed tissues.
[0133] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 343 as residues: Gln-30 to Lys-36, Pro-41 to
Arg-48.
[0134] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:34 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 423 of SEQ ID NO:34, b is an integer
of 15 to 437, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:34, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 25
[0135] This gene is expressed primarily in early stage human
brain.
[0136] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural or developmental
disorders, particularly neurodegenerative or behavioral disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the human brain development and related diseases, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., brain and other
tissue of the nervous system, developmental, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0137] The tissue distribution in brain suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system.
Alternatively, expression within fetal tissues and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0138] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 344 as residues: Pro-20 to Glu-25.
[0139] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:35 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 929 of SEQ ID NO:35, b is an integer
of 15 to 943, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:35, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 26
[0140] It has been discovered that this gene is expressed primarily
in human brain tissue.
[0141] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural disorders,
particularly brain diseases, such as neurodegenerative disorders
which may be caused by brain diseases. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the human brain diseases,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0142] The tissue distribution in brain suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0143] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 345 as residues: Glu-31 to Leu-36.
[0144] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:36 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 590 of SEQ ID NO:36, b is an integer
of 15 to 604, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:36, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 27
[0145] It has been discovered that this gene is expressed primarily
in anergic T-cells.
[0146] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune diseases, particularly
inflammatory diseases and diseases related to T lymph cells, such
as immunodeficiencies. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune diseases, inflammatory
diseases and diseases related to T lymph cells, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., blood cells, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0147] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of a
variety of immune system disorders. Expression of this gene product
in T-cells suggests a role in the regulation of the proliferation;
survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages, including blood stem cells. This gene
product may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g. by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0148] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 346 as residues: Gly-26 to Ser-35.
[0149] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:37 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 335 of SEQ ID NO:37, b is an integer
of 15 to 349, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:37, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 28
[0150] The translation product of this gene shares sequence
homology with Shigella flexneri positive transcriptional regulator
CriR (criR) gene which part of a two-component regulatory pathway
and is thought to be important in regulation of gene expression.
Since there are multiple examples of prokaryotic two-component
regulatory pathways that have significant parallels to signal
transduction pathways in eukaryotic tissues, it is anticipated that
this gene would have utility for treating human disorders. In
specific embodiments, polypeptides of the invention comprise the
following amino acid sequence: LLHELVQAH
YPGDVVFTTAASDMETVSEAVRCGVFDYLIKPIAYXXLGQTLTRFRQRKHMLESID
SASQKQIDEMFNAYARGEPKDELPTGIEPLTLNAVRKLFKXPGVQHTAXTVXQALNHQPHHCQALS
(SEQ ID NO: 643), or TISRTTARRYLEYCASRHLIIAEIVHGKVGR PQRIYHSG (SEQ
ID NO: 644). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0151] This gene is expressed primarily in human synovial sarcoma
and normal human brain tissues.
[0152] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: human brain diseases
particularly sarcomas of the synovium. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the human brain and synovium and
other related human brain diseases, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., synovial tissue, and brain
and other tissue of the nervous system, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0153] The tissue distribution in brain, combined with the homology
to a two-component regulatory protein suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0154] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:38 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 658 of SEQ ID NO:38, b is an integer
of 15 to 672, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:38, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 29
[0155] The gene encoding the disclosed cDNA is believed to reside
on the X chromosome. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for the X
chromosome.
[0156] This gene is expressed in bone marrow, infant brain, fetal
liver and spleen, prostate and to a lesser extent in pineal gland,
adipose tissue, kidney, adrenal gland, umbilical vein endothelial
cells, and T cells.
[0157] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of
diseases and conditions: diseases related to bone marrow or
hematoplastic tissues, prostate, kidney, neural, adrenal gland, and
cardiovascular tissue or organs. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the diseases related to hematoplastic tissues,
immune system, prostate, kidney, adrenal gland, and cardiovascular
tissue or organs, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., bone marrow, hematopoietic cells, pineal gland,
adipose tissue, neural, developing, kidney, adrenal gland,
endothelial cells, and blood cells, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0158] The tissue distribution and homology to the gene indicate
that polynucleotides and polypeptides corresponding to this gene
are useful for diagnosis and treatment of diseases related to
hematoplastic tissues, immune system, prostate, kidney, adrenal
gland, and cardiovascular tissue or organs. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0159] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:39 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1894 of SEQ ID NO:39, b is an integer
of 15 to 1908, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:39, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 30
[0160] In specific embodiments, polypeptides of the invention
comprise the following amino acid sequence:
MKISFVDLPKSIVVTRPSQNLLSSFTVQPCVLGPQPNSIVPHFTFHGQLPSLLDIKPHILNXVEI
(SEQ ID NO: 645). Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0161] This gene is expressed primarily in meningeal and to a
lesser extent in breast and adult brain.
[0162] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural disorders,
particularly diseases of the meningea and related brain diseases.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the meningea and related brain diseases, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., miningea, mammary tissue, and
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0163] The tissue distribution in meningeal tissue suggests that
the protein product of this clone would be useful for the
detection/treatment of neurodegenerative disease states,
behavioural disorders, or inflamatory conditions such as Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette
Syndrome, meningitis, encephalitis, demyelinating diseases,
peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, sexually-linked disorders,
or disorders of the cardiovascular system. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0164] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 349 as residues: Ser-19 to Glu-26.
[0165] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:40 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 444 of SEQ ID NO:40, b is an integer
of 15 to 458, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:40, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 31
[0166] The gene encoding the disclosed cDNA is believed to reside
on chromosome 12. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
12.
[0167] This gene is expressed in meningea, fetal spleen, osteoblast
and to a lesser extent in activated T-cells, endometrial stromal
cells, fetal lung, HL-60, thymus, testis and endothelial cells.
[0168] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: meningeal disease,
osteoporosis, immune diseases, and hematoplastic diseases.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
meningeal diseases, osteoporosis, immune diseases, and
hematoplastic diseases, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., blood cells, endometrium, lung, thymus,
testis, and endothelial cells, and cancerous and wounded tissues)
or bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid
or spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0169] The tissue distribution of this gene indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis and treatment of meningeal, osteoporosis,
immune diseases, hematoplastic diseases, reproductive or pulmonary
disorders. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0170] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 350 as residues: Gln-7 to Arg-12, Pro-69 to Glu-76,
Leu-119 to Trp-125.
[0171] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:41 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1139 of SEQ ID NO:41, b is an integer
of 15 to 1153, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:41, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 32
[0172] The translation product of this gene was shown to have
homology to the bovine leucine aminopeptidase (LAP), which is
thought to be involved in the intracellular degradation and
turn-over of cellular proteins (See Genbank Accession No.
bbs|137417). In specific embodiments, polypeptides of the invention
comprise the following amino acid sequence: PQFTSAGENF (SEQ ID NO:
646), LKAGKTRTFYGL (SEQ ID NO: 647),
AGIDEQENWHEGKENIRAAVAAGCRQIQDLE (SEQ ID NO: 648), SVEVDPCGDAQ (SEQ
ID NO: 649), EPPLVFVGKGITFDSGGISIKA (SEQ ID NO: 650),
ANMDLMRADMGGAATICSAIVSAAKL (SEQ ID NO: 651),
GLAPLCENMPSGKANKPGDVVRA (SEQ ID NO: 652),
NGKTIQVDNTDAEGRLILADALCYAHTFNPK (SEQ ID NO: 653),
ALGSGATGVFTNSSWLWNKLFEASIETGDRVWRMPLFEHYTRQV (SEQ ID NO: 654),
VNNIGKYRSAGACTAAAFLKEFVTHPKWAHLDIAGVMTNKDEVPYLRKGMAAAEGAVLGLYEYDDLKQ
K (SEQ ID NO: 655), or DMTKGLVLGIYSKEKE (SEQ ID NO: 656).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0173] This gene is expressed primarily in human thymus and to a
much lesser extent in infant brain, T-cells, smooth muscle,
endothelial cells, bone marrow, human ovarian tumor and
keratinocytes testes, osteoclastoma, breast, and tonsils.
[0174] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: Diseases involving the
thymus, particularly thymic cancer and diseases involving T-cell
maturation. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the thymus, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., thymus, brain, and other tissue of the nervous system,
blood cells, bone marrow, ovaries, and testes, and other
reproductive tissue, mammary tissue, tonsils, melanocytes and
cancerous and wounded tissues) or bodily fluids (e.g. lymph, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0175] The tissue distribution in thymus and immune tissues,
combined with the homology to the LAP gene indicate that
polynucleotides and polypeptides corresponding to this gene are
useful for diagnosis and treatment of diseases of the thymus
particularly thymic cancer and diseases involving T-cell
maturation. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0176] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 351 as residues: Ser-42 to Val-49, Lys-79 to Thr-85,
Asp-109 to Asn-120, Asp-163 to Lys-170, Tyr-178 to Trp-186, Pro-206
to Pro-212, His-265 to Glu-274, Met-338 to Gly-347, Asp-361 to
Glu-366.
[0177] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:42 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1969 of SEQ ID NO:42, b is an integer
of 15 to 1983, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:42, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 33
[0178] This gene is expressed primarily in human tonsils, and
placenta, and to a lesser extent in adipocytes, melanocyte, and
infant brain.
[0179] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: inflammatory diseases, immune
diseases, and metabolic disorders, particularly obesity. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
inflammatory diseases, immune diseases, and obesity, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., metabolic tissue,
tonsils, placenta, adipocytes, melanocytes, and brain and other
tissue of the nervous system, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, bile, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0180] The tissue distribution and homology to this gene indicate
that polynucleotides and polypeptides corresponding to this gene
are useful for diagnosis and treatment of diseases such as
inflammation, immune diseases, and obesity. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0181] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 352 as residues: Ser-26 to Ser-34.
[0182] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:43 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1392 of SEQ ID NO:43, b is an integer
of 15 to 1406, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:43, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 34
[0183] The translation product of this gene was found to have
homology to the human FGF-1 intracellular binding protein which is
thought to play a role in the regulation of fibroblast growth
factors (See Genbank Accession No. gi|2738520 (AF010187)). In
specific embodiments, polypeptides of the invention comprise the
following amino acid sequence:
MTSELDIFVGNTTLIDEDVYRLWLDGYSVTDAVALRVRSGILEQTGATAAVLQSDTMDHY (SEQ
ID NO: 657),
RTFHMLERLLHAPPKLLHQLIFQIPPSRQALLIERYYAFDEAFVREVLGKKLSKGTKKDL (SEQ
ID NO: 658),
DDISTKTGITLKSCRRQFDNFKRVFKVVEEMRGSLVDNIQQHFLLSDRLARDYAAIVFFA (SEQ
ID NO: 659), NNRFETGKKKLQYLSFGDFAFCAELMIQNWTLG (SEQ ID NO: 660),
AAKLTHNKDVRDLFVDLV (SEQ ID NO: 661), EKFVEPCRSDHWPLSDVRFFLNQYSASV
(SEQ ID NO: 662), SLDGFRHQA (SEQ ID NO: 663), RPPTLTIKLL (SEQ ID
NO: 664), IMTEVPPN (SEQ ID NO: 665), or
ISIQRLSNPSMASDTRPSGTATWAPSAAASCACIMTEVPPNVRPR (SEQ ID NO: 666).
Polynucleotides encoding these polypeptides are also encompassed by
the invention. When tested against Jurkat T-cell cell lines,
supernatants removed from cells containing this gene activated the
GAS (gamma activation site) promoter element. Thus, it is likely
that this gene activates T-cells through the Jaks-STAT signal
transduction pathway. GAS is a promoter element found upstream in
many genes which are involved in the Jaks-STAT pathway. The
Jaks-STAT pathway is a large, signal transduction pathway involved
in the differentiation and proliferation of cells. Therefore,
activation of the Jaks-STATs pathway, reflected by the binding of
the GAS element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. The gene encoding the
disclosed cDNA is believed to reside on chromosome 11. Accordingly,
polynucleotides related to this invention are useful as a marker in
linkage analysis for chromosome 11.
[0184] This gene is expressed in activated T cells, and to a lesser
extent in pituitary, testis, and breast lymph node.
[0185] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders,
particularly diseases relating to T-cells. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the disorders of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g. immune, hematopoietic, pituitary, testes and other
reproductive tissue, mammary tissue, and lymphoid tissue, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph,
seminal fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0186] The tissue distribution in T-cells and lymph nodes, combined
with its homology to a known regulatory protein for FGF-1, and the
detected GAS activity in T-cells, strongly suggests that the
protein product of this clone would be useful for the diagnosis and
treatment of a variety of immune system disorders. Specifically,
the expression of this gene product in T-cells suggests a role in
the regulation of the proliferation; survival; differentiation;
and/or activation of potentially all hematopoietic cell lineages,
including blood stem cells. This gene product may be involved in
the regulation of cytokine production, antigen presentation, or
other processes that may also suggest a usefulness in the treatment
of cancer (e.g. by boosting immune responses). Since the gene is
expressed in cells of lymphoid origin, the natural gene product may
be involved in immune functions. Therefore it may be also used as
an agent for immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersentivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0187] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 353 as residues: Lys-111 to Thr-125, Ser-133 to
Lys-142, Asn-182 to Lys-190, Asp-216 to Asp-221, Asp-227 to
Thr-233.
[0188] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:44 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1377 of SEQ ID NO:44, b is an integer
of 15 to 1391, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:44, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 35
[0189] This gene is expressed primarily in infant brain.
[0190] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural disorders,
particularly neurodegenerative or behavioral disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
diseases relating to neurological disorders, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., brain, and other
tissue of the nervous system, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0191] The tissue distribution in infant brain suggests that the
protein product of this clone would be useful for the
detection/treatment of neurodegenerative disease states,
behavioural disorders, or inflamatory conditions such as Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette
Syndrome, meningitis, encephalitis, demyelinating diseases,
peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, sexually-linked disorders,
or disorders of the cardiovascular system. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0192] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 354 as residues: Ser-67 to Glu-74, Ala-117 to
Leu-126, Gln-128 to Arg-137, Lys-158 to Gly-167.
[0193] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:45 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1555 of SEQ ID NO:45, b is an integer
of 15 to 1569, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:45, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 36
[0194] This gene is expressed primarily in infant brain.
[0195] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neurological disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the diseases relating to neurological disorders, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., brain and other
tissue of the nervous system, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0196] The tissue distribution in brain suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system.
Alternatively, expression within fetal tissue and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0197] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:46 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1910 of SEQ ID NO:46, b is an integer
of 15 to 1924, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:46, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 37
[0198] Polynucleotides of the invention do not comprise the nucleic
acid sequence shown as Genbank accession no. gb|G22195|G22195,
which is hereby incorporated herein by reference. The gene encoding
the disclosed cDNA is believed to reside on chromosome 10.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 10.
[0199] This gene is expressed primarily in human ovary.
[0200] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: ovarian cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
ovarian disorders such as those involving germ cells, ovarian
follicles, stromal cells, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., ovary and other reproductive tissue, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph,
amniotic fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0201] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
diagnosis, treatment, and/or prevention of a variety of
reproductive or endocrine disorders, particularly ovariopathies,
tumors, or dysfunctions. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0202] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 356 as residues: Met-1 to Gly-17, Glu-49 to
Ile-54.
[0203] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:47 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 461 of SEQ ID NO:47, b is an integer
of 15 to 475, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:47, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 38
[0204] This gene is expressed primarily in lymph node breast
cancer.
[0205] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune or reproductive
disorders, particularly breast cancer. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the breast cancer, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., mammary tissue
and lymphoid tissue, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0206] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for use as a
diagnostic marker for breast cancer.
[0207] Protein, as well as, antibodies directed against the protein
may show utility as a tumor marker and/or immunotherapy targets for
the above listed tissues.
[0208] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 357 as residues: Leu-16 to Asp-24.
[0209] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:48 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 332 of SEQ ID NO:48, b is an integer
of 15 to 346, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:48, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 39
[0210] The translation product of this gene was shown to have
homology to the type III adenylyl cyclase of Rattus norvegicus (See
Genbank Accession No gi|202715.), which is thought to play an
essential role in mediating signal transduction in the sensory
neuronal cilia of olfactory neurons. In specific embodiments,
polypeptides of the invention comprise the following amino acid
sequence: ERWQHLADLADFALAMKDTLTNINNQSFNN (SEQ ID NO: 667), or
HFLHGPLAQEDKSERERWQHLADLADFALAMKDTLTNINNQSFNNXHCA (SEQ ID NO: 668).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0211] This gene is expressed primarily in brain, and to a lesser
extent in other tissues.
[0212] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neuronal disorders such as
trauma, brain degeneration, and brain tumor. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the brain,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0213] The tissue distribution in brain suggests that the protein
product of this clone would be useful for the detection/treatment
of neurodegenerative disease states, behavioural disorders, or
inflamatory conditions such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0214] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 358 as residues: Gln-19 to Phe-25.
[0215] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:49 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1352 of SEQ ID NO:49, b is an integer
of 15 to 1366, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:49, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 40
[0216] Polynucleotides of the invention do not comprise the nucleic
acid sequence shown as Genbank accession no. gb|AB006624|AB006624,
which is hereby incorporated herein by reference.
[0217] This gene is expressed in early stage human embryo, adrenal
gland tumor, and immune tissues such as fetal liver, fetal spleen,
T-cell, and myoloid progenitor cell line and to a lesser extent in
ovary, colon cancer, and a few other tissues.
[0218] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: tumorigenesis including
adrenal gland tumor, colon cancer and various other tumors,
developmental and immune disorders. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the cancer tissues, early stage
human tissues, and immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., liver, spleen, blood cells,
developmental, bone marrow, ovary and other reproductive tissue,
and colon, and cancerous and wounded tissues) or bodily fluids
(e.g. lymph, amniotic fluid, bile, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0219] The tissue distribution in fetal tissues and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Alternatively, the tissue distribution in myeloid
progenitor cells, in addition to fetal spleen, suggests that the
protein product of this clone would be useful for the treatment and
diagnosis of hematopoetic related disorders such as anemia,
pancytopenia, leukopenia, thrombocytopenia or leukemia since
stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0220] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:50 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1391 of SEQ ID NO:50, b is an integer
of 15 to 1405, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:50, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 41
[0221] This gene is expressed primarily in fetal lung, endothelial
cells, liver, thymus and a few other immune tissues.
[0222] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders such as
immune deficiency and autoimmune diseases, pulmonary diseases,
liver diseases, developmental disorders, and tumor metastasis.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the fetal lung, liver, endothelial cells, and immune tissues,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
lung, endothelial cells, liver, thymus, and other tissue of the
immune system, and cancerous and wounded tissues) or bodily fluids
(e.g. lymph, amniotic fluid, pulmonary surfactant, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0223] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis of
immune disorders and pulmonary and hepatic diseases. Its promoter
may also be used for immune system and lung-specific gene
therapies. The expression of this gene in endothelial cells
indicates that it may also involve in angiogenesis which therefore
may play role in tumor metastasis. Similarly, expression within
fetal tissue and other cellular sources marked by proliferating
cells suggests that this protein may play a role in the regulation
of cellular division, and may show utility in the diagnosis and
treatment of cancer and other proliferative disorders. Similarly,
embryonic development also involves decisions involving cell
differentiation and/or apoptosis in pattern formation. Thus this
protein may also be involved in apoptosis or tissue differentiation
and could again be useful in cancer therapy. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0224] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:51 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2619 of SEQ ID NO:51, b is an integer
of 15 to 2633, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:51, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 42
[0225] This gene is expressed primarily in liver, thyroid,
parathyroid and to a lesser extent in fetal lung, stomach and early
embryos.
[0226] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: metabolic regulation,
obesity, heptic failure, heptacellular tumors, endocrine disorders,
particularly thyroiditis and thyroid tumors. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
digestive/endocrine system expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., liver, thyroid, parathyroid, lung, stomach,
and embryonic tissue, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, bile, amniotic fluid, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0227] The tissue distribution and the extracellular locations
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the detection and treatment of
digestive/endocrine disorders, including metabolic regulation,
heptic failure, malabsorption, gastritis and neoplasms. Similarly,
expression within thyroid and parathyroid tissues suggests that the
protein product of this clone would be useful for the detection,
treatment, and/or prevention of various endocrine disorders and
cancers, particularly Addison's disease, Cushing's Syndrome, and
disorders and/or cancers of the pancrease (e.g. diabetes mellitus),
adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism),
thyroid (e.g. hyper-, hypothyroidism), parathyroid (e.g. hyper-,
hypoparathyroidism), hypothalamus, and testes. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0228] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:52 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 763 of SEQ ID NO:52, b is an integer
of 15 to 777, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:52, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 43
[0229] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0230] This gene is expressed primarily in Schizophrenic adult
brain, pituitary, front cortex, hypothalmus and to a lesser extent
in retina, adipose and stomach cancer and placenta.
[0231] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: schizophrenia and other
neurological disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the central nervous system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.
neural, retinal tissue, adipose, stomach, and placenta, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0232] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful in
treatment/detection of disorders in the nerve system, including
schizophrenia, neurodegeneration, and neoplasia. Additionally, a
secreted protein in brain may serve as an endocrine. Similarly, the
secreted protein may also be used to determine biological activity,
to raise antibodies, as tissue markers, to isolate cognate ligands
or receptors, to identify agents that modulate their interactions
and as nutritional supplements. It may also have a very wide range
of biological activities. Typical of these are cytokine, cell
proliferation/differentiation modulating activity or induction of
other cytokines; immunostimulating/immunosuppressant activities
(e.g. for treating human immunodeficiency virus infection, cancer,
autoimmune diseases and allergy); regulation of hematopoiesis (e.g.
for treating anaemia or as adjunct to chemotherapy); stimulation or
growth of bone, cartilage, tendons, ligaments and/or nerves (e.g.
for treating wounds, stimulation of follicle stimulating hormone
(for control of fertility); chemotactic and chemokinetic activities
(e.g. for treating infections, tumors); hemostatic or thrombolytic
activity (e.g. for treating haemophilia, cardiac infarction etc.);
anti-inflammatory activity (e.g. for treating septic shock, Crohn's
disease); as antimicrobials; for treating psoriasis or other
hyperproliferative diseases; for regulation of metabolism, and
behaviour. Also contemplated is the use of the corresponding
nucleic acid in gene therapy procedures. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0233] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 362 as residues: Pro-14 to Ser-23, Ser-57 to Phe-65,
Asn-121 to Asn-131.
[0234] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:53 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 588 of SEQ ID NO:53, b is an integer
of 15 to 602, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:53, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 44
[0235] The translation product of this gene shares sequence
homology with a human GTP binding protein which are thought to be
important in signal transduction and protein transport (See Genbank
Accession No. dbj.parallel.D84488.sub.--1). In specific
embodiments, polypeptides of the invention comprise the following
amino acid sequence:
MGSRDHLFKVLVVGDAAVGKTSLVQDYSQDSFSKHYKSTVGVDFALKVLQWSDYEIVRLQ (SEQ
ID NO: 669),
LWDIAGQERFTSMTRLYYRDASACVIMFDVTNATTFSNSQRWKQDLDSKLTLPNGEPVPC (SEQ
ID NO: 670),
LLANKCDLSPWAVSRDQIDRFSKENGFTGWTETSVKENKNINEAMRVLIEKMMRNSTED (SEQ ID
NO: 671), or IMSLSTQGDYINLQTK (SEQ ID NO: 672). Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0236] This gene is expressed primarily in umbilical vein and
microvascular endothelial cells, GM-CSF treated macrophage, anergic
T cells, osteoblast, osteoclast, CD34+ cells and to a lesser extent
in gall bladder.
[0237] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: bone formation and growth,
osteonecrosis, osteoporosis, angiogenesis and/or hematopoiesis.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the skeletal and hematopoiesis systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., endothelial cells, blood
cells, bone, and gall bladder, and cancerous and wounded tissues)
or bodily fluids (e.g. lymph, amniotic fluid, bile, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0238] The tissue distribution in various immune tissues and cell
types combined with its homology to a conserved GTP binding protein
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for treatment/detection of bone formation and
growth, osteonecrosis, osteoporosis, and/or hematopoiesis because
its involvement in the growth signaling or angiogenesis. Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0239] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 363 as residues: Asp-26 to Ser-38, Leu-76 to Ala-81,
Ser-97 to Ser-108, Asp-137 to Phe-148, Thr-154 to Ile-162, Gln-194
to Ser-199.
[0240] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:54 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1735 of SEQ ID NO:54, b is an integer
of 15 to 1749, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:54, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 45
[0241] The translation product of this gene shares sequence
homology with a signal sequence receptor gamma subunit which is
thought to be important in protein translocation on the endoplasmic
reticulum.
[0242] This gene is expressed primarily in adrenal gland, salivary
gland, prostate, and to a lesser extent in endothelial cells and
smooth muscle.
[0243] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: endocrine or gastrointestinal
disorders, particularly disorders in protein secretion. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
secretory organs, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., adrenal gland, salivary gland, prostate,
endothelial cells, and smooth muscle, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0244] The tissue distribution in various endocrine tissues and
homology to SSR gamma subunit indicate that polynucleotides and
polypeptides corresponding to this gene are useful for endocrine
disorders, prostate cancer, xerostomia or sialorrhea. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0245] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 364 as residues: Lys-27 to Ser-32.
[0246] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:55 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1882 of SEQ ID NO:55, b is an integer
of 15 to 1896, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:55, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 46
[0247] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0248] This gene is expressed primarily in osteoclastoma cells, and
to a lesser extent in melanocyte, amygdala, brain, and stomach.
[0249] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: skeletal disorders,
particularly ossification, osteoporosis, fracture, osteonecrosis,
osteosarcoma. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the skeletal systems, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g. skeletal, melanocytes, amygdala, brain and
other tissue of the nervous system, and stomach, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0250] The tissue distribution in osteoclastoma cells suggests that
the protein product of this clone may play a role in the detection
and treatment of disorders and conditions affecting the skeletal
system, in particular osteoporosis as well as disorders afflicting
connective tissues (e.g. arthritis, trauma, tendonitis,
chrondomalacia and inflammation), such as in the diagnosis or
treatment of various autoimmune disorders such as rheumatoid
arthritis, lupus, scleroderma, and dermatomyositis as well as
dwarfism, spinal deformation, and specific joint abnormalities as
well as chondrodysplasias (ie. spondyloepiphyseal dysplasia
congenita, familial osteoarthritis, Atelosteogenesis type II,
metaphyseal chondrodysplasia type Schmid). Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed tissues.
indicates that polynucleotides and polypeptides corresponding to
this gene are useful in intervention of ossification, osteoporosis,
fracture, osteonecrosis and osteosarcoma.
[0251] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 365 as residues: Gln-19 to Tyr-27, Pro-47 to
Glu-59.
[0252] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:56 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1739 of SEQ ID NO:56, b is an integer
of 15 to 1753, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:56, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 48
[0253] It is likely that the open reading frame containing the
predicted signal peptide continues in the 5' direction. Therefore,
in specific embodiments, polypeptides of the invention comprise the
following amino acid sequence:
RACRHSIYSAWVTSVNITDCKPPSISGAAHQGPTAPGRMVRILANGEIVQDDDPRVRTTTQPPRGSIPRQSFF
NRGHGAPPGGPGPRQQQAGARLGAAQSPFNDLNRQLVN (SEQ ID NO: 673).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0254] The gene encoding the disclosed cDNA is believed to reside
on chromosome 10. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
10.
[0255] The translation product of this gene shares sequence
homology with proline rich proteins which is thought to be
important in protein-protein interactions.
[0256] This gene is expressed primarily in brain.
[0257] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neurological and
psychological disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the central nerve system and
endocrine system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., brain and other tissue of the nervous system, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0258] The tissue distribution and homology to proline-rich
proteins indicate that polynucleotides and polypeptides
corresponding to this gene are useful in intervention and detection
of neurological diseases, including trauma, neoplasia, degenerative
or metabolic conditions in the central nerve system. Additionally,
the gene product may be a secreted by the brain as an endocrine.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0259] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:58 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1035 of SEQ ID NO:58, b is an integer
of 15 to 1049, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:58, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 49
[0260] The translation product of this gene shares sequence
homology with the AOCB gene from Aspergillus nidulans which is
important in asexual development. The gene encoding the disclosed
cDNA is believed to reside on chromosome 12. Accordingly,
polynucleotides related to this invention are useful as a marker in
linkage analysis for chromosome 12.
[0261] This gene is expressed primarily in infant brain and to a
lesser extent in the developing embryo, trachea tumors, B-cell
lymphoma and synovial sarcoma.
[0262] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neurodegenerative diseases,
leukemia and sarcoma's. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the brain and immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
embryonic tissue, neural, blood cells, trachea, and synovial
tissue, and cancerous and wounded tissues) or bodily fluids (e.g.
amniotic fluid, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0263] The tissue distribution in infant brain and sarcoma's and
homology to a gene involved in a key step of eukaryotic development
(fungal spore formation) indicates that the protein product of this
clone could play a role in neurological diseases such as
schizophrenia, particularly in infants. The existence of the gene
in a B-cell lymphoma indicates the gene may be used in the
treatment and detection of leukemia. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0264] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 368 as residues: Thr-8 to Glu-13, Thr-89 to Leu-96,
Ser-144 to Leu-152, Arg-160 to Asp-166.
[0265] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:59 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1762 of SEQ ID NO:59, b is an integer
of 15 to 1776, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:59, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 50
[0266] This gene is expressed primarily in fetal lung.
[0267] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: pulmonary disorders including
lung cancer. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the pulmonary system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., lung, developing, and cancerous and wounded
tissues) or bodily fluids (e.g. amniotic fluid, pulmonary
surfactant, serum, plasma, urine, synovial fluid or spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder.
[0268] The tissue distribution of this gene only in fetal lung
indicates that it plays a key role in development of the pulmonary
system. This would suggest that misregulation of the expression of
this protein product in the adult could lead to lymphoma or sarcoma
formation, particularly in the lung. It may also be involved in
predisposition to certain pulmonary defects such as pulmonary edema
and embolism, bronchitis and cystic fibrosis. Similarly, expression
within fetal tissue and other cellular sources marked by
proliferating cells suggests that this protein may play a role in
the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0269] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:60 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 429 of SEQ ID NO:60, b is an integer
of 15 to 443, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:60, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 51
[0270] The gene encoding the disclosed cDNA is believed to reside
on chromosome 22. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
22.
[0271] It has been discovered that this gene is expressed primarily
in hematopoietic cell types and fetal cells and to a lesser extent
in all tissue types.
[0272] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects in the immune system and
hematopoiesis. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune and hematopoietic systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g. lypmh, serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0273] The tissue distribution of this gene predominantly in
hematopoeitic cells and in the developing embryo suggests that the
protein product of this clone would be useful for the detection and
treatment of lymphomas and disease states affecting the immune
system or hematopoiesis disorders. Expression of this gene product
in hematopoeitic cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0274] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:61 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2874 of SEQ ID NO:61, b is an integer
of 15 to 2888, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:61, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 52
[0275] It has been discovered that this gene is expressed primarily
in prostate and to a lesser extent in fetal spleen, fetal liver,
infant brain and T cell leukemias.
[0276] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: prostate disorders, prostate
cancer, leukemia. Similarly, polypeptides and antibodies directed
to those polypeptides are useful to provide immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the immune system, and/or prostate gland expression
of this gene at significantly higher or lower levels may be
detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue from an
individual not having the disorder.
[0277] The tissue distribution of this gene in prostate suggests
that the protein product of this clone would be useful for
detection or treatment of prostate disorders or prostate cancer.
Its distribution in fetal liver and fetal spleen suggests it may
play a role in the immune system. Expression of this gene product
in hematopoietic cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0278] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 371 as residues: Gln-70 to Asn-80, Leu-129 to
Gly-150, Pro-153 to Thr-166, Met-174 to Val-181, Thr-185 to
Ser-196, Pro-200 to Glu-216, Phe-225 to Ser-230, Ile-232 to
Lys-253, Cys-257 to Pro-271, Leu-293 to Pro-298, Ile-339 to
Asn-344.
[0279] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:62 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1837 of SEQ ID NO:62, b is an integer
of 15 to 1851, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:62, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 53
[0280] The translation product of this gene shares sequence
homology with dynein.
[0281] It has been discovered that this gene is expressed primarily
in brain.
[0282] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neuro-degenerative diseases of
the brain. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly
neuro-degenerative diseases expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0283] The predominant tissue distribution in the brain and
homology to dynein, a microtubule motor protein involved in the
positioning of cellular organelles and molecules suggests that the
protein product of this clone would be useful for
detection/treatment of neurodegenerative diseases, such as
Alzheimers, Huntingtons, Parkinsons diseases and schizophrenia.
[0284] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 372 as residues: Ser-89 to Thr-95.
[0285] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:63 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 3528 of SEQ ID NO:63, b is an integer
of 15 to 3542, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:63, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 54
[0286] The translation product of this gene shares sequence
homology with ubiquitin-conjugation protein, an enzyme which is
thought to be important in the processing of the Huntingtons
Disease causing gene.
[0287] It has been discovered that this gene is expressed primarily
in brain and to a lesser extent in activated macrophages.
[0288] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neurodegenerative disease states
including Huntingtons disease. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of brain
tissues. For a number of disorders of the above tissues or cells,
particularly of the neurological systems expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0289] The predominant tissue distribution of this gene in the
brain and its homology to a Huntington interacting protein suggests
that the protein product of this clone would be useful for the
regulation of the expression of the Huntington disease gene and
other neurodegenerative diseases including spinocerebullar ataxia
types I and III, dentatorubropallidoluysian and spinal bulbar
muscular atrophy. In addition, the existence of elevated levels of
free ubiquitin pools in Alzheimers disease, Parkinson's disease and
amylotrophic lateral sclerosis indicates that the ubiquitin pathway
of protein degradation plays a role in these disease states. Thus,
considering the gene described here is homologous to a
ubiquitin-conjugation protein it may play a general role in
neurodegenerative conditions.
[0290] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 373 as residues: Asn-19 to Arg-25, Arg-31 to Tyr-36,
Glu-44 to Asp-52, Glu-57 to Gly-67, Leu-102 to Lys-108, Ser-135 to
Gly-141.
[0291] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:64 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 869 of SEQ ID NO:64, b is an integer
of 15 to 883, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:64, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 56
[0292] The gene encoding the disclosed cDNA is believed to reside
on chromosome 9. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
9.
[0293] It has been discovered that this gene is expressed primarily
in T-cells (anergic T-cells, resting T-Cells, apoptotic T-cells)
and lymph node (breast), as well as brain (hypothalamus,
hippocampus, pituitary, infant brain, early-stage brain).
[0294] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: immune (e.g. immunodeficiencies,
autoimmunities, inflammation, leukemias & lymphomas) and
neurological (e.g. Alzheimer's disease, dementia, schizophrenia)
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous, hematopoietic and immune systems, expression
of this gene at significantly higher or lower levels may be
detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0295] The tissue distribution suggests that the protein product of
this clone would be useful in the intervention or detection of
pathologies associated with the hematopoietic and immune systems,
such as anemias (leukemias). Expression of this gene product in
immune cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. In addition, the expression in brain (including
fetal) might suggest a role in developmental brain defects,
neuro-degenerative diseases or behavioral abnormalities (e.g.
schizophrenia, Alzheimer's, dementia, depression, etc.). Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0296] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 375 as residues: Thr-48 to Ala-66.
[0297] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:66 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 718 of SEQ ID NO:66, b is an integer
of 15 to 732, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:66, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 57
[0298] It has been discovered that this gene is expressed primarily
in lung, and to a lesser extent in a variety of other hematological
cell types (e.g. Raji cells, bone marrow cell line, activated
monocytes).
[0299] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: pulmonary and/or hematological
disfunction. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the vasculo-pulmonary and hematopoietic systems, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues or cell types (e.g., cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0300] The tissue distribution suggests that the protein product of
this clone would be useful in the intervention and detection of
pathologies associated with the vasculo-pulmonary system. In
addition the expression of this gene in a variety of leukocytic
cell types and a bone marrow cell line might suggest a role in
hematopoietic and immune system disorders, such as leukemias &
lymphomas, inflammation, immunodeficiencies and autoimmunities.
[0301] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 376 as residues: Met-1 to Glu-7, Arg-39 to Ser-55,
Lys-59 to Glu-66, Leu-70 to Asn-77.
[0302] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:67 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 617 of SEQ ID NO:67, b is an integer
of 15 to 631, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:67, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 58
[0303] The translation product of this gene shares sequence
homology with adenylate kinase isozyme 3 (gi|163528 GTP:AMP
phosphotransferase (EC 2.7.4.10) [Bos taurus]), which is thought to
be important in catalyzing the phosphorylation of AMP to ADP in the
presence of ATP or inorganic triphosphate.
[0304] It has been discovered that this gene is expressed primarily
in fetal liver, heart and placenta, and to a lesser extent in many
other tissues.
[0305] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: hepatic, cardiovascular or
reproductive disorders. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the hepatic, cardiovascular and reproductive
systems, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0306] The tissue distribution suggests that the protein product of
this clone would be useful for the treatment and diagnosis of
conditions related to hepatic function and pathogenesis, in
particular, those dealing with liver development and the
differentiation of hepatocyte progenitor cells. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0307] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 377 as residues: Gln-160 to Glu-167, Glu-177 to
Pro-182, Ser-197 to Ile-204, Lys-215 to Ser-224.
[0308] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:68 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1737 of SEQ ID NO:68, b is an integer
of 15 to 1751, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:68, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 59
[0309] It has been discovered that this gene is expressed primarily
in CD34 positive cells (Cord Blood).
[0310] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: hematopoietic differentiation
and immune disorders. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of hematopoietic and immune systems, expression of
this gene at significantly higher or lower levels may be detected
in certain tissues or cell types (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0311] The tissue distribution suggests that the protein product of
this clone would be useful in the detection and treatment of
conditions associated with CD34-positive cells, and therefore as a
marker for cell differentiation in hematopoiesis, as well as
immunological disorders. Expression of this gene product in
hematopoietic cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0312] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 378 as residues: Lys-56 to Gly-71.
[0313] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:69 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 494 of SEQ ID NO:69, b is an integer
of 15 to 508, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:69, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 60
[0314] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 379 as residues: Asp-35 to Lys-49.
[0315] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:70 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 231 of SEQ ID NO:70, b is an integer
of 15 to 245, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:70, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 61
[0316] It has been discovered that this gene is expressed primarily
in schizophrenic frontal cortex.
[0317] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: nervous system and cognitive
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the frontal cortex and CNS expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0318] The tissue distribution suggests that the protein product of
this clone would be useful for study, treatment and diagnosis of
frontal cortex, neuro-degenerative and CNS disorders such as
Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,
Tourette Syndrome, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, sexually-linked disorders,
or disorders of the cardiovascular system. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0319] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 380 as residues: Thr-22 to Thr-27.
[0320] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:71 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 347 of SEQ ID NO:71, b is an integer
of 15 to 361, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:71, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 62
[0321] It has been discovered that this gene is expressed primarily
in human adrenal gland tumor, and to a lesser extent in human
kidney medulla and adult pulmonary tissue.
[0322] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: metabolic and endocrine
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the endocrine and nervous system disorders and neoplasia,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0323] The tissue distribution suggests that the protein product of
this clone would be useful for study, treatment and diagnosis of
neurological and endocrine disorders including neoplasia. Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0324] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 381 as residues: Ile-20 to Leu-25.
[0325] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:72 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 699 of SEQ ID NO:72, b is an integer
of 15 to 713, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:72, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 63
[0326] It has been discovered that this gene is expressed primarily
in human adipocytes, and to a lesser extent in spleen, 12-week old
human, and testes.
[0327] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: immune, metabolic and growth
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0328] The tissue distribution suggests that the protein product of
this clone would be useful for study, diagnosis and treatment of
immune, developmental and metabolic disorders. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0329] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:73 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 848 of SEQ ID NO:73, b is an integer
of 15 to 862, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:73, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 64
[0330] One translated product of this clone is homologous to the
mouse zinc finger protein PZF. (See Accession No. 453376; see also
Gene 152 (2), 233-238 (1995).) Preferred polypeptide fragments
correspond to the highly conserved domains shared between mouse and
man. For example, preferred polypeptide fragments comprise the
amino acid sequence:
LQCEICGFTCRQKASLNWHMKKHDADSFYQFSCNICGKKFEKKDSVVAHKAKSHPEV (SEQ ID
NO: 674), ITSTDILGTNPESLTQPSD (SEQ ID NO: 675), NSTSGECLLLEAEGMSKSY
(SEQ ID NO: 676), CSGTERVSLMADGKIFVGSGSSG
GTEGLVMNSDILGATTEVLIEDSDSAGP (SEQ ID NO: 677),
IQYVRCEMEGCGTVLAHPRYLQHHIKYQHLLKKKYVCPHPSCGRLFRLQKQLLRHAKHHT (SEQ
ID NO: 678), DQRDYICEYCARAFKSSHNLAVHRMIHTGEK (SEQ ID NO: 679),
SALPQEVSIAASRPSRGWRSSRTSVSRHRDTENTRSSRSKTGSLQLICK SEPNTDQLDY (SEQ
ID NO: 680), PFKDDPRDETYKPHLERETPKPRRKSG (SEQ ID NO: 681),
QYVRCEMEGCGTVLAHPRYLQHHIKYQHLLKKKYVCPHPSCGRLFRL QKQLLRHAKHHTD (SEQ
ID NO: 682), and QRDYICEYCARAFKSSHNLAVHRMI HTGEKHY (SEQ ID NO:
683). Also preferred are polynucleotide fragments encoding these
polypeptide fragments. When tested against renal mesangial cell
lines, supernatants removed from cells containing this gene induced
a calcium flux in the renal cells tested in the FLIPR assay (small
molecule concentration and membrane permeability assay). Thus, it
is likely that this gene activates renal mesangial cells via the
binding of a ligand to a receptor. The FLIPR assay indicates the
binding of a ligand to a receptor, which is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
Alterations in small molecule concentration can be measured to
identify supernatants which bind to receptors of a particular
cell.
[0331] It has been discovered that this gene is expressed primarily
in Rhabdomyosarcoma, Melanocyte and colon cancer tissue and to a
lesser extent in smooth muscle, pancreatic tumor, and apoptotic T
cells.
[0332] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: cancer. Similarly, polypeptides
and antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune and hemopoetic,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0333] The tissue distribution suggests that the protein product of
this clone would be useful for study, diagnosis and treatment of
cancer and hematopoietic disorders. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0334] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 383 as residues: Phe-10 to Ile-46, Val-54 to Pro-91,
Lys-123 to Pro-129, His-150 to Tyr-156, Thr-179 to Asn-185.
[0335] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:74 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 4588 of SEQ ID NO:74, b is an integer
of 15 to 4602, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:74, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 65
[0336] It has been discovered that this gene is expressed primarily
in human adipose and salivary gland tissue and to a lesser extent
in human bone marrow and fetal kidney.
[0337] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: metabolic and immune disorders.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the metabolic and hematopoietic systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0338] The tissue distribution suggests that the protein product of
this clone would be useful for study, diagnosis of metabolic and
immune disorders. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0339] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:75 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1241 of SEQ ID NO:75, b is an integer
of 15 to 1255, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:75, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 66
[0340] This translated product of this gene was recently identified
as oxytocinase splice variant 1. (See Accession Nos. 2209276 and
d1010078.) Preferred polypeptide fragments comprise the amino acid
sequence:
EMFDSLSYFKGSSLLLMLKTYLSEDVFQHAVVLYLHNHSYASIQSDDLWDSFNEVTNQTLDVKRMMKTW
TLQKGFPLVTVQKKGKELFIQQERFFLNMKPEIQPSDTRYM (SEQ ID NO: 739). Also
preferred are polynucleotide fragments encoding this polypeptide
fragment. The gene encoding the disclosed cDNA is believed to
reside on chromosome 5. Accordingly, polynucleotides related to
this invention are useful as a marker in linkage analysis for
chromosome 5.
[0341] This gene is expressed primarily in tonsils, and to a lesser
extent, in fetal liver spleen.
[0342] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: immune, hematopoietic, or
developmental disorders. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune or hematopoietic system, expression of
this gene at significantly higher or lower levels may be detected
in certain tissues or cell types (e.g. immune, hematopoietic,
developmental, hepatic, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, amniotic fluid, bile, serum, plasma,
urine, synovial fluid or spinal fluid) taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue from an
individual not having the disorder.
[0343] The tissue distribution in fetal liver spleen and tonsils
suggests that the protein product of this clone would be useful for
the treatment and diagnosis of hematopoetic related disorders such
as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Similarly, expression
within fetal tissue and other cellular sources marked by
proliferating cells suggests that this protein may play a role in
the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0344] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 385 as residues: Pro-99 to Tyr-108.
[0345] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:76 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 461 of SEQ ID NO:76, b is an integer
of 15 to 475, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:76, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 67
[0346] It has been discovered that this gene is expressed primarily
in hematopoietic cells, particularly apoptotic T-cells, and to
lesser extent in primary dendritic cells and adipose tissue.
[0347] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of apoptotic T-cells,
primary dendritic cells, and adipose tissue present in a biological
sample and for diagnosis of the following diseases and conditions:
hematopoietic diseases including cancer and general immune
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the oral and intestinal mucosa as well as hematopoietic and immune
systems, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0348] The tissue distribution suggests that the protein product of
this clone would be useful for treatment of diseases of the immune
system, including cancer, hematopoietic and infectious diseases.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0349] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 386 as residues: Ala-2 to Ala-9, Ser-36 to
Trp-41.
[0350] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:77 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 451 of SEQ ID NO:77, b is an integer
of 15 to 465, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:77, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 68
[0351] It has been discovered that this gene is expressed primarily
in kidney cortex and to a lesser extent in infant brain, heart,
uterus, and blood.
[0352] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of kidney tissue present
in a biological sample and for diagnosis of the following diseases
and conditions: soft tissue cancer, inflammation, kidney fibrosis.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous and endocrines systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0353] The tissue distribution suggests that the protein product of
this clone would be useful for study and treatment of cancer and
fibroses. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0354] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 387 as residues: Arg-8 to Lys-20, Asp-28 to Glu-33,
Val-42 to Thr-47, Glu-52 to Ser-57, Thr-65 to Phe-73, Gly-75 to
Tyr-85, Ala-90 to Arg-95, Arg-111 to Gln-124, Lys-134 to Ser-140,
Phe-165 to Asp-170, Pro-199 to Cys-205, Gly-239 to Val-244, Glu-247
to Trp-253, Tyr-319 to Gln-326, Leu-423 to Asn-433.
[0355] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:78 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1893 of SEQ ID NO:78, b is an integer
of 15 to 1907, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:78, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 69
[0356] The translation product of this gene shares strong sequence
homology with vertebrate and invertebrate protein tyrosine
phosphatases. The gene encoding the disclosed cDNA is thought to
reside on chromosome 1. Accordingly, polynucleotides related to
this invention are useful as a marker in linkage analysis for
chromosome 1.
[0357] It has been discovered that this gene is expressed primarily
in endometrial tumors, melanocytes, myeloid progenitors and to a
lesser extent in infant brain, adipocytes, and several
hematopoietic stem cells. Therefore, nucleic acids of the invention
are useful as reagents for differential identification of
transformed hematopoietic and epithelial cells present in a
biological sample and for diagnosis of the following diseases and
conditions: cancer of skin and endometrium, leukemia. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
nervous and hematopoietic systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0358] The tissue distribution and sequence similarity with
tyrosine phosphatases suggests that the protein product of this
clone would be useful for study and treatment of cancer and
hematopoietic disorders. Expression within embryonic tissue and
other cellular sources marked by proliferating cells suggests that
this protein may play a role in the regulation of cellular
division. Additionally, the expression in hematopoietic cells and
tissues suggests that this protein may play a role in the
proliferation, differentiation, and/or survival of hematopoietic
cell lineages. In such an event, this gene may be useful in the
treatment of lymphoproliferative disorders, and in the maintenance
and differentiation of various hematopoietic lineages from early
hematopoietic stem and committed progenitor cells. Similarly,
embryonic development also involves decisions involving cell
differentiation and/or apoptosis in pattern formation. Thus, this
protein may also be involved in apoptosis or tissue differentiation
and could again be useful in cancer therapy. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0359] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 388 as residues: Met-1 to Gly-6.
[0360] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:79 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1154 of SEQ ID NO:79, b is an integer
of 15 to 1168, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:79, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 70
[0361] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2. Thus, polynucleotides related to this invention are
useful as a marker in linkage analysis for chromosome 2.
[0362] It has been discovered that this gene is expressed primarily
in osteoclastoma, breast, and infant brain and to a lesser extent
in various fetal and transformed bone, ovarian, and neuronal
cells.
[0363] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: degenerative conditions of the
brain and skeleton. Similarly, polypeptides and antibodies directed
to those polypeptides are useful to provide immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the nervous and skeletal system, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues or cell types (e.g., cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0364] The tissue distribution suggests that the protein product of
this clone would be useful for study and treatment of degenerative,
neurological and skeletal disorders. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0365] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:80 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1271 of SEQ ID NO:80, b is an integer
of 15 to 1285, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:80, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 71
[0366] This gene was originally cloned from tumor cell lines.
Recently another group has also cloned this gene, calling it the
human malignant melanoma metastasis-suppressor (KiSS-1) gene. (See
Accession No. U43527). Preferred polypeptide fragments comprise the
amino acid sequence: LEKVASVGNSRPTGQQLESLGLLA (SEQ ID NO: 685),
VHREEASCYCQAEPSGDL (SEQ ID NO: 686), RPALRQAGGGTREPRQKRWAGL (SEQ ID
NO: 687), and AVNFRPQRSQSM (SEQ ID NO: 688). Any frame shifts can
easily be resolved using known molecular biology techniques. The
gene encoding the disclosed cDNA is thought to reside on chromosome
1. Accordingly, polynucleotides related to this invention are
useful as a marker in linkage analysis for chromosome 1.
[0367] This gene is expressed primarily in many types of carcinomas
and to a lesser extent in many normal organs.
[0368] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissues(s) or cell type(s) present in a biological sample and for
diagnosis of the following diseases and conditions: cancer,
particularly melanomas, and other hyperproliferative disorders.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
transformed organ tissue, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder. As a tumor suppressor gene,
increase amounts of the polypeptide can be used to treat patients
having a particular cancer.
[0369] The tissue distribution indicates that this gene and the
translated product is useful for the diagnosis and study of cancer.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0370] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 390 as residues: Gly-29 to Leu-38, Pro-52 to Ala-59,
Ser-65 to Ser-70, Pro-73 to Gly-85, Pro-89 to Pro-95, Arg-105 to
Trp-114, Pro-128 to Arg-137.
[0371] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:81 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1276 of SEQ ID NO:81, b is an integer
of 15 to 1290, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:81, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 72
[0372] It has been discovered that this gene is expressed primarily
in striatum and to a lesser extent in adipocytes and
hemangioperiocytoma.
[0373] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of striatal cells present
in a biological sample and for diagnosis of the following diseases
and conditions: neurological disorders as well as fat and lysosomal
storage diseases. Similarly, polypeptides and antibodies directed
to those polypeptides are useful to provide immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the nervous and immune systems, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues (e.g., cancerous and wounded tissues) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0374] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis, study and treatment of
neurodegenerative and growth disorders, such as Alzheimers Disease,
Parkinsons Disease, Huntingtons Disease, Tourette Syndrome, and
schizophrenia. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0375] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:82 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 670 of SEQ ID NO:82, b is an integer
of 15 to 684, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:82, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 73
[0376] It has been discovered that this gene is expressed primarily
in bone marrow stromal cells and to a lesser extent in smooth
muscle, testes, endothelium, and brain.
[0377] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of bone marrow present in
a biological sample and for diagnosis of the following diseases and
conditions: connective tissue and hematopoietic diseases.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the skeletal and hematopoietic systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0378] The tissue distribution suggests that the protein product of
this clone would be useful for study, diagnosis, and treatment of
connective tissue and blood diseases. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0379] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 392 as residues: Thr-17 to Glu-24, Gly-28 to Pro-45,
Ser-47 to Pro-59, Lys-62 to Asp-79, Gly-91 to Gly-99, Ser-144 to
Leu-157, Gln-199 to Thr-210, Thr-215 to Ser-221, Pro-231 to
Ser-247.
[0380] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:83 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2010 of SEQ ID NO:83, b is an integer
of 15 to 2024, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:83, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 74
[0381] The gene encoding the disclosed cDNA is believed to reside
on chromosome 12. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
12.
[0382] It has been discovered that this gene is expressed primarily
in brain, fetal liver and lung and to a lesser extent in retina,
spinal chord, activated T-cells and endothelial cells.
[0383] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of brain and regenerating
liver present in a biological sample and for diagnosis of the
following diseases and conditions: CNS and spinal chord injuries,
immune disorders. Similarly, polypeptides and antibodies directed
to those polypeptides are useful to provide immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the nervous and immune system, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues or cell types (e.g., cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0384] The tissue distribution suggests that the protein product of
this clone would be useful for study and treatment of hematopoietic
disorders, such as anemia, pancytopenia, leukopenia,
thrombocytopenia or leukemia. Furthermore, the tissue distribution
also suggests that the protein product of this clone would be
useful for the study and treatment of neurological disorders such
as Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,
Tourette Syndrome, and schizophrenia.
[0385] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 393 as residues: Pro-55 to Lys-63.
[0386] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:84 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 917 of SEQ ID NO:84, b is an integer
of 15 to 931, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:84, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 75
[0387] The translation product of this gene shares sequence
homology with GTP binding proteins (intracellular).
[0388] It has been discovered that this gene is expressed primarily
in bone marrow, brain, and melanocytes and to a lesser extent in
various endocrine and hematopoietic tissues.
[0389] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: hematopietic and nervous system
conditions. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous and immune, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0390] The tissue distribution and homology to nucleotide binding
factors suggests that the protein product of this clone would be
useful for study, diagnosis, and treatment of brain degenerative,
skin and blood diseases. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0391] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 394 as residues: Ala-3 to Glu-12, Glu-36 to Thr-41,
Val-49 to Leu-60.
[0392] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:85 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 811 of SEQ ID NO:85, b is an integer
of 15 to 825, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:85, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 76
[0393] It has been discovered that this gene is expressed primarily
in activated T-cells and to a lesser extent in retina, brain, and
fetal bone.
[0394] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of activated T-cells and
developing brain present in a biological sample and for diagnosis
of the following diseases and conditions: immune deficiencies and
skeletal and neuronal growth disorders. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the nervous, immune, and
skeletomuscular systems, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0395] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis, study and treatment of
cancer, urogenital, and brain degenerative diseases. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0396] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:86 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1224 of SEQ ID NO:86, b is an integer
of 15 to 1238, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:86, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 77
[0397] It has been discovered that this gene is expressed primarily
in fetal liver, activated monocytes, osteoblasts and to a lesser
extent in synovial, brain, and lymphoid tissues.
[0398] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of myeloid and lymphoid
present in a biological sample and for diagnosis of the following
diseases and conditions: inflammation, immune deficiencies, cancer.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system and skeleton, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0399] The tissue distribution suggests that the protein product of
this clone would be useful for study, diagnosis, and treatment of
lymphoid and mesenchymal cancers and nervous system diseases.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0400] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 396 as residues: Asp-129 to Ser-134, Arg-159 to
Ala-168, Arg-258 to Pro-264.
[0401] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:87 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1446 of SEQ ID NO:87, b is an integer
of 15 to 1460, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:87, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 78
[0402] The translation product of this gene shares sequence
homology with polymerase polyprotein precursor which is thought to
be important in DNA repair and replication
[0403] It has been discovered that this gene is expressed primarily
in infant brain and to a lesser extent in tumors and tumor cell
lines
[0404] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: cancer, especially of the neural
system and developing organs. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the neural system expression of
this gene at significantly higher or lower levels may be detected
in certain tissues or cell types (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0405] The tissue distribution and homology to polymerase
polyprotein precursor suggests that the protein product of this
clone would be useful for diagnosis and treatment of cancers,
especially of the neural system and developing organs. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0406] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:88 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1381 of SEQ ID NO:88, b is an integer
of 15 to 1395, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:88, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 79
[0407] It has been discovered that this gene is expressed primarily
in muscle and endothelial cells and to a lesser extent in
brain.
[0408] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: vascular diseases. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
vascular system, expression of this gene at significantly higher or
lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0409] The tissue distribution suggests that the protein product of
this clone would be useful for treatment and diagnosis of disorders
of the vascular and neural system including cardiovascular and
endothelial. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0410] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 398 as residues: Tyr-5 to Glu-13.
[0411] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:89 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1172 of SEQ ID NO:89, b is an integer
of 15 to 1186, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:89, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 80
[0412] This gene appears to be a human homolog of a mouse
metalloproteinase/disintegrin protein, which is thought to play a
role in skeletal muscle development involving the formation of
multi-nucleated myotubes. The gene encoding the disclosed cDNA is
believed to reside on chromosome 10. Accordingly, polynucleotides
related to this invention are useful as a marker in linkage
analysis of chromosome 10.
[0413] It has been discovered that this gene is expressed primarily
in placenta and to a lesser extent in fetal liver
[0414] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: developmental disorders and
disorder of the haemopoietic system, fetal liver and placenta.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
developmental disorders and disorder of the hematopoietic system,
fetal liver and placenta, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0415] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis and treatment of
developmental disorders and disorders of the hematopoietic system,
fetal liver and placenta. Alternatively, the tissue distribution
and nucleotide homology to metalloproteinase/disintegrin protein
suggest that the translation product of this gene may be important
in the fetal development of skeletal muscle. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0416] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 399 as residues: Cys-34 to Tyr-41, Lys-53 to
Lys-68.
[0417] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:90 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1807 of SEQ ID NO:90, b is an integer
of 15 to 1821, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:90, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 81
[0418] It has been discovered that this gene is expressed primarily
in bone marrow, placenta and tissues and organs of the
hematopoietic system
[0419] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: disorders of the bone and
hematopoietic system. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune, bone and hematopoietic system,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0420] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis and treatment of disorders
of the immune, bone and hematopoietic system, such as anemia,
pancytopenia, leukopenia, thrombocytopenia or leukemia. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0421] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:91 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 848 of SEQ ID NO:91, b is an integer
of 15 to 862, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:91, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 82
[0422] The translation product of this gene shares sequence
homology with secretory carrier membrane protein which is thought
to be important in protein transport and export. Any frame shifts
in coding sequence can be easily resolved using standard molecular
biology techniques. Another group recently cloned this gene,
calling it SCAMP. (See Accession No. 2232243.). The gene encoding
the disclosed cDNA is thought to reside on chromosome 1.
Accordingly, the polynucleotides related to this invention are
useful as a marker in linkage analysis of chromosome 1.
[0423] It has been discovered that this gene is expressed primarily
in prostate, breast and spleen, and to a lesser extent in several
other tissues and organs
[0424] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: disorders of the breast prostate
and spleen. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly
disorders of the breast prostate and spleen, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues or cell types (e.g., cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0425] The tissue distribution and homology to secretory carrier
membrane protein suggests that the protein product of this clone
would be useful for diagnosis and treatment of disorders of the
breast, prostate and spleen. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0426] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 401 as residues: Ala-2 to Glu-13, Ser-15 to Gln-22,
Pro-43 to Ala-57, Gln-70 to Gln-89, Leu-100 to Ser-119.
[0427] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:92 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 682 of SEQ ID NO:92, b is an integer
of 15 to 696, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:92, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 83
[0428] It has been discovered that this gene is expressed primarily
in developing organs and tissue like placenta and infant brain and
to a lesser extent in developed organs and tissue like cerebellum
and heart
[0429] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neurological diseases.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the neural system, expression of this gene at significantly higher
or lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0430] The tissue distribution suggests that the protein product of
this clone would be useful for treatment and diagnosis of diseases
of the neural system including neurological disorders and cancer.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0431] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:93 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1872 of SEQ ID NO:93, b is an integer
of 15 to 1886, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:93, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 84
[0432] The translation product of this gene shares sequence
homology with ATPase 6 in Trypanosoma brucei which is thought to be
important in metabolism.
[0433] It has been discovered that this gene is expressed primarily
in tumor and fetal tissues and to a lesser extent in melanocytes,
kidney cortex, monocytes and ovary.
[0434] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: metabolism disorders. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the fetal
systems, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0435] The tissue distribution and homology to ATPase indicates
that the protein product of this clone would be useful for
treatment and diagnosis of metabolism disorders, especially in
fetal and tumor tissue growth. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0436] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 403 as residues: Gln-17 to Ile-22, Gln-54 to
Ser-60.
[0437] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:94 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1760 of SEQ ID NO:94, b is an integer
of 15 to 1774, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:94, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 85
[0438] The translation product of this gene shares sequence
homology with the immunoglobulin superfamily of proteins which are
known to be important in immune response and immunity.
[0439] It has been discovered that this gene is expressed primarily
in stromal cells, colon cancer, lung, amygdala, melanocyte and to a
lesser extent in a variety of other tissues.
[0440] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of stromal cell
development and cancer. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the stromal cells, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0441] The tissue distribution and homology to immunoglobulin
indicates that the protein product of this clone would be useful
for treatment and diagnosis of immune system disorders. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0442] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:95 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1765 of SEQ ID NO:95, b is an integer
of 15 to 1779, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:95, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 86
[0443] The translation product of this gene shares sequence
homology with transcription initiation factor eIF-4 gamma which is
thought to be important in gene transcription. Additionally, the
translation product of this gene shares significant homology to a
Homo sapiens polyadenylate binding protein-interacting protein-1
(PAIP1), which could play a possible role as an RNA editing enzyme
or polypeptide, the defect of which could lead to translational
errors or cancer. One embodiment of this clone comprises
polypeptides of the following amino acid sequence: HQPPQPKAPGF (SEQ
ID NO: 689),
GAQCEVPASPQRPSRPGALPEQTRPLRAPPSSQDKIPQQNSESAMAKPQVVVAPVLMSKLSVNAPEF
(SEQ ID NO: 690), EDGCEDYPTLSEYVQDFLNHLTEQPGSFETEI
EQFAETLNGCVTTDDALQELVELIYQQATSIPNFSYMGARLCNYLSHHLTISPQSGNFRQLLLQRCRTEYEV
KDQAAKGDEVTRKRFHAFVLFLGELYLNLEIKGTNGQVTRADILQVGLRELLNALFSNPMDDNLICAVKL
LKLTGSVLEDAWKEKGKMDMEEIIQRIENVVLDANCSRDVKQMLLKLVELRSSNWGRVHATSTYREATP
ENDPNYFMNEPTFYTSDGVPFTAADPDYQEKYQELLEREDFFPDYEENGTDLSGAGDPYL (SEQ
ID NO: 691), or AYEKFCLESERKRKQ (SEQ ID NO: 692). Recently another
group published a paper in which they described a Homo sapiens
polyadenylate binding protein-interacting protein-1 (PAIP1) mRNA,
including the complete coding sequence (Genbank accession AF013758,
Nature 392, 520-523 (1998)).
[0444] It has been discovered that this gene is expressed primarily
in tumor tissues.
[0445] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumorigenesis. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly in tumor
tissues, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0446] The tissue distribution and homology to transcription
initiation factor eIF-4 gamma and Homo sapiens polyadenylate
binding protein-interacting protein-1 (PAIP1) suggests that the
protein product of this clone would be useful for gene regulation
in tumorigenesis. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0447] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 405 as residues: Met-1 to Arg-15.
[0448] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:96 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2787 of SEQ ID NO:96, b is an integer
of 15 to 2801, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:96, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 87
[0449] The translation product of this gene shares sequence
homology at low level in prolines with secreted basic proline-rich
peptide II-2 which is thought to be important in protein structure
or inhibiting hydroxyapatite formation in vitro.
[0450] It has been discovered that this gene is expressed primarily
in endometrial tumor and fetal lung.
[0451] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: endometrial tumors. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
muscular/skeletal and reproductive systems, expression of this gene
at significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0452] The tissue distribution and homology to secreted basic
proline-rich peptide II-2 suggests that the protein product of this
clone would be useful for inhibiting hydroxyapatite formation or
establishing cell/tissue structure. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0453] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 406 as residues: Glu-175 to Glu-193.
[0454] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:97 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1617 of SEQ ID NO:97, b is an integer
of 15 to 1631, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:97, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 88
[0455] It has been discovered that this gene is expressed primarily
in: amniotic cells induced with TNF in culture; and to a lesser
extent in colon tissue from a patient with Crohn's Disease;
parathyroid tumor; activated T-cells; cells of the human Caco-2
cell line; adenocarcinoma; colon; corpus colosum; fetal kidney;
pancreas tumor; fetal brain; early stage brain, and anergic
T-cells.
[0456] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumors. Similarly, polypeptides
and antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system; e.g., tumors,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0457] The tissue distribution indicates that the protein product
of this clone is useful for modulating tumorigenesis and other
immune system conditions such as disorders in immune response.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0458] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 407 as residues: Pro-61 to Glu-75.
[0459] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:98 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 490 of SEQ ID NO:98, b is an integer
of 15 to 504, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:98, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 89
[0460] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis of chromosome
1.
[0461] It has been discovered that this gene is expressed primarily
in fetal liver/spleen and hematopoietic cells and to a lesser
extent in brain, osteosarcoma, and testis tumor.
[0462] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: leukemia and hematopoietic
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hematopoietic and immune systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0463] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis and treatment of
hematopoietic and immune disorders. Expression of this gene product
in hematopoietic cells suggests a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g. by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0464] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 408 as residues: Gly-13 to Cys-18, Arg-30 to Ser-36,
Ala-53 to Phe-58.
[0465] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:99 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1402 of SEQ ID NO:99, b is an integer
of 15 to 1416, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:99, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 90
[0466] The translation product of this gene shares weak sequence
homology with mouse Gcap1 protein which is developmentally
regulated in brain. The gene encoding the disclosed cDNA is thought
to reside on chromosome 11. Accordingly, polynucleotides of this
invention are useful as a marker in linkage analysis for chromosome
11.
[0467] It has been discovered that this gene is expressed primarily
in infant and adult brain and fetal liver/spleen and to a lesser
extent in smooth muscle, T cells, and a variety of other
tissues.
[0468] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neurological or hematopoietic
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous, hematopoietic, immune, and endocrine systems,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0469] The tissue distribution and its homology to Gcap1 protein
suggests that the protein product of this clone would be useful for
treating and diagnosis of disorders in neuronal, hematopoietic,
immune, and endocrine systems. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0470] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:100 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2833 of SEQ ID NO:100, b is an
integer of 15 to 2847, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:100, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 91
[0471] It has been discovered that this gene is expressed primarily
in brain and hematopoietic cells and to a lesser extent in tumor
tissues.
[0472] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: disorder in nervous,
hematopoietic, immune systems and tumorigenesis. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the in
nervous, hematopoietic, immune systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0473] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of disorders in
the nervous, hematopoietic, and immune systems. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0474] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:101 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1380 of SEQ ID NO:101, b is an
integer of 15 to 1394, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:101, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 92
[0475] The translation product of this gene shares sequence
homology with neuroendocrine-specific protein A which is thought to
be important in neurologic systems.
[0476] It has been discovered that this gene is expressed primarily
in brain tissues.
[0477] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neural disorders and
degeneration disease. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the central or peripheral nervous systems,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0478] The tissue distribution and homology to
neuroendocrine-specific protein A suggests that the protein product
of this clone would be useful for treatment or diagnosis of neural
disorders and degeneration disease. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0479] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 411 as residues: Glu-65 to Gln-70.
[0480] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:102 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 780 of SEQ ID NO:102, b is an integer
of 15 to 794, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:102, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 93
[0481] The translation product of this gene shares sequence
homology with collagen-like protein and prolin-rich protein which
are thought to be important in connective tissue function and
tissue structure.
[0482] It has been discovered that this gene is expressed primarily
in fetal liver/spleen and brain tissues.
[0483] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: neuronal or hematopoietic
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous and hematopoietic systems, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0484] The tissue distribution and homology to collagen-like
protein and proline-rich proteins suggests that the protein product
of this clone would be useful for supporting brain and
hematopoietic tissue function and diagnosis and treatment of
disorders in these functions. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0485] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:103 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1530 of SEQ ID NO:103, b is an
integer of 15 to 1544, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:103, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 94
[0486] It has been discovered that this gene is expressed primarily
in embryonic tissues and tumor tissues.
[0487] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: cancer. Similarly, polypeptides
and antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system (e.g., tumors),
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0488] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis and treatment of cancer.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0489] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 413 as residues: Pro-39 to Leu-46, Pro-96 to Arg-103,
Pro-117 to Ser-124.
[0490] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:104 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 857 of SEQ ID NO:104, b is an integer
of 15 to 871, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:104, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 95
[0491] It has been discovered that this gene is expressed primarily
in brain tumor, placenta, and melanoma.
[0492] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: brain tumor or melanoma.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the brain or melanocytes, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0493] The tissue distribution indicates that the translation
product of this gene is useful in the diagnosis and treatment of
brain tumors and melanoma. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0494] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 414 as residues: Ser-44 to Glu-50, Pro-53 to
Gly-60.
[0495] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:105 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 390 of SEQ ID NO:105, b is an integer
of 15 to 404, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:105, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 96
[0496] The translation product of this gene shares sequence
homology with a yeast membrane protein, SUR4, which encodes for
APA1 that acts on a glucose-signaling pathway that controls the
expression of several genes that are transcriptionally regulated by
glucose. The gene encoding the disclosed cDNA is thought to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
1.
[0497] It has been discovered that this gene is expressed primarily
in fetal liver, and to a lesser extent in placenta and breast
tissue.
[0498] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of fetal liver or
defects of glucose-regulated ATPase activities in tissues.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the fetal immune/hematopoietic system, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0499] The tissue distribution and homology to yeast SUR4 membrane
protein suggests that the protein product of this clone would be
useful for diagnosis and treatment of defects of fetal liver or
defects of glucose-regulated ATPase activities. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0500] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 415 as residues: Ala-15 to Gln-20, Tyr-89 to Glu-103,
His-253 to Leu-261.
[0501] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:106 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1528 of SEQ ID NO:106, b is an
integer of 15 to 1542, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:106, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 97
[0502] The gene encoding the disclosed cDNA is thought to reside on
chromosome 18. Accordingly, polynucleotides related to the
invention are useful as a marker in linkage analysis for chromosome
18.
[0503] It has been discovered that this gene is expressed primarily
in fetal liver, brain, and amniotic fluid.
[0504] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of the fetal immune
system and adult brain. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the fetal immune system and adult brain, expression
of this gene at significantly higher or lower levels may be
detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0505] The tissue distribution indicates that the protein product
of this clone is useful for detecting defects of the fetal immune
and hematopoietic systems since fetal liver is the predominant
organ responsible for hematopoiesis in the fetus. In addition, the
gene product of this gene is thought to be useful for detecting
certain neurological defects of the brain. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0506] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:107 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2313 of SEQ ID NO:107, b is an
integer of 15 to 2327, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:107, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 98
[0507] The translation product of this gene shares sequence
homology with an yolk protein precursor, Vitellogenin which is
thought to be important in binding lipids such as phosvitin. The
gene encoding the disclosed cDNA is thought to reside on chromosome
10. Accordingly, polynucleotides related to the invention are
useful as a marker in linkage analysis for chromosome 10.
[0508] It has been discovered that this gene is expressed primarily
in amniotic cells and fetal liver.
[0509] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects in amniotic cells, fetal
liver development and the fetal immune system. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the fetal
liver and developing tissues, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0510] The tissue distribution and homology to vitellogenin
indicates that the protein product of this clone is useful for
treatment and diagnosis of defects in amniotic cells, fetal liver
development and the fetal immune system. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0511] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 417 as residues: Pro-24 to Ala-32.
[0512] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:108 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1048 of SEQ ID NO:108, b is an
integer of 15 to 1062, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:108, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 99
[0513] It has been discovered that this gene is expressed primarily
in placenta, endometrial tumor, osteosarcoma and stromal cells.
[0514] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumor of the endometrium or
bone, and osteosarcoma. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the obsteric system (i.e. placenta, endometrium)
and the bones, expression of this gene at significantly higher or
lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0515] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis and treatment of tumors
and abnormalities of the endometrium, and the bones because of its
abundance in the aforementioned tissues. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0516] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 418 as residues: Leu-3 to Arg-8, His-11 to Glu-16,
Leu-19 to Glu-27, Lys-67 to Glu-73, Tyr-79 to Asp-87, Lys-101 to
Ile-107, Val-143 to Leu-155, Thr-162 to Ser-169.
[0517] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:109 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2525 of SEQ ID NO:109, b is an
integer of 15 to 2539, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:109, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 100
[0518] It has been discovered that this gene is expressed primarily
in hepatocellular tumor.
[0519] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: hepatocellular tumor. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the liver,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0520] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of
hepatocellular cancer because of its abundant expression in this
tissue. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0521] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 419 as residues: Ala-100 to Ser-109, His-138 to
His-145, Glu-171 to Ser-182.
[0522] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:110 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1737 of SEQ ID NO:110, b is an
integer of 15 to 1751, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:110, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 101
[0523] The gene encoding the disclosed cDNA is thought to reside on
chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0524] It has been discovered that this gene is expressed primarily
in Corpus Colosum, fetal lung and infant brain.
[0525] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of the Corpus Colosum or
defects of the fetal lung. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the Corpus Colosum and brain in general, and fetal
lung, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0526] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of defects of
the Corpus Colosum and brain in general, and defects of fetal lung.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0527] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:111 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1103 of SEQ ID NO:111, b is an
integer of 15 to 1117, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:111, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 102
[0528] It has been discovered that this gene is expressed primarily
in T cells and stromal cells, and to a lesser extent in adrenal
gland.
[0529] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of T cell immunity and
stromal cell development. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune system, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0530] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of defects of T
cell immunity and stromal cell development because of its abundant
expression in these tissues. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0531] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 421 as residues: Tyr-12 to Glu-17.
[0532] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:112 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1299 of SEQ ID NO:112, b is an
integer of 15 to 1313, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:112, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 103
[0533] It has been discovered that this gene is expressed primarily
in infant brain and placenta.
[0534] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: defects of the brain and nervous
system. Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous system, especially brain, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0535] The tissue distribution indicates that the protein product
of this clone is useful for detecting defects of the brain,
especially in young children. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0536] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:113 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1640 of SEQ ID NO:113, b is an
integer of 15 to 1654, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:113, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 105
[0537] It has been discovered that this gene is expressed primarily
in human osteoclastoma and to a lesser extent in human pancreas
tumor.
[0538] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: cancer, particularly
osteoclastoma and pancreatic tumor. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly in transformed tissues, expression
of this gene at significantly higher or lower levels may be
detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0539] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of some types
of tumors, particularly pancreatic cancer and osteoclastoma.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0540] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 424 as residues: Glu-17 to Leu-23, Ala-148 to
Leu-173.
[0541] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:115 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 828 of SEQ ID NO:115, b is an integer
of 15 to 842, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:115, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 106
[0542] The gene encoding the disclosed cDNA is thought to reside on
chromosome 11. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
11.
[0543] It has been discovered that this gene is expressed primarily
in fetal liver/spleen, and to a lesser extent in activated T-Cells,
8 hrs.
[0544] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: immune disorders. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0545] The tissue distribution suggests that the protein product of
this clone would be useful for diagnosis or treatment of immune
disorders. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0546] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 425 as residues: Leu-31 to Lys-37.
[0547] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:116 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1626 of SEQ ID NO:116, b is an
integer of 15 to 1640, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:116, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 107
[0548] It has been discovered that this gene is expressed primarily
in human embryo and to a lesser extent in spleen and chronic
lymphocytic leukemia.
[0549] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: leukemia. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
or hemopoietic systems, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0550] The tissue distribution indicates that the protein product
of this clone is useful for the diagnosis and treatment of
leukemia. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0551] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 426 as residues: Gly-26 to Asn-31, Glu-53 to
Gly-62.
[0552] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:117 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 938 of SEQ ID NO:117, b is an integer
of 15 to 952, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:117, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 108
[0553] The gene encoding the disclosed cDNA is thought to reside on
chromosome 17. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
17.
[0554] It has been discovered that this gene is expressed primarily
in placenta, and to a lesser extent in early stage human brain and
in lung.
[0555] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: fetal developmental
abnormalities. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly in
fetal and amniotic tissue, expression of this gene at significantly
higher or lower levels may be detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0556] The tissue distribution indicates that the protein product
of this is useful for production of growth factor(s) associated
with fetal development. Preferred polypeptides comprise the
full-length polypeptide shown in the sequence listing, truncated
however, at the amino terminus and beginning with QTIE. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0557] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 427 as residues: Pro-12 to Gly-22, Ile-57 to Cys-63,
Leu-87 to Met-96, Ala-109 to Gln-118, Glu-144 to Phe-150.
[0558] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:118 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1242 of SEQ ID NO:118, b is an
integer of 15 to 1256, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:118, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 109
[0559] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
2.
[0560] It has been discovered that this gene is expressed primarily
in fetal spleen, and to a lesser extent in B-Cell lymphoma and
T-Cell lymphoma.
[0561] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: lymphoma. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be detected in certain tissues (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0562] The tissue distribution indicates that the protein product
of this clone is useful for the treatment and diagnosis of human
lymphomas. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0563] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 428 as residues: Glu-9 to Arg-15, Pro-71 to
Lys-79.
[0564] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:119 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1129 of SEQ ID NO:119, b is an
integer of 15 to 1143, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:119, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 110
[0565] The translation product of this gene shares sequence
homology with sarcoma amplified sequence (SAS), a tetraspan
receptor which is thought to be important in malignant fibrous
histiocytoma and liposarcoma. The translation product of this clone
also shares sequence homology with Transmembrane 4 superfamily
proteins. The transmembrane 4 superfamily (TM4SF), or tetraspan
superfamily, is the second biggest subfamily among CD antigen
superfamilies. Members of this family appear to serve a role in the
activation of T-cells, for example as an activation antigen of
T-cells. All TM4SF members contain four putative transmembrane
domains, two extracellular loops, and two short cytoplasmic tails.
One embodiment of this clone comprises polypeptides of the
following amino acid sequence: GPGPPPTSALLPRLGXAPKARTKQLSGNLRRS
RIYVQLPATTGSKMVCGGFACSKNCLCALNLLYTLVSLLLIGIAAWGIGFGLISSLRVVGVVIAVGIFLFLIA
LVGLIGAVKHHQVLLFFY (SEQ ID NO: 693). An additional embodiment would
be the polynucleotides encoding these polypeptides.
[0566] It has been discovered that this gene is expressed primarily
in human osteoclastoma, and to a lesser extent in pineal gland and
infant brain.
[0567] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: malignant fibrous histiocytoma
and liposarcoma. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0568] The tissue distribution and homology to sarcoma amplified
sequence (SAS) indicates that the protein product of this clone is
useful for treatment of, osteosarcoma, malignant fibrous
histiocytoma and liposarcoma and related cancers, particularly
sarcomas. Alternatively, the homology to TM4SF proteins indicates
that the translation product of this clone may function as an
activating agent of T-cells and their immune response in the body.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0569] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 429 as residues: Asn-100 to Gly-105, Asn-114 to
Leu-126, Ser-133 to Thr-139, Lys-146 to Cys-151, Tyr-188 to
Pro-200.
[0570] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:120 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1768 of SEQ ID NO:120, b is an
integer of 15 to 1782, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:120, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 111
[0571] The translation product of this gene shares sequence
homology with 6.8K proteolipid protein, mitochondrial--bovine.
[0572] It has been discovered that this gene is expressed primarily
in Wilm's tumor and to a lesser extent in cerebellum and
placenta.
[0573] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: Wilm's tumor. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
or renal systems, expression of this gene at significantly higher
or lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0574] The tissue distribution and homology to 6.8K proteolipid
protein indicates that the protein product of this clone is useful
for diagnostic and therapeutics associated with tumors,
particularly Wilm's tumor disease. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0575] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 430 as residues: Asp-42 to Ala-47.
[0576] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:121 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 596 of SEQ ID NO:121, b is an integer
of 15 to 610, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:121, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 112
[0577] It has been discovered that this gene is expressed primarily
in embryonic tissue and to a lesser extent in osteoblasts,
endothelial cells, macrophages (GM-CSF treated), and bone
marrow.
[0578] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: immune disorders. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0579] The tissue distribution suggests that the protein product of
this clone would be useful for treatment or diagnosis of immune
disorders. Expression of this gene product in immune cells suggests
a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g. by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the
natural gene product may be involved in immune functions. Therefore
it may be also used as an agent for immunological disorders
including arthritis, asthma, immune deficiency diseases such as
AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease,
sepsis, acne, and psoriasis. In addition, this gene product may
have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Protein, as well as, antibodies directed against the protein may
show utility as a tissue-specific marker and/or immunotherapy
target for the above listed tissues. Preferred polypeptides encoded
by this gene comprise the following amino acid sequence:
MITDVQLAIFANMLGVSLFLLVVLYHYVAVNNPKKQE (SEQ ID NO: 694).
[0580] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 431 as residues: Asn-20 to Glu-25.
[0581] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:122 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 512 of SEQ ID NO:122, b is an integer
of 15 to 526, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:122, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 113
[0582] The gene encoding the disclosed cDNA is thought to reside on
chromosome 5. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
5.
[0583] It has been discovered that this gene is expressed primarily
in hepatocellular tumor, and to a lesser extent in fetal
liver/spleen.
[0584] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumors, particularly
hepatocellular tumors. Similarly, polypeptides and antibodies
directed to those polypeptides are useful to provide immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the hepatic system, expression of this gene at
significantly higher or lower levels may be detected in certain
tissues or cell types (e.g., cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue from an individual not having the disorder.
[0585] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of tumors,
particularly hepatocellular tumors. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0586] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:123 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2067 of SEQ ID NO:123, b is an
integer of 15 to 2081, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:123, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 114
[0587] The translation product of this gene exhibits a very high
degree of sequence identity with the human Pig8 gene which is
thought to be important in p53 mediated apoptosis. The sequence of
this gene has since been published by Polyak and colleagues (Nature
389, 300-306 (1997)). In addition, the predicted translation
product of this contig exhibits very high sequence homology with a
murine gene denoted as EI24 which is also thought to be important
in p53 mediated apoptosis.
[0588] It has been discovered that this gene is expressed primarily
in infant brain and activated T-cells and to a lesser extent in
bone marrow, fetal liver, and prostate.
[0589] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: cancer and tissue damage by
radiation and anti-cancer drugs. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the nervous and immune systems,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0590] The tissue distribution and homology to human Pig8 and
murine EI24 genes suggests that the protein product of this clone
would be useful for preventing apoptosis in patients being treated
with anti-oncogenic drugs such as etoposide,
hydroperoxycyclophosphamide, and X-irradiation, since this protein
product is upregulated in cells undergoing such treatment where p53
was overexpressed. It may also be useful in the treatment of
hematopoietic disorders and in boosting numbers of hematopoietic
stem cells by interfering with the apoptosis of progenitor cells.
The mature polypeptide is predicted to comprise the following amino
acid sequence: EEMADSVKTFLQDLARGIKD
SIWGICTISKLDARIQQKREEQRRRRASSVLAQRRAQSIERKQESEPRIVSRIFQCCAWNGGVFWFSLLLFYR
VFIPVLQSVTARIIGDPSLHGDVWSWLEFFLTSIFSALWVLPLFVLSKVVNAIWFQDIADLAFEVSGRKPHPF
PSVSKIIADMLFNLLLQALFLIQGMFVSLFPIHLVGQLVSLLHMSLLYSLYCFEYRWFNKGIEMHQRLSNIER
NWPYYFGFGLPLAFLTAMQSSYIISGCLFSILFPLFIISANEAKTPGKAYLFQLRLFSLVVFLSNRLFHKTVY-
L QSALSSSTSAEKFPSPHPSPAKLKATAGH (SEQ ID NO: 695). Accordingly,
polypeptides comprising the foregoing amino acid sequence are
provided as are polynucleotides encoded such polypeptides. Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0591] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 433 as residues: Asn-24 to Gly-30, Thr-65 to
Ala-78.
[0592] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:124 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1703 of SEQ ID NO:124, b is an
integer of 15 to 1717, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:124, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 115
[0593] It has been discovered that this gene is expressed primarily
in stromal cells and to a lesser extent in multiple sclerosis.
[0594] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: affecting the nervous system.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the nervous system, expression of this gene at significantly higher
or lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0595] The tissue distribution suggests that the protein product of
this clone would be useful for treatment and diagnosis of multiple
sclerosis and other autoimmune diseases. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0596] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 434 as residues: Pro-28 to Ile-33, Lys-88 to Ser-93,
Glu-182 to Pro-192.
[0597] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:125 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 790 of SEQ ID NO:125, b is an integer
of 15 to 804, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:125, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 116
[0598] It has been discovered that this gene is expressed primarily
in the gall bladder.
[0599] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: gall stones or infection of the
digestive system. Similarly, polypeptides and antibodies directed
to those polypeptides are useful to provide immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the digestive system or renal system, expression of
this gene at significantly higher or lower levels may be detected
in certain tissues or cell types (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0600] The tissue distribution suggests that the protein product of
this clone would be useful for possible prevention of digestive
disorders where there may be a lack of digestive enzymes produced
or in the detection and possible prevention of gall stones.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0601] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 435 as residues: Lys-32 to Val-37.
[0602] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:126 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 417 of SEQ ID NO:126, b is an integer
of 15 to 431, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:126, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 117
[0603] The translation product of this gene shares sequence
homology with the dystrophin gene which is thought to be important
in the building and maintenance of muscles. The gene encoding the
disclosed cDNA is thought to reside on chromosome 1. Accordingly,
polynucleotides related to this invention are useful as a marker in
linkage analysis for chromosome 1.
[0604] It has been discovered that this gene is expressed primarily
in placenta and to a lesser extent in fetal brain and fetal liver
spleen.
[0605] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: muscular dystrophy, Duchenne and
Becker's muscular dystrophies. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the skeletal muscle system,
expression of this gene at significantly higher or lower levels may
be detected in certain tissues or cell types (e.g., cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder.
[0606] The tissue distribution and homology to the dystrophin gene
suggests that the protein product of this clone would be useful for
diseases related the degenerative myopathies that are characterized
by the weakness and atrophy of muscles without neural degradation;
such as Duchenne and Becker's muscular dystrophies. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0607] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 436 as residues: Lys-2 to Val-9, Lys-52 to Leu-58,
Gln-88 to Asp-99, Met-115 to Val-122, Arg-124 to Glu-135, Glu-143
to Pro-159, Ser-167 to Ile-174, Glu-190 to Leu-195, Arg-237 to
Arg-248, Asp-275 to Tyr-281, Pro-293 to Glu-308, Ile-329 to
Arg-335, Gln-341 to Gln-347, Arg-355 to Trp-362.
[0608] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:127 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 3738 of SEQ ID NO:127, b is an
integer of 15 to 3752, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:127, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 118
[0609] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
2.
[0610] It has been discovered that this gene is expressed primarily
in olfactory and to a lesser extent in cartilage.
[0611] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: connective tissue diseases;
chondrosarcomas. Similarly, polypeptides and antibodies directed to
those polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the connective tissue, expression of this gene at significantly
higher or lower levels may be detected in certain tissues (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0612] The tissue distribution and suggests that the protein
product of this clone would be useful for tumors of connective
tissues, osteoarthritis and the treatment and diagnosis of
chondrosarcoma. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0613] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:128 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1130 of SEQ ID NO:128, b is an
integer of 15 to 1144, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:128, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 119
[0614] The gene encoding the disclosed cDNA is thought to reside on
chromosome 20. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
20.
[0615] It has been discovered that this gene is expressed primarily
in Activated Neutrophils and to a lesser extent in fetal spleen,
and CD34 positive cells from cord blood.
[0616] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: allergies, defects in
hematopoesis and inflammation. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system and
hematopoesis system the, expression of this gene at significantly
higher or lower levels may be detected in certain tissues (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0617] The tissue distribution suggests that the protein product of
this clone would be useful for reducing the allergic effects felt
by allergy suffers by neutralizing the activity of the immune
system, especially since neutrophils are abundant in persons
suffering from allergies and other inflammatory conditions.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0618] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:129 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1816 of SEQ ID NO:129, b is an
integer of 15 to 1830, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:129, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 120
[0619] The translation product of this gene shares sequence
homology with poly A binding protein II which is thought to be
important in RNA binding for transcription of RNA to DNA. In
specific embodiments, polypeptides of the invention comprise the
following amino acid sequence:
MATPASAPDTRALVADFVGYKLRQKGYVCGAGPGEGPAADPLHQAMRAAGDEFETRFRRTFSDLAAQL
HVTPGSAQQRFTQVSDELFQGGPNWGRLVAFFVFGAALCAESVNKEMEPLVGQVQEWMVAYLETRLAD
WIHSSGGWLSQITEAEMADEVICSEILSDCDSAASSPDLEELEAIKARVREMEEEAEKLKELQNEVEKQMN
MSPPPGNAGPVIMSIEEKMEADARSIYVGNVDYGATAEELEAHFHGCGSVNRVTILCDKFSGHPKGFAYIE
FSDKESVRTSLALDESLFRGRQIKVIPKRTNRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRV
YRGRARATSWYSPY (SEQ ID NO: 696), VEKQMNMSPPPGNAGPVIMSIEEKM (SEQ ID
NO: 697), ELEAIKARVRC (SEQ ID NO: 698),
VDYGATAEELEAHFHGCGSVNRVTILCDKFSGHP (SEQ ID NO: 699), or
ALDESLFRGRQIKVIPKRTNRPGISTTDRG (SEQ ID NO: 700). Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0620] This gene is expressed primarily in colon and to a lesser
extent in brain and immune system.
[0621] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: colon cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and digestive system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., colon, tissue and cells of the immune system,
and brain or other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, serum, plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0622] The tissue distribution and homology to poly A binding
protein II indicate that polynucleotides and polypeptides
corresponding to this gene are useful for detection and treatment
of colon cancer and other disorders of the digestive system.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0623] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:130 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1850 of SEQ ID NO:130, b is an
integer of 15 to 1864, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:130, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 121
[0624] The translation product of this gene shares sequence
homology with thymidine diphosphoglucose 4.6 dehydrase which is
thought to be important in the metabolism of sugar. In specific
embodiments, polypeptides of the invention comprise the following
amino acid sequence:
PRRVEPLYIEVDQIYHLASPASPPNYMYNPIKTLKTNTIGTLNMLGLAKRVGARLLLASTSEVYGDPEVHPQ
SEDYWGHVNPIGPRACYDEGKRVAETMCYAYMKQEGVEVRVARIFNTFGPRMHMNDGRVVSNFILQAL
QGEPLTVYGSGSQTRAFQYVSDLVNGLVALMNSNVSSPVNLGNPEEHTILEFAQLIKNLVGSGSEIQFLSEA
QDDPQKRKPDIKKAKLMLGWEPVVPLEEGLNKAIHYFRKELEYQANNQYIPKPKPARIKKGRTRHS
(SEQ ID NO: 701), MLGLAKRVGAR (SEQ ID NO: 702), LLASTSEVYGDP (SEQ
ID NO: 703), IGPRACYDEGKRVAET (SEQ ID NO: 704), RVARIFNT (SEQ ID
NO: 705), or NDGRVVSNFI (SEQ ID NO: 706). Polynucleotides encoding
these polypeptides are also encompassed by the invention. The gene
encoding the disclosed cDNA is believed to reside on chromosome 2.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 2.
[0625] This gene is expressed primarily in fetal liver and spleen
and to a lesser extent in infant brain.
[0626] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: diabetes. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
endocrine system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., liver, spleen, and brain and other tissue of the
nervous system, and cancerous and wounded tissues) or bodily fluids
(e.g. bile, amniotic fluid, lymph, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0627] The tissue distribution and homology to thymidine
diphosphoglucose 4.6 dehydrase indicate that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
persons with diabetes since it appears that this protein is needed
in the metabolism of sugar to its more basic components.
Alternatively, expression within fetal tissues and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0628] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 440 as residues: Ser-42 to Gly-47, Leu-62 to Pro-79,
Ser-84 to Lys-89, Phe-122 to Asn-128, Pro-148 to Thr-154.
[0629] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:131 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2027 of SEQ ID NO:131, b is an
integer of 15 to 2041, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:131, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 122
[0630] The translation product of this gene shares sequence
homology with ceruloplasmin which is thought to be important in the
metabolism and transport of iron and copper. Ceruloplasmin also
contains domains with homology to clotting factors V and VIII.
Defects in the circulating levels of ceruloplasmin
(aceruloplasminemia) have been associated with certain disease
conditions such as Wilson disease, and the accompanying
hepatolenticular degeneration. In specific embodiments,
polypeptides of the invention comprise the following amino acid
sequence:
YQAARIYYIMAEEVEWDYCPDRSWEREWHNQSEKDSYGYIFLSNKDGLLGSRYKKAVFREYTDGTFRXP
RPRTGPEEHLGILGPLIKGEVGDILTVVFKNNASRPYSVHAHGVLESTTVWPLAAEPGEVVTYQWNIPERS
GPGPMTLLVFPGSIILQWIPSRTCIVAWWGPWLSAKRASWXPHGGRXDMDREFALLFLIFDENKSWYLEE
NVATHGSQDPGSINLQDETFLESNKMHAINGKLYANLRGLTMYQGERVAWYMLAMGQDVDLHTIHFHA
ESFLYRNGENYRADVVDLFPGTFEVVEMVASNPGTWLMHCHVTDHVHAGMETLFTVFSRTEHLSPLTVIT
KETEKAVPPRDIEEGNVKMLGMQIPIKNVEMLASVLVAISVTLLLVVLALGGVVWYQHRQRKLRRNRRSI
LDDSFKLLSFKQ (SEQ ID NO: 707), or
GGHLSVEHPREVWPWANDSACVSWIYYSAVDPIKDMYSGLVGPLA ICQKGILXAPWRT (SEQ ID
NO: 708). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0631] This gene is expressed primarily in brain and retina and to
a lesser extent in endothelial cells.
[0632] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: diseases marked by defects in
iron metabolism; aceruloplasminemia not characterized by defects in
the known ceruloplasmin gene locus; nonclassical Wilson disease;
movement disorders; and tumors derived from a brain tissue origin.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the brain, retina, and nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., brain and other tissue of the
nervous system, retinal tissue, and endothelial cells, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0633] The tissue distribution and homology to ceruloplasmin
indicate that polynucleotides and polypeptides corresponding to
this gene are useful for treatment of patients with
aceruloplasminemia, or other defects in iron and/or copper
metabolism. Mutations in this locus could also be diagnostic for
patients currently experiencing or predicted to experience
aceruloplasminemia. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0634] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 441 as residues: Asn-30 to Asp-37.
[0635] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:132 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1998 of SEQ ID NO:132, b is an
integer of 15 to 2012, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:132, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 123
[0636] The gene encoding the disclosed cDNA is believed to reside
on chromosome 9. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
9.
[0637] This gene is expressed primarily in brain and B cell
lymphoma and to a lesser extent in fetal liver and spleen.
[0638] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune, or developmental
disorders, particularly B cell lymphoma; tumors and diseases of the
brain and/or spleen; hematopoietic defects. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the brain and hematopoietic
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., brain and other tissue of the nervous system, blood cells,
liver, and spleen, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0639] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
disorders in neuronal, hematopoietic, and immune systems. It could
potentially be useful for neurodegenerative disorders and neuronal
and/or hematopoietic cell survival or proliferation. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0640] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:133 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1655 of SEQ ID NO:133, b is an
integer of 15 to 1669, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:133, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 124
[0641] Polynucleotides of the invention do not comprise the nucleic
acid sequence shown as Genbank Accession No. gb|G20858|G20858,
which is hereby incorporated herein by reference.
[0642] This gene is expressed primarily in osteoclastoma,
dermatofibrosarcoma, and B cell lymphoma and to a lesser extent in
endothelial cells.
[0643] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancers, in particular osteoclastoma,
dermatofibrosarcoma, and B cell lymphoma. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the bone, immune, and
circulatory system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., bone, epidermis, blood cells, muscle, immune, and
endothelial cells, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0644] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis
and treatment of cancers and lymphoma; osteoporosis; and the
control of cell proliferation and/or differentiation. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0645] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:134 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1551 of SEQ ID NO:134, b is an
integer of 15 to 1565, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:134, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 125
[0646] This gene is expressed primarily in immune tissues and
hematopoietic cells, particularly in activated T cells and
neutrophils, spleen, and fetal liver, and to a lesser extent in
infant adrenal gland.
[0647] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders,
particularly defects in T cell activation; hematopoietic disorders,
particularly tumors of hematopoietic and/or adrenal gland origin.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hematopoietic and/or endocrine systems, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues and cell types (e.g., cells and tissues of the
immune system, hematopoietic cells, blood cells, liver, and adrenal
gland, and cancerous and wounded tissues) or bodily fluids (e.g.
lymph, amniotic fluid, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0648] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for immune
and/or hematopoietic disorders; diseases related to proliferation
and/or differentiation of hematopoietic cells; defects in T cell
and neutrophil activation and responsiveness; and endocrine and/or
metabolic disorders, particularly of early childhood and
development. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0649] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 444 as residues: Met-1 to His-6.
[0650] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:135 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1993 of SEQ ID NO:135, b is an
integer of 15 to 2007, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:135, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 126
[0651] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
1.
[0652] This gene is expressed primarily in placenta and endothelial
cells and to a lesser extent in melanocytes and embryonic
tissues.
[0653] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: tumors of an endothelial cell
origin; angiogenesis associated with tumor development and
metastasis. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the vascular system and developing embryo, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues and cell types (e.g., placenta, endothelial
cells, melanocytes, and embryonic tissues, and cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0654] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
developmental disorders; inhibition of angiogenesis; and vascular
patterning. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0655] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 445 as residues: Pro-16 to Gln-21.
[0656] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:136 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1277 of SEQ ID NO:136, b is an
integer of 15 to 1291, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:136, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 127
[0657] This gene is expressed primarily in endothelial cells and
hematopoietic tissues, including spleen, tonsils, leukocytes, and
both B- and T-cell lymphomas.
[0658] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders,
particularly tumors of an endothelial cell and/or hematopoietic
origin; leukemias and lymphomas. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune and vascular systems,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
endothelial cells, hematopoietic cells, spleen, tonsils, and blood
cells, and cancerous and wounded tissues) or bodily fluids (e.g.
lymph, serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0659] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
manipulation of angiogenesis; the differentiation and morphogenesis
of endothelial cells; the proliferation and/or differentiation of
hematopoietic cells; and the commitment of hematopoietic cells to
distinct cell lineages. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0660] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:137 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1892 of SEQ ID NO:137, b is an
integer of 15 to 1906, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:137, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 128
[0661] The gene encoding the disclosed cDNA is thought to reside on
chromosome 11. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
11.
[0662] It has been discovered that this gene is expressed primarily
in kidney medulla and to a lesser extent in spleen from chronic
myelogenous leukemia patients, prostate cancer, and some other
tissues.
[0663] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumors of a kidney origin;
chromic myelogenous leukemia; prostate cancer. Similarly,
polypeptides and antibodies directed to those polypeptides are
useful to provide immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the kidney
and spleen, expression of this gene at significantly higher or
lower levels may be detected in certain tissues or cell types
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue from
an individual not having the disorder.
[0664] The tissue distribution suggests that the protein product of
this clone would be useful for the diagnosis and treatment of
kidney disorders and cancer, particularly chronic myelogenous
leukemia and prostate cancer. It may also be useful for the
enhancement of kidney tubule regeneration in the treatment of acute
renal failure. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0665] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:138 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1921 of SEQ ID NO:138, b is an
integer of 15 to 1935, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:138, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 129
[0666] The sequence shares homology with a rat neuroligin protein,
which is thought to play a role in recognition between neurons. All
neuroligins contain an N-terminal hydrophobic sequence with the
characteristics of a cleaved signal peptide followed by a large
esterase homology domain, a highly conserved single transmembrane
region, and a short cytoplasmic domain. Neuroligins constitute a
multigene family of brain-specific proteins with distinct isoforms
that may have overlapping functions in mediating recognition
processes between neurons. The gene encoding the disclosed cDNA is
thought to reside on chromosome 17. Accordingly, polynucleotides
related to this invention are useful as a marker in linkage
analysis for chromosome 17.
[0667] It has been discovered that this gene is expressed primarily
in adult and infant brain and to a lesser extent in mesenchymal or
fibroblast cells, as well as tissues with a mesenchymal origin.
[0668] Therefore, nucleic acids of the invention are useful as
reagents for differential identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of the
following diseases and conditions: tumors of a brain and/or
mesenchymal origin; neurodegenerative disorders; cancer; fibrosis.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the brain and of mesenchymal cells and tissues, expression of this
gene at significantly higher or lower levels may be detected in
certain tissues or cell types (e.g., cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue from an individual not having
the disorder.
[0669] The tissue distribution and homology to neuroligins suggests
that the protein product of this clone would be useful for the
diagnosis of tumors of a brain and/or mesenchymal origin;
neurodegenerative disorders; cancer; and fibrosis, based upon the
expression of this gene within those tissues. Fibrosis is
considered as mesenchymal cells and fibroblasts are the primary
cellular targets involved in this pathological condition.
[0670] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 448 as residues: Ser-42 to Pro-47.
[0671] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:139 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1432 of SEQ ID NO:139, b is an
integer of 15 to 1446, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:139, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 130
[0672] This gene is expressed primarily in hepatocellular cancer
and to a lesser extent in fetal tissues as well as testes
tumor.
[0673] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: hepatic, developmental, or
reproductive disorders, particularly liver cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
digestive system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., liver, fetal tissue, and testes and other
reproductive tissue, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0674] The tissue distribution suggests that the protein product of
this clone would be useful for the detection and treatment of liver
disorders and cancers (e.g. hepatoblastoma, jaundice, hepatitis,
liver metabolic diseases and conditions that are attributable to
the differentiation of hepatocyte progenitor cells). Similarly,
expression within fetal tissues and other cellular sources marked
by proliferating cells suggests that this protein may play a role
in the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0675] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:140 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1095 of SEQ ID NO:140, b is an
integer of 15 to 1109, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:140, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 131
[0676] This gene is expressed only in infant early brain.
[0677] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: development and neural
disorders, particularly neurodegenerative disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the brain
and nervous system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., brain and other tissue of the nervous system and
cancerous and wounded tissues) or bodily fluids (e.g. amniotic
fluid, serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0678] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
diseases of the brain in children and in treating nervous system
disorders such as Alzheimer's disease, schizophrenia, dementia,
depression, etc. Similarly, expression within fetal tissues and
other cellular sources marked by proliferating cells suggests that
this protein may play a role in the regulation of cellular
division, and may show utility in the diagnosis and treatment of
cancer and other proliferative disorders. Similarly, embryonic
development also involves decisions involving cell differentiation
and/or apoptosis in pattern formation. Thus this protein may also
be involved in apoptosis or tissue differentiation and could again
be useful in cancer therapy. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0679] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:141 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 483 of SEQ ID NO:141, b is an integer
of 15 to 497, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:141, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 132
[0680] The gene encoding the disclosed cDNA is believed to reside
on chromosome 14. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
14.
[0681] This gene is expressed primarily in brain and to a lesser
extent in glioblastoma.
[0682] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neural disorders,
particularly Alzheimer's disease, schizophrenia, depression, mania,
and dementia. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the brain and nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., brain and other tissue of the
nervous system, and cancerous and wounded tissues) or bodily fluids
(e.g., serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0683] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
brain disorders such as Alzheimer's disease, schizophrenia,
depression, mania, and dementia. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0684] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 451 as residues: Met-1 to Cys-8, Pro-10 to Gly-16,
Gln-76 to Lys-89.
[0685] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:142 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 255 of SEQ ID NO:142, b is an integer
of 15 to 269, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:142, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 133
[0686] The translation product of this gene shares sequence
homology with ribitol dehydrogenase of Caenorhabditis elegans which
is thought to be important in metabolism of sugars, in addition to
being a key enzyme in biosynthesis pathways (See Genbank Accession
No. gi|1125838).
[0687] This gene is expressed primarily in macrophage and to a
lesser extent in T-cell lymphoma and lung.
[0688] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders,
particularly tissue destruction in inflammation. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g. immune, blood cells and lung, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, pulmonary surfactant, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0689] The tissue distribution and homology to ribitol
dehydrogenase indicate that polynucleotides and polypeptides
corresponding to this gene are useful for altering macrophage
metabolism in diseases such as inflammation where macrophages are
known to cause excess tissue destruction. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0690] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 452 as residues: Thr-87 to Lys-93, Thr-247 to
Ser-253, Pro-299 to Ser-311, Ser-315 to Arg-320.
[0691] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:143 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1255 of SEQ ID NO:143, b is an
integer of 15 to 1269, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:143, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 134
[0692] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0693] This gene is expressed primarily in pancreatic tumors, and
to a lesser extent in synovial sarcoma.
[0694] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, endocrine, gastrointestinal, or skeletal disorders,
particularly those involving proliferating tissues, such as tumors
or cancers. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the endocrine and connective tissue systems, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., pancreas, and
synovial tissue, and cancerous and wounded tissues) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder.
[0695] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating and
diagnosing various cancers. Similarly, expression within pancreatic
tumors and other cellular sources marked by proliferating cells
suggests that this protein may play a role in the regulation of
cellular division, and may show utility in the diagnosis and
treatment of cancer and other proliferative disorders. Similarly,
embryonic development also involves decisions involving cell
differentiation and/or apoptosis in pattern formation. Thus this
protein may also be involved in apoptosis or tissue differentiation
and could again be useful in cancer therapy. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0696] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:144 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1930 of SEQ ID NO:144, b is an
integer of 15 to 1944, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:144, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 135
[0697] This gene is expressed primarily in T cell lines such as
Raji and to a lesser extent in infant brain.
[0698] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune, developmental, or
neural disorders, particularly inflammation. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., blood cells, and brain and other tissue of the nervous
system, and cancerous and wounded tissues) or bodily fluids (e.g.
lymph, amniotic fluid, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0699] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating and
diagnosing inflammatory diseases such as rheumatoid arthritis,
sepsis, inflammatory bowel disease, and psoriasis, as well as
neutropenia. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0700] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 454 as residues: Pro-16 to Arg-34, Gly-45 to
Asn-50.
[0701] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:145 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1007 of SEQ ID NO:145, b is an
integer of 15 to 1021, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:145, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 136
[0702] The translation product of this gene shares high sequence
homology with SAR1 subfamily of GTP-binding proteins which is
thought to be important in vesicular transport in mammalian cells.
In specific embodiments, polypeptides of the invention comprise the
following amino acid sequence:
ARGGPRQPSRFAVRSPGVRPTPSRGLRSLQSQTLSDLD (SEQ ID NO: 709),
GFSSVLQFLGLYKK (SEQ ID NO: 710),
GKLVFLGLDNAGKTTLLHMLKDDRLGQHVPTLHPT (SEQ ID NO: 711),
SEELTIAGMTFTTFDLGGH (SEQ ID NO: 712), QARRVWKNYLPAINGIVFLVDCADH
(SEQ ID NO: 713), NVPILILGNKIDR (SEQ ID NO: 714), or
EVFMCSVLKRQGYGEGFRW (SEQ ID NO: 715). Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0703] This gene is expressed primarily in serum-stimulated smooth
muscle cells and to a lesser extent in a T-cell lymphoma.
[0704] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: vascular, muscular, immune,
or metabolic disorders or diseases, particularly those affecting
vesicular transport. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the muscular system, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues and cell types (e.g., blood
cells, and smooth muscle, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0705] The tissue distribution and homology to GTP-binding proteins
indicate that polynucleotides and polypeptides corresponding to
this gene are useful for gene therapy in treating the large number
of diseases involved in defective vesicular transport within cells.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0706] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 455 as residues: Lys-46 to Gln-52, Leu-108 to
Leu-115, Gly-155 to Lys-160, Lys-182 to Phe-190.
[0707] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:146 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1271 of SEQ ID NO:146, b is an
integer of 15 to 1285, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:146, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 137
[0708] The translation product of this gene shares sequence
homology with a protein found in C. elegans cosmid F25B5. The gene
encoding the disclosed cDNA is believed to reside on chromosome 17.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 17.
[0709] This gene is expressed primarily in a fetal tissues and to a
lesser extent in melanocytes.
[0710] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: abnormal fetal development,
especially of the pulmonary or integumentary system. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the fetal
pulmonary system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., fetal tissue, pulmonary tissue, and melanocytes,
and cancerous and wounded tissues) or bodily fluids (e.g. lymph,
amniotic fluid, pulmonary surfactant, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0711] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment
and diagnosis of diseases affecting the pulmonary system, such as
emphysema. Similarly, expression within fetal tissue and other
cellular sources marked by proliferating cells suggests that this
protein may play a role in the regulation of cellular division, and
may show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0712] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 456 as residues: Ala-7 to Ser-15, Asp-47 to Lys-55,
Tyr-160 to Val-166.
[0713] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:147 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1372 of SEQ ID NO:147, b is an
integer of 15 to 1386, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:147, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 138
[0714] The gene encoding the disclosed cDNA is believed to reside
on chromosome 11. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
11.
[0715] This gene is expressed primarily in gall bladder and to a
lesser extent in smooth muscle.
[0716] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: gastrointestinal, digestive
system, or vascular disorders, particularly atherosclerosis,
vasculitis, aneurysm, and gall bladder problems. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
digestive system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., gall bladder and tissue of the digestive system,
and smooth muscle, and cancerous and wounded tissues) or bodily
fluids (e.g. bile, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0717] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
diseases of the digestive or cardiopulmonary system. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0718] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 457 as residues: Leu-50 to Ala-59, Pro-75 to
Thr-80.
[0719] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:148 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2084 of SEQ ID NO:148, b is an
integer of 15 to 2098, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:148, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 139
[0720] Polypeptides of the invention do not comprise the amino acid
sequence shown as Genbank accession no. W42028, which is hereby
incorporated herein by reference. It is likely that the open
reading frame containing the predicted signal peptide continues in
the 5' direction. In specific embodiments, polypeptides of the
invention comprise the following amino acid sequence:
STHASELRRRREPVGVWSSRRPRGPDAAPGAGRRWCXAASGPPRAATSEPSATAGVRRAAGPAGPGARX
ARRGGVELIRI (SEQ ID NO: 716). Polynucleotides encoding these
polypeptides are also encompassed by the invention. The gene
encoding the disclosed cDNA is believed to reside on chromosome 7.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 7.
[0721] This gene is expressed primarily in placenta and to a lesser
extent in brain.
[0722] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: developmental, reproductive,
or neural disorders, particularly abnormal fetal development.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
developing tissues, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., placenta, and brain and other tissue of the
nervous system, and cancerous and wounded tissues) or bodily fluids
(e.g. lymph, amniotic fluid, serum, plasma, urine, synovial fluid
or spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0723] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating and
diagnosing abnormal fetal development, reproductive, or neural
disorders. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0724] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 458 as residues: Leu-78 to Thr-88, Gly-92 to
Gly-108.
[0725] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:149 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1833 of SEQ ID NO:149, b is an
integer of 15 to 1847, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:149, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 140
[0726] In specific embodiments, polypeptides of the invention
comprise the following amino acid sequence:
ECLDCHLHCCLLDQVVPKHYTLDASLPLRLRPESMEKLRCLRACVIRSLYHMYEPFAARISKNPAIPESTPS
TLKNSKCLLFWCRKIVGNRQEPMWEFNFKFKKQSPRLKSKCTGGLQPPVQYEDVHTNPDQDCCLLQVTTL
NFIFIPIVMGMIFTLFTINVSTDMRHHRVRLVFQDSPVHGGRKLRSEQGVQVILDPVHSVRLFDWWHPQYP
FSLRA (SEQ ID NO: 717). Polynucleotides encoding these polypeptides
are also encompassed by the invention.
[0727] This gene is expressed primarily in smooth muscle and to a
lesser extent in ovary, prostate cancer, and activated
monocytes.
[0728] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: vascular disorders,
particularly hypertension and atherosclerosis. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
circulatory system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., smooth muscle, ovary and other reproductive
tissue, prostate, and blood cells, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0729] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
diseases of the circulatory system, such as hypertension,
atherosclerosis, etc. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0730] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:150 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1555 of SEQ ID NO:150, b is an
integer of 15 to 1569, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:150, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 141
[0731] Polynucleotides of the invention do not comprise the nucleic
acid sequence shown as Genbank accession no. gb|G11389|G11389 which
is hereby incorporated herein by reference.
[0732] This gene is expressed primarily in fetal spleen, and to a
lesser extent in placenta and bone marrow.
[0733] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: developmental, immune,
hematopoietic, or reproductive disorders, particularly anemia and
other diseases affecting blood cells. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the circulatory and pulmonary
systems, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., spleen, placenta, reproductive, developmental,
hematopoietic, bone marrow, and blood cells, and cancerous and
wounded tissues) or bodily fluids (e.g. amniotic fluid, lymph,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0734] The tissue distribution in fetal spleen and bone marrow
suggests that the protein product of this clone would be useful for
the treatment and diagnosis of hematopoetic related disorders such
as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Alternatively,
expression within fetal tissues and other cellular sources marked
by proliferating cells suggests that this protein may play a role
in the regulation of cellular division, and may show utility in the
diagnosis and treatment of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0735] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 460 as residues: Leu-50 to Lys-58, Lys-64 to Leu-71,
His-89 to Thr-94, Pro-102 to Trp-110, Tyr-162 to Cys-169, Asp-367
to Ala-377.
[0736] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:151 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1526 of SEQ ID NO:151, b is an
integer of 15 to 1540, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:151, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 142
[0737] The predicted translation product of this contig is a human
homolog of the murine tetracycline/sugar transporter molecule
recently reported by Matsuo and colleagues (Biochem. Biophys. Res.
Commun. 238 (1), 126-129 (1997), incorporated herein by reference).
In specific embodiments, polypeptides of the invention comprise the
following amino acid sequence: LMRSIGNKNT (SEQ ID NO: 718),
AWYGFGSEPC (SEQ ID NO: 719), SSITFPAVSALVSRTAD (SEQ ID NO: 720),
ADQQGVVQGMITGIRGLCNGLGPALYGFIFYIFHVELKELPITGTDLGTNTSPQHHFEQN (SEQ
ID NO: 721), SIIPGPPFLFGACS (SEQ ID NO: 722),
FIPEHTNLSLRSSSWRKHCGSHSHPHNTQAPGEAKEP (SEQ ID NO: 723), or LLQDTNV
(SEQ ID NO: 724). Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0738] This gene is expressed primarily in synovium and to a lesser
extent in endothelial cells.
[0739] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: skeletal disorders,
particularly rheumatoid arthritis and inflammation. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and lymphatic systems, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., synovial tissue, and endothelial cells, and
cancerous and wounded tissues) or bodily fluids (e.g. lymph, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0740] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment
and diagnosis of inflammatory diseases, such as rheumatoid
arthritis, leukemia, neutropenia, inflammatory bowel disease,
psoriasis, sepsis, and the like. In addition, the protein product
of this clone may also be useful for the treatment and diagnosis of
a variety of autoimmune disorders, as rheumatoid arthritis, lupus,
scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation, and specific joint abnormalities as well as
chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita,
familial osteoarthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0741] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 461 as residues: Leu-106 to Gln-113, Arg-153 to
Lys-177.
[0742] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:152 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1705 of SEQ ID NO:152, b is an
integer of 15 to 1719, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:152, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 143
[0743] It is likely that the open reading frame containing the
predicted signal peptide continues in the 5' direction. In specific
embodiments, polypeptides of the invention comprise the following
amino acid sequence: SKGNSSHSKELEASPSVVGRQPGAVFWELWDVPLGARENRRK
(SEQ ID NO: 725). Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0744] This gene is expressed primarily in placenta and to a lesser
extent in melanocyte, fetal liver and spleen, and bone marrow.
[0745] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: abnormal early development.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, lower levels may
be routinely detected in certain tissues and cell types (e.g.,
placenta, hematopoietic, integumentary, developmental, liver,
spleen, and bone marrow, and cancerous and wounded tissues) or
bodily fluids (e.g. amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0746] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
treatment and diagnosis of abnormal early development phenomena and
diseases. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0747] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:153 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 849 of SEQ ID NO:153, b is an integer
of 15 to 863, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:153, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 144
[0748] Polynucleotides of the invention do not comprise the nucleic
acid sequence shown as Genbank accession no. gb|G15249|G15249,
which is hereby incorporated herein by reference.
[0749] This gene is expressed primarily in fetal liver and
spleen.
[0750] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: developmental or immune
disorders, particularly anemia and neutropenia. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and blood systems, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., liver and spleen, developmental, immune,
hematopoietic, and cancerous and wounded tissues) or bodily fluids
(e.g. amniotic fluid, lymph, bile, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0751] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful in hematopoiesis
and bone marrow regeneration as it is most abundant in fetal
tissues responsible for the generation of hematopoeitic cells.
Similarly, expression within fetal tissues and other cellular
sources marked by proliferating cells suggests that this protein
may play a role in the regulation of cellular division, and may
show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0752] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 463 as residues: Ser-35 to Lys-46.
[0753] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:154 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1087 of SEQ ID NO:154, b is an
integer of 15 to 1101, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:154, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 145
[0754] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0755] The translation product of this gene shares sequence
homology with protein tyrosine phosphatase which is thought to be
important in transducing signals to activate cells such as T cell,
B cell and other cell types.
[0756] This gene is expressed primarily in T cells and tissues in
early stages of development and to a lesser extent in cancers.
[0757] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immuno-related diseases and
cancer. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., embryonic and fetal tissue, undifferentiated
cells, and blood cells, immune tissues, and cancerous and wounded
tissues) or bodily fluids (e.g. lymph, amniotic fluid, serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0758] The tissue distribution and homology to the protein tyrosine
phosphatase suggests that the protein product of this clone would
be useful for the diagnosis and treatment of a variety of immune
system disorders. Expression of this gene product in T-cells
suggests a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g. by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the
natural gene product may be involved in immune functions. Therefore
it may be also used as an agent for immunological disorders
including arthritis, asthma, immunodeficiency diseases such as
AIDS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated cytotoxicity; immune reactions to transplanted organs and
tissues, such as host-versus-graft and graft-versus-host diseases,
or autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
scleroderma and tissues. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0759] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:155 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2017 of SEQ ID NO:155, b is an
integer of 15 to 2031, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:155, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 146
[0760] This gene is expressed primarily in T cell and to a lesser
extent in B cell, macrophages and tumor tissues.
[0761] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immuno-disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g. immune, blood cells, and cancerous and wounded tissues) or
bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0762] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for regulating
the immune system and therefore can be used in treating diseases
such as autoimmune diseases and cancers. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0763] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:156 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1967 of SEQ ID NO:156, b is an
integer of 15 to 1981, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:156, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 147
[0764] This gene is expressed primarily in placenta, and to a
lesser extent in endothelial cells, testis tumor, ovarian cancer,
uterine cancer.
[0765] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to cancer. Similarly, polypeptides and antibodies directed
to these polypeptides are useful in providing immunological probes
for differential identification of the tissue(s) or cell type(s).
For a number of disorders of the above tissues or cells,
particularly of the immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., placenta, endothelial cells,
testis and ovary and other reproductive tissue, and cancerous and
wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0766] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis
and treatment of cancers or reproductive disorders. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0767] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 466 as residues: Lys-136 to Thr-145.
[0768] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:157 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 901 of SEQ ID NO:157, b is an integer
of 15 to 915, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:157, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 148
[0769] This sequence has significant homology to mouse torsin A.
Recently, another group cloned the human Torsin A gene. (See,
Accession No. 2358279; see also Nature Genet. 17, 40-48 (1997),
incorporated herein by reference.) In specific embodiments,
polypeptides of the invention comprise the following amino acid
sequence: VEPISLGLALAGVLTGYIYP (SEQ ID NO: 726),
PLTLSLHGWTGTGKNFVSKIIAENIYE (SEQ ID NO: 727), NITLYKDQLQLW
IRGNVSACARSI (SEQ ID NO: 728), DFWRSGKQREDIKLKDIEHALSVSVFN (SEQ ID
NO: 729), LIDYFVPFLPLEYKHLKMCIRV (SEQ ID NO: 730), VSRVAEEMTF
FPKEERVF (SEQ ID NO: 731). Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0770] This gene is expressed primarily in osteoclastoma, T-cell,
and placenta and to a lesser extent in fetal lung, fetal liver,
fetal brain, adult brain and tumor tissues
[0771] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: disease conditions in
hematopoiesis and cancers. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the hematopoiesis system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
blood cells, bone, placenta, lung, liver, and brain and other
tissues of the nervous system, and cancerous and wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0772] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treating
blood related diseases such as deficiencies in red blood cell,
white blood cell, platelet and other hematopoiesis cells. Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0773] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 467 as residues: Cys-50 to Ser-57, Asn-88 to Pro-95,
Trp-222 to Lys-232, Asn-246 to Phe-252, Gln-286 to Ile-292, Asp-327
to Asp-332.
[0774] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:158 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2103 of SEQ ID NO:158, b is an
integer of 15 to 2117, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:158, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 149
[0775] The gene encoding the disclosed cDNA is believed to reside
on chromosome 6. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
6.
[0776] This gene is expressed primarily in T cell, prostate and
prostate cancer, endothelial cells and to a lesser extent in
monocyte, dendritic cell, bone marrow, salivary gland, colon
cancer, stomach cancer, pancreatic tumor, uterine cancer, fetal
spleen and osteoclastoma.
[0777] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immuno-related diseases and
cancers. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., blood cells, prostate, endothelial cells,
dendritic cells, bone marrow, salivary gland, colon, stomach,
pancreas, uterus, spleen and bone, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0778] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
cancers. Similarly, the tissue distribution within T-cells,
dendritic cells, and bone marrow suggests that the protein product
of this clone would be useful for the treatment and diagnosis of
hematopoetic related disorders such as anemia, pancytopenia,
leukopenia, thrombocytopenia or leukemia since stromal cells are
important in the production of cells of hematopoietic lineages. The
uses include bone marrow cell ex vivo culture, bone marrow
transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis, therefore, it can be used in immune disorders such
as infection, inflammation, allergy, immunodeficiency etc. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0779] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:159 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2381 of SEQ ID NO:159, b is an
integer of 15 to 2395, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:159, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 150
[0780] This gene was recently cloned by another group, calling it
eIF3-p66. (See Accession No. 2351378.) This gene plays a role in
RNA binding and macromolecular assembly, and therefore, any
mutations in this gene would likely result in a diseased phenotype.
Preferred polypeptide fragments comprise the amino acid sequence:
MAKFMTPVIQDNPSGWGPCAVPEQFRDMPYQPFSKGDRLGKVADWTGATYQDKRYTNKYSSQFGGGSQ
YAYFHEEDESSFQLVDTARTQKTAYQRNRMRFAQRNLRRDKDRRNMLQFNLQILPKSAKQKERERIRLQK
KFQKQFGVRQKWDQKSQKPRDSSVEVRSDWEVKEEMDFPQLMKMRYLEVSEPQDIECCGALEYYDKAF
DRITTRSEKPLRXXKRIFHTVTTTDDPVIRKLAKTQGNVFATDAILATLMSCTRSVYSWDIVVQRVGSKLFF
DKRDNSDFDLLTVSETANEPPQDEGNSFNSPRNLAMEATYINHNFSQQCLRMGKERYNFPNPNPFVEDDM
DKNEIASVAYRYRSGKLGDDIDLIVRCEHDGVMTGANGEVSFINIKTLNEWDSRHCNGVDWRQKLDSQR
GAVIATELKNNSYKLARWTCCALLAGSEYLKLGYVSRYHVKDSSRHVILGTQQFKPNEFASQINLSVENA
WGILRCVIDICMKLEEGKYLILKDPNKQVIRVYSLPDGTFSS (SEQ ID NO: 732), or
CQSHLSRLGASEANEDAWRNAVTDFRVDLRFTAREFFRPTQRKRESSLRQRRSCLRRRPLRAHVSSFPGFS
RVLLLTRSPARTICCHPGRPRHCRFWK (SEQ ID NO: 733), as well as N-terminal
and C-terminal deletions of this polypeptide fragment.
[0781] This gene is expressed primarily in T cell, bone marrow,
embryo and endothelial cells and to a lesser extent in testis tumor
and endometrial tumor.
[0782] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders,
particularly inflammatory or immunodeficiency disorders, and
tumors. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system and reproductive system, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues and cell types (e.g. immune, developmental,
hematopoietic, and cancerous and wounded tissues) or bodily fluids
(e.g. lymph, amniotic fluid, serum, plasma, urine, synovial fluid
or spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0783] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for immune
disorders and cancers. Similarly, expression within embryonic
tissue and other cellular sources marked by proliferating cells
suggests that this protein may play a role in the regulation of
cellular division, and may show utility in the diagnosis and
treatment of cancer and other proliferative disorders. Similarly,
embryonic development also involves decisions involving cell
differentiation and/or apoptosis in pattern formation. Thus this
protein may also be involved in apoptosis or tissue differentiation
and could again be useful in cancer therapy. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0784] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 469 as residues: Gln-10 to Trp-16, Pro-32 to Leu-39,
Tyr-50 to Gln-68, Ala-86 to Phe-100, Arg-103 to Asn-113, Ser-125 to
Arg-133, Gln-137 to Lys-142, Arg-147 to Ser-161, Arg-165 to
Glu-173, Ile-209 to Arg-218.
[0785] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:160 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2106 of SEQ ID NO:160, b is an
integer of 15 to 2120, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:160, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 151
[0786] The translation product of this gene was found to have
homology to the human VRK1 and VRK2 proteins (See Genbank Accession
No. gi|1827450 (AB000449), and gi|1827452 (AB000450), respectively)
which are thought to encode novel serine/threonine kinases. Based
upon the role of such proteins in regulation of various cellular
processes, namely regulation of cellular proliferation, an
important function for this clone would be realized by a skilled
artisan. In specific embodiments, polypeptides of the invention
comprise the following amino acid sequence:
KLKSFQTRDNQGILYEAAPTSTLTCXSGPQKQKFSLKLDAKDGRLFNEQNFFQRAAKPLQVNKWKKLYST
PLLAIPTCMGFGVHQDKYRFLVLPSLGRSLQSALDVSPKHVLCREVCAAGGLPAAGCPGVPP (SEQ
ID NO: 734). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0787] This gene is expressed primarily in testes and to a lesser
extent in T cell, spinal cord, placenta, neutrophil and
monocyte.
[0788] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: male reproductive and
endocrine disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the reproductive, immune and
endocrine systems, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., testis and other reproductive tissue, blood
cells, tissue of the nervous system, and placenta, and cancerous
and wounded tissues) or bodily fluids (e.g. lymph, seminal fluid,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0789] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for regulating
immune and reproductive functions. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0790] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 470 as residues: Lys-2 to Trp-24, Arg-35 to Gly-40,
Lys-46 to Cys-58, Arg-71 to Trp-77, Glu-164 to Gly-172.
[0791] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:161 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 886 of SEQ ID NO:161, b is an integer
of 15 to 900, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:161, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 152
[0792] The translation product of this gene shares sequence
homology with tyrosyl-tRNA synthetase which is thought to be
important in cell growth.
[0793] This gene is expressed primarily in brain, liver,
keratinocytes, tonsils, and heart.
[0794] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancer and/or autoimmune diseases. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the brain,
liver, keratinocytes, tonsils and heart, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., brain and other tissues of
the nervous system, liver, keratinocytes, tonsils and heart,
immune, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0795] The tissue distribution and homology to tyrosyl-tRNA
synthetase indicate that polynucleotides and polypeptides
corresponding to this gene are useful for modulating cell growth.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0796] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 471 as residues: Thr-2 to Glu-17, Cys-74 to
Ser-79.
[0797] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:162 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 989 of SEQ ID NO:162, b is an integer
of 15 to 1003, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:162, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 153
[0798] This gene is homologous to the Drosophila transcriptional
regulator dre4. (See Genbank Accession No. 2511745.) Dre4 is a gene
required for steroidogenesis in Drosophila melanogaster and encodes
a developmentally expressed homologue of the yeast transcriptional
regulator CDC68. Preferred polypeptide fragments comprise the amino
acid sequence:
KKRHTDVQFYTEVGEITTDLGKHQHMHDRDDLYAEQMEREMRHKLKTAFKNFIEKVEALTKEELEFEVP
FRDLGFNGAPYRSTCLLQPTSSALVNATEWPPFVVTLDEVELIHFXRVQFHLKNFDMVIVYKDYSKKVTMI
NAIPVASLDPIKEWLNSCDLKYTEGVQSLNWTKIMKTIVDDPEGFFEQGGWSFL (SEQ ID NO:
735), as well as N-terminal and C-terminal deletions of this
fragments. Also preferred are polynucleotide fragments encoding
this polypeptide fragment.
[0799] This gene is expressed primarily in fetal liver, spleen,
placenta, lung, T cell, thyroid, testes.
[0800] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: brain tumor, heart and liver
diseases. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the fetal liver, spleen, placenta, lung, T cell, thyroid and
testes, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., liver, spleen, placenta, lung, blood cells, thyroid, and
testes and other reproductive tissue, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0801] The tissue distribution in fetal liver, spleen, T-cells, and
thyroid suggests that the protein product of this clone would be
useful for the diagnosis and treatment of a variety of immune
system disorders. Expression of this gene product in immune cells
suggests a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g. by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the
natural gene product may be involved in immune functions. Therefore
it may be also used as an agent for immunological disorders
including arthritis, asthma, immunodeficiency diseases such as
AIDS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated cytotoxicity; immune reactions to transplanted organs and
tissues, such as host-versus-graft and graft-versus-host diseases,
or autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
scleroderma and tissues. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0802] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:163 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2182 of SEQ ID NO:163, b is an
integer of 15 to 2196, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:163, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 154
[0803] The translation product of this gene was found to have
homology to both the human cAMP-dependent protein kinase and
phospholemman chloride channel, which are thought to be involved in
regulation of signal transduction pathways, potentially for cell
cycle regulation (See Genbank Accession No. pir|A40533|A40533 and
gi|1916010, respectively). The gene encoding the disclosed cDNA is
believed to reside on chromosome 11. Accordingly, polynucleotides
related to this invention are useful as a marker in linkage
analysis for chromosome 11.
[0804] This gene is expressed primarily in brain and to a lesser
extent in fetal heart, testis, spleen, lung.
[0805] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: heart, liver and spleen
diseases, immunological diseases. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the brain, fetal heart, testis,
spleen and lung, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., brain and other tissue of the nervous system, heart,
testes and other reproductive tissue, spleen, and lung, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0806] The tissue distribution in brain combined with the homology
to the conserved cAMP-dependent protein kinase and phospholemman
proteins suggests that the protein product of this clone would be
useful for the detection/treatment of neurodegenerative disease
states, behavioural disorders, or inflamatory conditions such as
Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, sexually-linked disorders,
or disorders of the cardiovascular system.
[0807] Alternatively, expression within fetal tissues and other
cellular sources marked by proliferating cells suggests that this
protein may play a role in the regulation of cellular division, and
may show utility in the diagnosis and treatment of cancer and other
proliferative disorders. Similarly, embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus this protein may also be involved in
apoptosis or tissue differentiation and could again be useful in
cancer therapy. Protein, as well as, antibodies directed against
the protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0808] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 473 as residues: Glu-22 to Phe-30, Arg-59 to Ala-77,
Ala-87 to Asn-95.
[0809] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:164 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1931 of SEQ ID NO:164, b is an
integer of 15 to 1945, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:164, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 155
[0810] Activation of T cells through the T cell antigen receptor
(TCR) results in the rapid tyrosine phosphorylation of a number of
cellular proteins, one of the earliest being a 100 kDa protein.
This gene is the human equivalent of murine valosin containing
protein (VCP). VCP is a member of a family of ATP binding,
homo-oligomeric proteins, and the mammalian homolog of
Saccharomyces cerevisiae cdc48p, a protein essential to the
completion of mitosis in yeast. Both endogenous and expressed
murine VCP are tyrosine phosphorylated in response to T cell
activation. Thus we have identified a novel component of the TCR
mediated tyrosine kinase activation pathway that may provide a link
between TCR activation and cell cycle control. In specific
embodiments, polypeptides of the invention comprise the following
amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 736)
MASGADSKGDDLSTAILKQKNRPNRLIVDEAINEDN, (SEQ ID NO: 737)
GKKRREAVCIVLSDDTCSDEKIRM, (SEQ ID NO: 738)
GDVISIQPCPDVKYGKRIHVLPID, (SEQ ID NO: 739)
ITGNLFEVYLKPYFLEAYRPIRKGDIFLVRGGMRA, (SEQ ID NO: 740)
GYDDIGGCRKQLAQIKEMVELPLRHPALFKAI, (SEQ ID NO: 741)
PPGTGKTLIARAVANETGAFFFLINGPEIMSKLAGES, (SEQ ID NO: 742)
RRIVSQLLTLMDGLKQRAHVIVMAATNRPNSIDPA, (SEQ ID NO: 743)
VDIGIPDATGRLEILQIHTKNMKLADDVD, (SEQ ID NO: 744)
EAALQAIRKKMDLIDLEDETI (SEQ ID NO: 745)
TVVEVPQVTWEDIGGLEDVKRELQELVQYPVEHPD, (SEQ ID NO: 746)
NECQANFISIKGPELLTMWFGESEANVREIFDKAR, (SEQ ID NO: 747)
ILTEMDGMSTKKNVFIIGATNRPDIIDPAI, (SEQ ID NO: 748)
IYIPLPDEKSRVAILKANLRKSPVAKDVDLEFL, (SEQ ID NO: 749)
DPVPEIRRDHFEEAMRFARRSVSDNDI, (SEQ ID NO: 750) QTLQQSRGFGSFRFP, or
(SEQ ID NO: 751)
CGPGTVANETHGHVGADLAALCSEAALQAIRKKMDLIDLEDETIDAEVMNSLAVTMDDFRWALSQSNPS
ALRETVVEVPQVTWEDIGGLEDVKRELQELVQYPVEHPDKFLKFGMTPSKGVLFYGPPGCGKTLLAKAIA
NECQANFISIKGPELLTMWFGESEANVREIFDKARQAAPCVLFFDELDSIAKARGGNIGDGGGAADRVINQI
LTEMDGMSTKKNVFIIGATNRPDIIDPAILRPGRLDQLIYIPLPDEKSRVAILKANLRKSPVAKDVDLEFLAK
MTNGFSGADLTEICQRACKLAIRESIESEIRRERERQTNPSAMEVEEDDPVPEIRRDHFEEAMRFARRSVSD
NDIRKYEMFAQTLQQSRGFGSFRFPSGNQGGAGPSQGSGGGTGGSVYTEDNDDDLYG.
Polynucleotides encoding these polypeptides are also encompassed by
the invention. The gene encoding the disclosed cDNA is believed to
reside on chromosome 9. Accordingly, polynucleotides related to
this invention are useful as a marker in linkage analysis for
chromosome 9.
[0811] This gene is expressed primarily in brain, liver, spleen,
placenta.
[0812] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancer and immunological disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the brain,
liver, spleen, placenta expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., brain and other tissue of the nervous system,
liver, spleen, and placenta, and cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0813] The tissue distribution and homology to VCR indicate that
polynucleotides and polypeptides corresponding to this gene are
useful for treating cancer. The secreted protein can also be used
to determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions and as nutritional
supplements. It may also have a very wide range of biological
activities. Typical of these are cytokine, cell
proliferation/differentiation modulating activity or induction of
other cytokines; immunostimulating/immunosuppressant activities
(e.g. for treating human immunodeficiency virus infection, cancer,
autoimmune diseases and allergy); regulation of hematopoiesis (e.g.
for treating anaemia or as adjunct to chemotherapy); stimulation or
growth of bone, cartilage, tendons, ligaments and/or nerves (e.g.
for treating wounds, stimulation of follicle stimulating hormone
(for control of fertility); chemotactic and chemokinetic activities
(e.g. for treating infections, tumors); hemostatic or thrombolytic
activity (e.g. for treating haemophilia, cardiac infarction etc.);
anti-inflammatory activity (e.g. for treating septic shock, Crohn's
disease); as antimicrobials; for treating psoriasis or other
hyperproliferative diseases; for regulation of metabolism, and
behaviour. Also contemplated is the use of the corresponding
nucleic acid in gene therapy procedures. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0814] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 474 as residues: Ala-5 to Asp-11, Lys-18 to Asn-24,
Asp-75 to Ile-82, Pro-106 to Lys-112, Arg-144 to Gly-149, Glu-187
to Ser-197, Gly-202 to Cys-209, Ser-282 to Lys-288, Glu-291 to
Asn-296, Pro-311 to His-317, Thr-347 to Ser-352, Arg-359 to
Val-367.
[0815] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:165 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2919 of SEQ ID NO:165, b is an
integer of 15 to 2933, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:165, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 156
[0816] The translation product of this gene shares sequence
homology with rat growth response protein which is thought to be
important in cell growth. A group recently cloned the human homolog
of this gene, calling it insulin induced protein 1. (See Accession
No. 2358269, see also, Genomics 43 (3), 278-284 (1997).) Preferred
polypeptide fragments comprise the amino acid sequence:
RSGLGLGITIAFLATLITQFLVYNGVYQYTSPDFLYIRSWLPCIFFSGGVTVGNIGRQLAMGVPEKPHSD
(SEQ ID NO: 752), as well as N-terminal and C-terminal deletions of
this polypeptide fragment. Also preferred are polynucleotide
fragments encoding these polypeptide fragments. The gene encoding
the disclosed cDNA is believed to reside on chromosome 7.
Accordingly, polynucleotides related to this invention are useful
as a marker in linkage analysis for chromosome 7.
[0817] This gene is expressed primarily in brain, liver, placenta,
heart, spleen, lymphoma.
[0818] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancer and immunological disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the brain,
liver, placenta, heart, spleen. expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., brain and other tissue of the
nervous system, liver, placenta, heart, spleen, and lymphoid
tissue, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0819] The tissue distribution in liver and homology to
growth-response protein indicate that polynucleotides and
polypeptides corresponding to this gene are useful for modulating
cell growth and may play a role in growth and differentiation of
tissues involved in metabolic control or development. Protein, as
well as, antibodies directed against the protein may show utility
as a tumor marker and/or immunotherapy targets for the above listed
tissues.
[0820] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:166 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2229 of SEQ ID NO:166, b is an
integer of 15 to 2243, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:166, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 157
[0821] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
1.
[0822] This gene is expressed primarily in Glioblastoma,
endometrial tumor, lymphoma and pancreas tumor.
[0823] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: Glioblastoma, Endometrial
tumor, lymphoma and pancreas tumor, and other proliferative
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues and cell
types (e.g., endometrium, lymphoid tissue, pancreas, and tissue of
the nervous system, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, bile, serum, plasma, urine, synovial fluid or
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0824] The tissue distribution in tumors of pancreas, endometrium,
and other proliferative tissues suggests that the protein product
of this clone would be useful for diagnosis and intervention of
these tumors, in addition to other tumors where expression has been
indicated. Protein, as well as, antibodies directed against the
protein may show utility as a tissue-specific marker and/or
immunotherapy target for the above listed tissues.
[0825] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 476 as residues: Thr-60 to Glu-66, Tyr-104 to
Tyr-111.
[0826] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:167 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1802 of SEQ ID NO:167, b is an
integer of 15 to 1816, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:167, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 158
[0827] The translation product of this gene shares sequence
homology with IGE receptor which is thought to be important in
allergy and asthma. In specific embodiments, polypeptides of the
invention comprise the following amino acid sequence:
ETRVKTSLELLRTQLEPTGTVGNTINFSQAEKPEPTNQGQDSLKKHLHAEIKVIGTIQILCG (SEQ
ID NO: 753). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0828] This gene is expressed primarily in T cell, and fetal
liver.
[0829] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: allergy and asthma and other
immunological disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g. immune, blood
cells, and liver, and cancerous and wounded tissues) or bodily
fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0830] The tissue distribution and homology to IgE receptor
indicate that polynucleotides and polypeptides corresponding to
this gene are useful for allergy and asthma. Similarly, The
secreted protein can also be used to determine biological activity,
to raise antibodies, as tissue markers, to isolate cognate ligands
or receptors, to identify agents that modulate their interactions
and as nutritional supplements. It may also have a very wide range
of biological activities. Typical of these are cytokine, cell
proliferation/differentiation modulating activity or induction of
other cytokines; immunostimulating/immunosuppressant activities
(e.g. for treating human immunodeficiency virus infection, cancer,
autoimmune diseases and allergy); regulation of hematopoiesis (e.g.
for treating anaemia or as adjunct to chemotherapy); stimulation or
growth of bone, cartilage, tendons, ligaments and/or nerves (e.g.
for treating wounds, stimulation of follicle stimulating hormone
(for control of fertility); chemotactic and chemokinetic activities
(e.g. for treating infections, tumors); hemostatic or thrombolytic
activity (e.g. for treating haemophilia, cardiac infarction etc.);
anti-inflammatory activity (e.g. for treating septic shock, Crohn's
disease); as antimicrobials; for treating psoriasis or other
hyperproliferative diseases; for regulation of metabolism, and
behaviour. Also contemplated is the use of the corresponding
nucleic acid in gene therapy procedures. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0831] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 477 as residues: Glu-94 to Thr-102.
[0832] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:168 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 931 of SEQ ID NO:168, b is an integer
of 15 to 945, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:168, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 159
[0833] For purposes of this application, this gene and its
corresponding translation product are known as the B7-H5 gene and
B7-H5 protein. This protein is believed to reside as a cell-surface
molecule, and the transmembrane domain of this protein is believed
to embody the following preferred amino acid residues:
IRVPVFNIVILLAGGF (SEQ ID NO: 758). Polynucleotides encoding these
polypeptides are also encompassed by the invention, as are
antibodies that bind one or more of these peptides. The B7-H5 gene
shares sequence homology with members of the B7 family of ligands
(i.e., B7-1 (See Genbank Accession 507873)). These proteins and
their corresponding receptors play vital roles in the growth,
differentiation and death of T cells. For example, some members of
this family (i.e., B7-H1) are involved in costimulation of the T
cell response, as well as inducing increased cytokine production.
Therefore, antagonists such as antibodies or small molecules
directed against the B7-H5 gene are useful for treating T cell
mediated immune system disorders. The gene encoding the disclosed
cDNA is thought to reside on chromosome 6. Accordingly,
polynucleotides related to this invention have uses, such as, for
example, as a marker in linkage analysis for chromosome 6.
[0834] It has been discovered that this gene is expressed in
activated neutrophils and activated T cells, and to a lesser extent
in monocytes and heart tissue.
[0835] Preferred polypeptides of the present invention comprise, or
alternatively consist of, one, two, three, four, five, six, seven
or all seven of the immunogenic epitopes of the extracellular
portion of the B7-H5 protein shown in SEQ ID NO: 478 as residues:
Leu-24 to Gln-35, Arg-59 to Pro-64, Glu-71 to His-78, Asp-89 to
Gly-94, Pro-141 to Val-151, Thr-167 to Val-172, Ala-175 to Thr-180.
Polynucleotides encoding these polypeptides are also encompassed by
the invention, as are antibodies that bind one or more of these
peptides.
[0836] In additional nonexclusive embodiments, polypeptides of the
invention comprise, or alternatively consist of, one or more of the
following amino acid sequences: 1.) The extracellular domain of the
B7-H5 protein:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMP-
KTLAC
TERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGF-
SGT PGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDI (SEQ
ID NO: 759), 2.) The mature extracellular domain of the B7-H5
protein:
EKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLR
VRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTS
PRTVTQAPPKSTADVSTPDSEINLTNVTDI (SEQ ID NO: 760), and/or 3.) The
leader sequence of the B7-H5 protein: MRKTRLWGLLWMLFVSELRAATKLTE
(SEQ ID NO: 761).
[0837] Polynucleotides encoding these polypeptides are also
encompassed by the invention, as are antibodies that bind one or
more of these polypeptides.
[0838] Also preferred are polypeptides comprising, or alternatively
consisting of, fragments of the mature extracellular portion of the
B7-H5 protein demonstrating functional activity (SEQ ID NO: 760).
Polynucleotides encoding these polypeptides are also encompassed by
the invention. By functional activity is meant, a polypeptide
fragment capable of displaying one or more known functional
activities associated with the full-length (complete) B7-H5
protein. Such functional activities include, but are not limited
to, biological activity (e.g., T cell costimulatory activity,
ability to bind ICOS, and ability to induce or inhibit cytokine
production), antigenicity [ability to bind (or compete with a B7-H5
polypeptide for binding) to an anti-B7-H5 antibody], immunogenicity
(ability to generate antibody which binds to a B7-H5 polypeptide),
ability to form multimers with B7-H5 polypeptides of the invention,
and ability to bind to a receptor or ligand for a B7-H5
polypeptide.
[0839] FIG. 1 shows the nucleotide (SEQ ID NO: 169) and deduced
amino acid sequence (SEQ ID NO: 478) corresponding to this
gene.
[0840] FIG. 2 shows an analysis of the amino acid sequence (SEQ ID
NO: 478). Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown, and all were generated
using the default settings of the recited computer algorithms. In
the "Antigenic Index or Jameson-Wolf" graph, the positive peaks
indicate locations of the highly antigenic regions of the protein,
i.e., regions from which epitope-bearing peptides of the invention
can be obtained. Polypeptides comprising, or alternatively
consisting of, domains defined by these graphs are contemplated by
the present invention, as are polynucleotides encoding these
polypeptides.
[0841] The data presented in FIG. 2 are also represented in tabular
form in Table 3. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIV. The column headings refer to
the following features of the amino acid sequence presented in FIG.
2, and Table 3: "Res": amino acid residue of SEQ ID NO: 478 and
FIGS. 1A and 1B; "Position": position of the corresponding residue
within SEQ ID NO: 478 and FIGS. 1A and 1B; I: Alpha,
Regions--Garnier-Robson; II: Alpha, Regions--Chou-Fasman; III:
Beta, Regions--Garnier-Robson; W: Beta, Regions--Chou-Fasman; V:
Turn, Regions--Garnier-Robson; VI: Turn, Regions--Chou-Fasman; VII:
Coil, Regions--Garnier-Robson; VIII: Hydrophilicity
Plot--Kyte-Doolittle; IX: Hydrophobicity Plot--Hopp-Woods; X:
Alpha, Amphipathic Regions--Eisenberg; XI: Beta, Amphipathic
Regions--Eisenberg; XII: Flexible Regions--Karplus-Schulz; XIII:
Antigenic Index--Jameson-Wolf; and XIV: Surface Probability
Plot--Emini.
[0842] Preferred embodiments of the invention in this regard
include fragments that comprise, or alternatively consisting of,
one or more of the following regions: alpha-helix and alpha-helix
forming regions ("alpha-regions"), beta-sheet and beta-sheet
forming regions ("beta-regions"), turn and turn-forming regions
("turn-regions"), coil and coil-forming regions ("coil-regions"),
hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta amphipathic regions, flexible regions,
surface-forming regions and high antigenic index regions. The data
representing the structural or functional attributes of the protein
set forth in FIG. 2 and/or Table 3, as described above, was
generated using the various modules and algorithms of the DNA*STAR
set on default parameters. In a preferred embodiment, the data
presented in columns VIII, IX, XIII, and XIV of Table 3 can be used
to determine regions of the protein which exhibit a high degree of
potential for antigenicity. Regions of high antigenicity are
determined from the data presented in columns VIII, IX, XIII,
and/or XIV by choosing values which represent regions of the
polypeptide which are likely to be exposed on the surface of the
polypeptide in an environment in which antigen recognition may
occur in the process of initiation of an immune response.
[0843] Certain preferred regions in these regards are set out in
FIG. 2, but may, as shown in Table 3, be represented or identified
by using tabular representations of the data presented in FIG. 2.
The DNA*STAR computer algorithm used to generate FIG. 2 (set on the
original default parameters) was used to present the data in FIG. 2
in a tabular format (See Table 3). The tabular format of the data
in FIG. 2 is used to easily determine specific boundaries of a
preferred region.
[0844] The present invention is further directed to fragments of
the polynucleotide sequences described herein. By a fragment of,
for example, the polynucleotide sequence of a deposited cDNA or the
nucleotide sequence shown in SEQ ID NO: 169, is intended
polynucleotide fragments at least about 15 nt, and more preferably
at least about 20 nt, at least about 25 nt, still more preferably
at least about 30 nt, at least about 35 nt, and even more
preferably, at least about 40 nt in length, at least about 45 nt in
length, at least about 50 nt in length, at least about 60 nt in
length, at least about 70 nt in length, at least about 80 nt in
length, at least about 90 nt in length, at least about 100 nt in
length, at least about 125 nt in length, at least about 150 nt in
length, at least about 175 nt in length, which are useful as
diagnostic probes and primers as discussed herein. Of course,
larger fragments 200-1500 nt in length are also useful according to
the present invention, as are fragments corresponding to most, if
not all, of the nucleotide sequence of a deposited cDNA or as shown
in SEQ ID NO: 169. By a fragment at least 20 nt in length, for
example, is intended fragments which include 20 or more contiguous
bases from the nucleotide sequence of a deposited cDNA or the
nucleotide sequence as shown in SEQ ID NO: 169. In this context
"about" includes the particularly recited size, an sizes larger or
smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at both termini. Representative examples of
polynucleotide fragments of the invention include, for example,
fragments that comprise, or alternatively, consist of, a sequence
from about nucleotide 1 to about 50, from about 51 to about 100,
from about 101 to about 150, from about 151 to about 200, from
about 201 to about 250, from about 251 to about 300, from about 301
to about 350, from about 351 to about 400, from about 401 to about
450, from about 451 to about 500, and from about 501 to about 550,
and from about 551 to about 600, from about 601 to about 650, from
about 651 to about 700, from about 701 to about 750, from about 751
to about 800, and from about 801 to about 860, of SEQ ID NO: 169,
or the complementary strand thereto, or the cDNA contained in a
deposited clone. In this context "about" includes the particularly
recited ranges, and ranges larger or smaller by several (5, 4, 3,
2, or 1) nucleotides, at either terminus or at both termini. In
additional embodiments, the polynucleotides of the invention encode
functional attributes of the corresponding protein.
[0845] Preferred polypeptide fragments of the invention comprise,
or alternatively consist of, the secreted protein having a
continuous series of deleted residues from the amino or the carboxy
terminus, or both. Particularly, N-terminal deletions of the
polypeptide can be described by the general formula m-234 where m
is an integer from 2 to 228, where m corresponds to the position of
the amino acid residue identified in SEQ ID NO: 478. More in
particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, an amino
acid sequence selected from the group: R-2 to P-234; K-3 to P-234;
T-4 to P-234; R-5 to P-234; L-6 to P-234; W-7 to P-234; G-8 to
P-234; L-9 to P-234; L-10 to P-234; W-11 to P-234; M-12 to P-234;
L-13 to P-234; F-14 to P-234; V-15 to P-234; S-16 to P-234; E-17 to
P-234; L-18 to P-234; R-19 to P-234; A-20 to P-234; A-21 to P-234;
T-22 to P-234; K-23 to P-234; L-24 to P-234; T-25 to P-234; E-26 to
P-234; E-27 to P-234; K-28 to P-234; Y-29 to P-234; E-30 to P-234;
L-31 to P-234; K-32 to P-234; E-33 to P-234; G-34 to P-234; Q-35 to
P-234; T-36 to P-234; L-37 to P-234; D-38 to P-234; V-39 to P-234;
K-40 to P-234; C-41 to P-234; D-42 to P-234; Y-43 to P-234; T-44 to
P-234; L-45 to P-234; E-46 to P-234; K-47 to P-234; F-48 to P-234;
A-49 to P-234; 5-50 to P-234; 5-51 to P-234; Q-52 to P-234; K-53 to
P-234; A-54 to P-234; W-55 to P-234; Q-56 to P-234; I-57 to P-234;
I-58 to P-234; R-59 to P-234; D-60 to P-234; G-61 to P-234; E-62 to
P-234; M-63 to P-234; P-64 to P-234; K-65 to P-234; T-66 to P-234;
L-67 to P-234; A-68 to P-234; C-69 to P-234; T-70 to P-234; E-71 to
P-234; R-72 to P-234; P-73 to P-234; S-74 to P-234; K-75 to P-234;
N-76 to P-234; S-77 to P-234; H-78 to P-234; P-79 to P-234; V-80 to
P-234; Q-81 to P-234; V-82 to P-234; G-83 to P-234; R-84 to P-234;
I-85 to P-234; I-86 to P-234; L-87 to P-234; E-88 to P-234; D-89 to
P-234; Y-90 to P-234; H-91 to P-234; D-92 to P-234; H-93 to P-234;
G-94 to P-234; L-95 to P-234; L-96 to P-234; R-97 to P-234; V-98 to
P-234; R-99 to P-234; M-100 to P-234; V-101 to P-234; N-102 to
P-234; L-103 to P-234; Q-104 to P-234; V-105 to P-234; E-106 to
P-234; D-107 to P-234; S-108 to P-234; G-109 to P-234; L-110 to
P-234; Y-111 to P-234; Q-112 to P-234; C-113 to P-234; V-114 to
P-234; I-115 to P-234; Y-116 to P-234; Q-117 to P-234; P-118 to
P-234; P-119 to P-234; K-120 to P-234; E-121 to P-234; P-122 to
P-234; H-123 to P-234; M-124 to P-234; L-125 to P-234; F-126 to
P-234; D-127 to P-234; R-128 to P-234; I-129 to P-234; R-130 to
P-234; L-131 to P-234; V-132 to P-234; V-133 to P-234; T-134 to
P-234; K-135 to P-234; G-136 to P-234; F-137 to P-234; S-138 to
P-234; G-139 to P-234; T-140 to P-234; P-141 to P-234; G-142 to
P-234; S-143 to P-234; N-144 to P-234; E-145 to P-234; N-146 to
P-234; S-147 to P-234; T-148 to P-234; Q-149 to P-234; N-150 to
P-234; V-151 to P-234; Y-152 to P-234; K-153 to P-234; I-154 to
P-234; P-155 to P-234; P-156 to P-234; T-157 to P-234; T-158 to
P-234; T-159 to P-234; K-160 to P-234; A-161 to P-234; L-162 to
P-234; C-163 to P-234; P-164 to P-234; L-165 to P-234; Y-166 to
P-234; T-167 to P-234; S-168 to P-234; P-169 to P-234; R-170 to
P-234; T-171 to P-234; V-172 to P-234; T-173 to P-234; Q-174 to
P-234; A-175 to P-234; P-176 to P-234; P-177 to P-234; K-178 to
P-234; S-179 to P-234; T-180 to P-234; A-181 to P-234; D-182 to
P-234; V-183 to P-234; S-184 to P-234; T-185 to P-234; P-186 to
P-234; D-187 to P-234; S-188 to P-234; E-189 to P-234; I-190 to
P-234; N-191 to P-234; L-192 to P-234; T-193 to P-234; N-194 to
P-234; V-195 to P-234; T-196 to P-234; D-197 to P-234; I-198 to
P-234; I-199 to P-234; R-200 to P-234; V-201 to P-234; P-202 to
P-234; V-203 to P-234; F-204 to P-234; N-205 to P-234; I-206 to
P-234; V-207 to P-234; I-208 to P-234; L-209 to P-234; L-210 to
P-234; A-211 to P-234; G-212 to P-234; G-213 to P-234; F-214 to
P-234; L-215 to P-234; S-216 to P-234; K-217 to P-234; S-218 to
P-234; L-219 to P-234; V-220 to P-234; F-221 to P-234; S-222 to
P-234; V-223 to P-234; L-224 to P-234; F-225 to P-234; A-226 to
P-234; V-227 to P-234; T-228 to P-234; and/or L-229 to P-234 of SEQ
ID NO:478. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
[0846] Additionally, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the following group of
C-terminal deletions: M-1 to V-233; M-1 to F-232; M-1 to S-231; M-1
to R-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to A-226;
M-1 to F-225; M-1 to L-224; M-1 to V-223; M-1 to S-222; M-1 to
F-221; M-1 to V-220; M-1 to L-219; M-1 to S-218; M-1 to K-217; M-1
to S-216; M-1 to L-215; M-1 to F-214; M-1 to G-213; M-1 to G-212;
M-1 to A-211; M-1 to L-210; M-1 to L-209; M-1 to I-208; M-1 to
V-207; M-1 to I-206; M-1 to N-205; M-1 to F-204; M-1 to V-203; M-1
to P-202; M-1 to V-201; M-1 to R-200; M-1 to I-199; M-1 to I-198;
M-1 to D-197; M-1 to T-196; M-1 to V-195; M-1 to N-194; M-1 to
T-193; M-1 to L-192; M-1 to N-191; M-1 to I-190; M-1 to E-189; M-1
to S-188; M-1 to D-187; M-1 to P-186; M-1 to T-185; M-1 to S-184;
M-1 to V-183; M-1 to D-182; M-1 to A-181; M-1 to T-180; M-1 to
S-179; M-1 to K-178; M-1 to P-177; M-1 to P-176; M-1 to A-175; M-1
to Q-174; M-1 to T-173; M-1 to V-172; M-1 to T-171; M-1 to R-170;
M-1 to P-169; M-1 to S-168; M-1 to T-167; M-1 to Y-166; M-1 to
L-165; M-1 to P-164; M-1 to C-163; M-1 to L-162; M-1 to A-161; M-1
to K-160; M-1 to T-159; M-1 to T-158; M-1 to T-157; M-1 to P-156;
M-1 to P-155; M-1 to I-154; M-1 to K-153; M-1 to Y-152; M-1 to
V-151; M-1 to N-150; M-1 to Q-149; M-1 to T-148; M-1 to S-147; M-1
to N-146; M-1 to E-145; M-1 to N-144; M-1 to S-143; M-1 to G-142;
M-1 to P-141; M-1 to T-140; M-1 to G-139; M-1 to S-138; M-1 to
F-137; M-1 to G-136; M-1 to K-135; M-1 to T-134; M-1 to V-133; M-1
to V-132; M-1 to L-131; M-1 to R-130; M-1 to I-129; M-1 to R-128;
M-1 to D-127; M-1 to F-126; M-1 to L-125; M-1 to M-124; M-1 to
H-123; M-1 to P-122; M-1 to E-121; M-1 to K-120; M-1 to P-119; M-1
to P-118; M-1 to Q-117; M-1 to Y-116; M-1 to I-115; M-1 to V-114;
M-1 to C-113; M-1 to Q-112; M-1 to Y-111; M-1 to L-110; M-1 to
G-109; M-1 to S-108; M-1 to D-107; M-1 to E-106; M-1 to V-105; M-1
to Q-104; M-1 to L-103; M-1 to N-102; M-1 to V-101; M-1 to M-100;
M-1 to R-99; M-1 to V-98; M-1 to R-97; M-1 to L-96; M-1 to L-95;
M-1 to G-94; M-1 to H-93; M-1 to D-92; M-1 to H-91; M-1 to Y-90;
M-1 to D-89; M-1 to E-88; M-1 to L-87; M-1 to I-86; M-1 to I-85;
M-1 to R-84; M-1 to G-83; M-1 to V-82; M-1 to Q-81; M-1 to V-80;
M-1 to P-79; M-1 to H-78; M-1 to S-77; M-1 to N-76; M-1 to K-75;
M-1 to S-74; M-1 to P-73; M-1 to R-72; M-1 to E-71; M-1 to T-70;
M-1 to C-69; M-1 to A-68; M-1 to L-67; M-1 to T-66; M-1 to K-65;
M-1 to P-64; M-1 to M-63; M-1 to E-62; M-1 to G-61; M-1 to D-60;
M-1 to R-59; M-1 to I-58; M-1 to I-57; M-1 to Q-56; M-1 to W-55;
M-1 to A-54; M-1 to K-53; M-1 to Q-52; M-1 to S-51; M-1 to S-50;
M-1 to A-49; M-1 to F-48; M-1 to K-47; M-1 to E-46; M-1 to L-45;
M-1 to T-44; M-1 to Y-43; M-1 to D-42; M-1 to C-41; M-1 to K-40;
M-1 to V-39; M-1 to D-38; M-1 to L-37; M-1 to T-36; M-1 to Q-35;
M-1 to G-34; M-1 to E-33; M-1 to K-32; M-1 to L-31; M-1 to E-30;
M-1 to Y-29; M-1 to K-28; M-1 to E-27; M-1 to E-26; M-1 to T-25;
M-1 to L-24; M-1 to K-23; M-1 to T-22; M-1 to A-21; M-1 to A-20;
M-1 to R-19; M-1 to L-18; M-1 to E-17; M-1 to S-16; M-1 to V-15;
M-1 to F-14; M-1 to L-13; M-1 to M-12; M-1 to W-11; M-1 to L-10;
M-1 to L-9; M-1 to G-8; and/or M-1 to W-7; of SEQ ID NO: 478.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
[0847] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein (e.g., ability to inhibit the Mixed Lymphocyte Reaction),
other functional activities (e.g., biological activities, ability
to multimerize, ability to bind ligand, ability to generate
antibodies, ability to bind antibodies) may still be retained. For
example, the ability of the shortened polypeptide to induce and/or
bind to antibodies which recognize the complete or mature forms of
the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature polypeptide are
removed from the C-terminus. Whether a particular polypeptide
lacking C-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that a polypeptide with a large number of deleted C-terminal amino
acid residues may retain some biological or immunogenic activities.
In fact, peptides composed of as few as six amino acid residues may
often evoke an immune response. Accordingly, the present invention
further provides polypeptides having one or more residues deleted
from the carboxy terminus of the amino acid sequence of the
polypeptide shown in FIGS. 1A-B (SEQ ID NO: 478), as described by
the general formula 1-n, where n is an integer from 6 to 228, where
n corresponds to the position of the amino acid residue identified
in SEQ ID NO: 478.
[0848] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group of N-terminal
deletions of the mature extracellular portion of the B7-H5 protein
(SEQ ID NO: 760): K-28 to I-198; Y-29 to I-198; E-30 to I-198; L-31
to 1-198; K-32 to I-198; E-33 to I-198; G-34 to I-198; Q-35 to
I-198; T-36 to I-198; L-37 to I-198; D-38 to I-198; V-39 to I-198;
K-40 to I-198; C-41 to I-198; D-42 to I-198; Y-43 to I-198; T-44 to
I-198; L-45 to I-198; E-46 to I-198; K-47 to I-198; F-48 to I-198;
A-49 to I-198; 5-50 to I-198; 5-51 to I-198; Q-52 to I-198; K-53 to
I-198; A-54 to I-198; W-55 to I-198; Q-56 to I-198; I-57 to I-198;
I-58 to I-198; R-59 to I-198; D-60 to I-198; G-61 to I-198; E-62 to
1-198; M-63 to I-198; P-64 to I-198; K-65 to I-198; T-66 to I-198;
L-67 to I-198; A-68 to I-198; C-69 to I-198; T-70 to I-198; E-71 to
I-198; R-72 to I-198; P-73 to I-198; S-74 to I-198; K-75 to I-198;
N-76 to I-198; S-77 to I-198; H-78 to I-198; P-79 to I-198; V-80 to
I-198; Q-81 to I-198; V-82 to I-198; G-83 to I-198; R-84 to I-198;
I-85 to I-198; I-86 to I-198; L-87 to I-198; E-88 to I-198; D-89 to
I-198; Y-90 to I-198; H-91 to I-198; D-92 to I-198; H-93 to I-198;
G-94 to I-198; L-95 to I-198; L-96 to I-198; R-97 to I-198; V-98 to
I-198; R-99 to I-198; M-100 to I-198; V-101 to I-198; N-102 to
I-198; L-103 to I-198; Q-104 to I-198; V-105 to I-198; E-106 to
I-198; D-107 to I-198; 5-108 to I-198; G-109 to I-198; L-110 to
I-198; Y-111 to I-198; Q-112 to I-198; C-113 to I-198; V-114 to
I-198; I-115 to I-198; Y-116 to I-198; Q-117 to I-198; P-118 to
I-198; P-119 to I-198; K-120 to I-198; E-121 to I-198; P-122 to
I-198; H-123 to I-198; M-124 to I-198; L-125 to I-198; F-126 to
I-198; D-127 to I-198; R-128 to I-198; I-129 to 1-198; R-130 to
I-198; L-131 to I-198; V-132 to I-198; V-133 to I-198; T-134 to
I-198; K-135 to I-198; G-136 to I-198; F-137 to I-198; S-138 to
I-198; G-139 to I-198; T-140 to I-198; P-141 to I-198; G-142 to
I-198; S-143 to I-198; N-144 to I-198; E-145 to I-198; N-146 to
I-198; S-147 to I-198; T-148 to I-198; Q-149 to I-198; N-150 to
I-198; V-151 to I-198; Y-152 to I-198; K-153 to I-198; I-154 to
I-198; P-155 to I-198; P-156 to I-198; T-157 to I-198; T-158 to
I-198; T-159 to I-198; K-160 to I-198; A-161 to I-198; L-162 to
I-198; C-163 to I-198; P-164 to I-198; L-165 to I-198; Y-166 to
I-198; T-167 to I-198; S-168 to I-198; P-169 to I-198; R-170 to
I-198; T-171 to I-198; V-172 to I-198; T-173 to I-198; Q-174 to
I-198; A-175 to I-198; P-176 to I-198; P-177 to I-198; K-178 to
I-198; S-179 to I-198; T-180 to I-198; A-181 to I-198; D-182 to
I-198; V-183 to I-198; S-184 to I-198; T-185 to I-198; P-186 to
I-198; D-187 to I-198; S-188 to I-198; E-189 to I-198; I-190 to
I-198; N-191 to I-198; L-192 to I-198; T-193 to I-198; and/or K-23
to A-29 of SEQ ID NO: 478. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0849] Additionally, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
an amino acid sequence selected from the group of C-terminal
deletions of the mature extracellular portion of the B7-H5 protein
(SEQ ID NO: 760): E-27 to D-197; E-27 to T-196; E-27 to V-195; E-27
to N-194; E-27 to T-193; E-27 to L-192; E-27 to N-191; E-27 to
I-190; E-27 to E-189; E-27 to S-188; E-27 to D-187; E-27 to P-186;
E-27 to T-185; E-27 to S-184; E-27 to V-183; E-27 to D-182; E-27 to
A-181; E-27 to T-180; E-27 to S-179; E-27 to K-178; E-27 to P-177;
E-27 to P-176; E-27 to A-175; E-27 to Q-174; E-27 to T-173; E-27 to
V-172; E-27 to T-171; E-27 to R-170; E-27 to P-169; E-27 to S-168;
E-27 to T-167; E-27 to Y-166; E-27 to L-165; E-27 to P-164; E-27 to
C-163; E-27 to L-162; E-27 to A-161; E-27 to K-160; E-27 to T-159;
E-27 to T-158; E-27 to T-157; E-27 to P-156; E-27 to P-155; E-27 to
I-154; E-27 to K-153; E-27 to Y-152; E-27 to V-151; E-27 to N-150;
E-27 to Q-149; E-27 to T-148; E-27 to S-147; E-27 to N-146; E-27 to
E-145; E-27 to N-144; E-27 to S-143; E-27 to G-142; E-27 to P-141;
E-27 to T-140; E-27 to G-139; E-27 to S-138; E-27 to F-137; E-27 to
G-136; E-27 to K-135; E-27 to T-134; E-27 to V-133; E-27 to V-132;
E-27 to L-131; E-27 to R-130; E-27 to I-129; E-27 to R-128; E-27 to
D-127; E-27 to F-126; E-27 to L-125; E-27 to M-124; E-27 to H-123;
E-27 to P-122; E-27 to E-121; E-27 to K-120; E-27 to P-119; E-27 to
P-118; E-27 to Q-117; E-27 to Y-116; E-27 to I-115; E-27 to V-114;
E-27 to C-113; E-27 to Q-112; E-27 to Y-111; E-27 to L-110; E-27 to
G-109; E-27 to S-108; E-27 to D-107; E-27 to E-106; E-27 to V-105;
E-27 to Q-104; E-27 to L-103; E-27 to N-102; E-27 to V-101; E-27 to
M-100; E-27 to R-99; E-27 to V-98; E-27 to R-97; E-27 to L-96; E-27
to L-95; E-27 to G-94; E-27 to H-93; E-27 to D-92; E-27 to H-91;
E-27 to Y-90; E-27 to D-89; E-27 to E-88; E-27 to L-87; E-27 to
I-86; E-27 to I-85; E-27 to R-84; E-27 to G-83; E-27 to V-82; E-27
to Q-81; E-27 to V-80; E-27 to P-79; E-27 to H-78; E-27 to S-77;
E-27 to N-76; E-27 to K-75; E-27 to S-74; E-27 to P-73; E-27 to
R-72; E-27 to E-71; E-27 to T-70; E-27 to C-69; E-27 to A-68; E-27
to L-67; E-27 to T-66; E-27 to K-65; E-27 to P-64; E-27 to M-63;
E-27 to E-62; E-27 to G-61; E-27 to D-60; E-27 to R-59; E-27 to
I-58; E-27 to I-57; E-27 to Q-56; E-27 to W-55; E-27 to A-54; E-27
to K-53; E-27 to Q-52; E-27 to 5-51; E-27 to 5-50; E-27 to A-49;
E-27 to F-48; E-27 to K-47; E-27 to E-46; E-27 to L-45; E-27 to
T-44; E-27 to Y-43; E-27 to D-42; E-27 to C-41; E-27 to K-40; E-27
to V-39; E-27 to D-38; E-27 to L-37; E-27 to T-36; E-27 to Q-35;
E-27 to G-34; and/or E-27 to E-33 of SEQ ID NO: 478. Polypeptides
encoded by these polynucleotides are also encompassed by the
invention.
[0850] In addition, any of the above listed N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
polypeptide. The invention also provides polypeptides comprising,
or alternatively consisting of, one or more amino acids deleted
from both the amino and the carboxyl termini, which may be
described generally as having residues m-n of SEQ ID NO: 478, where
n and m are integers as described above. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0851] The present invention is also directed to proteins
containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to a polypeptide sequence set forth
herein as m-n. In preferred embodiments, the application is
directed to proteins containing polypeptides at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the
amino acid sequence of the specific N- and C-terminal deletions
recited herein. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0852] Also included are polynucleotide sequences encoding a
polypeptide consisting of a portion of the complete amino acid
sequence encoded by a cDNA clone contained in ATCC.TM. Deposit Nos.
97903 and/or 209049, where this portion excludes any integer of
amino acid residues from 1 to about 228 amino acids from the amino
terminus of the complete amino acid sequence encoded by a cDNA
clone contained in ATCC.TM. Deposit Nos. 97903 and/or 209049, or
any integer of amino acid residues from 1 to about 228 amino acids
from the carboxy terminus, or any combination of the above amino
terminal and carboxy terminal deletions, of the complete amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
Nos. 97903 and/or 209049. Polypeptides encoded by these
polynucleotides also are encompassed by the invention.
[0853] As described herein or otherwise known in the art, the
polynucleotides of the invention have uses that include, but are
not limited to, serving as probes or primers in chromosome
identification, chromosome mapping, and linkage analysis.
[0854] Polynucleotides and polypeptides of the invention are useful
as reagents for differential identification of immune system
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, diseases and/or disorders involving immune system
activation, stimulation and/or surveillance, particularly involving
T cells and/or neutrophils. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). Particularly contemplated are the use of
antibodies directed against the extracellular portion of this
protein which act as antagonists for the activity of the B7-H5
protein. Such antagonistic antibodies would be useful for the
prevention and/or inhibition of such biological activities as are
disclosed herein (e.g. T cell modulated activities).
[0855] For a number of disorders of the above tissues or cells,
particularly of the immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0856] The tissue distribution in immune cells (e.g., T-cells,
neutrophils), and the homology to members of the B7 family of
ligands, indicates that the polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis, detection
and/or treatment of diseases and/or disorders involving immune
system activation, stimulation and/or surveillance, particularly as
relating to T cells and/or neutrophils. In particular, the
translation product of the B7-H5 gene may be involved in the
costimulation of T cells, binding to ICOS, and/or may play a role
in modulation of the expression of particular cytokines.
Representative uses are also described in the "Immune Activity"
section below and elsewhere herein.
[0857] More generally, the tissue distribution in immune system
cells indicates that this gene product may be involved in the
regulation of cytokine production, antigen presentation, or other
processes that may also suggest a usefulness in the treatment of
cancer (e.g. by boosting immune responses). Since the gene is
expressed in cells of lymphoid origin, the gene or protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0858] Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement.
[0859] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO: 169 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 888 of SEQ ID NO: 169, b is an
integer of 15 to 902, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO: 169, and where
b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 160
[0860] The translation product of this gene shares sequence
homology with mouse X inactive specific transcript protein which is
thought to be important in X chromosome inactivation.
[0861] This gene is expressed primarily in HSA172 cell and to a
lesser extent in normal ovary tissue, ovarian cancer, frontal
cortex and brain.
[0862] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: ovarian tumor, schizophrenia
and other neurological disorders. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune and neural system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
ovary and other reproductive tissue, and brain and other tissue of
the nervous system, and cancerous and wounded tissues) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder.
[0863] The tissue distribution and homology to X inactive specific
transcript protein indicate that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment
of reproductive system tumors and CNS tumors.
[0864] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 479 as residues: His-48 to Trp-56.
[0865] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:170 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1869 of SEQ ID NO:170, b is an
integer of 15 to 1883, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:170, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 161
[0866] This gene is expressed primarily in adipose cells and to a
lesser extent in liver and prostate tissue.
[0867] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: obesity and liver disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the adipose cell, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., adipose cells, liver, and prostate, and cancerous
and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0868] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for treatment of
obesity and liver disorder.
[0869] This gene is believed to reside on chromosome 3.
Polynucleotides related to this gene are useful, therefore, in
linkage analysis for chromosome 3.
[0870] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:171 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2086 of SEQ ID NO:171, b is an
integer of 15 to 2100, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:171, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 162
[0871] The translation product of this gene shares sequence
homology with yeast ubiquitin activating enzyme homolog which is
thought to be important in protein post translation processing.
Mammalian cells contain two distinct proteolytic pathways that are
involved in different aspects of protein breakdown. One of these is
ubiquitin-dependent, it is a major pathway in eukaryotes involved
in the selective degradation of abnormal and short-lived proteins.
Ubiquitin is a highly conserved 76 amino acid residue protein
present in eukaryotic cells either free or covalently attached to a
great variety of proteins.
[0872] The ubiquitin gene is one of the genes known to be
stimulated during the apoptotic death program and ubiquitin of
nuclear proteins might be involved in chromatin disorganization and
oligonucleosomal fragmentation, which are among the key events
occurring in apoptosis. Apoptosis, the classical type of programmed
cell death, can be triggered in many cell types by widely diverse
stimuli, for example, gamma rays at low doses can induce apoptosis
in vitro in interphase human lymphocytes. In this type of apoptosis
induction, activated gene expression is necessary for the
fulfillment of the death program. It has been reported (Delic, J.,
et al., Mol. Cell. Biol., 13:4875, 83 (1993)) that there is a
relationship between ubiquitin gene expression or ubiquitination
and gamma-irradiation-mediated apoptosis in normal circulating
human lymphocytes. In this report it has been demonstrated that the
ubiquitin mRNA level is increased as a consequence of the
activation of ubiquitin gene transcription 15 to 90 minutes after
initiation of apoptosis; specifically, in apoptotic cells, and not
in all irradiated cells, nuclear proteins are highly ubiquitinated;
and ubiquitin sequence-specific antisense oligonucleotide
inhibition results in a decreased level of ubiquitinated nuclear
proteins and considerably diminishes the proportion of cells
exhibiting the apoptotic death pattern.
[0873] Perturbations of ubiquitin system can also induce a
programmed necrotic response in plants such as leaf curling,
vascular tissue alterations and necrotic lesions.
[0874] Ubiquitin can inhibit the cytotoxic properties of platelets
and the production of oxygen metabolites by these cells. Moreover,
this molecule is able to act as a proaggregating factor and seems
of a great interest in pathologies involving defects in platelet
aggregation. Ubiquitin also plays a role in the regulation of
immunological disorders in which platelets seem to be implicated
such as hymenoptera venom hypersensitivity and aspirin-sensitive
asthma, since in both situations, ubiquitin is able to inhibit the
cytotoxic function of platelets. Ubiquitin has also been shown to
be increased in patients with Alzheimer's disease (Taddei, N., et
al., Neurosci. Lett., 151:158-61 (1993)). This study concerned the
amount of soluble ubiquitin in different cortical and subcortical
regions of brains from patients with Alzheimer's disease compared
to the amount in a normal brain. The soluble ubiquitin content was
significantly higher in pathological tissue than in normal tissue.
The primary structure of ubiquitin isolated from brain tissue
affected by Alzheimer's degenerative processes was determined and
resulted to be identical to normal human ubiquitin. This report
suggests that an impairment of the process of intracellular,
ubiquitin-dependent proteolysis might play an important role in the
pathogenesis of this neurodegenerative disease.
[0875] Ubiquitin-proteasome system also plays a major role in
specific processing and subsequent presentation of MHC class
I-restricted antigens.
[0876] Maturation of the p105 NF-kB precursor into the active p50
subunit of the transcriptional activator also proceeds in a
ubiquitin and proteasome-dependent manner. Furthermore, inhibitors
to the proteasome block degradation of IkBa and thus prevent tumor
necrosis factor alpha induced activation of NF-kB and its entry
into the nucleus.
[0877] The unstable c-Jun, but not the stable v-Jun, is
multi-ubiquitinated and degraded. The escape of the oncogenic v-Jun
from ubiquitin-dependent degradation suggests a route to the
malignant transformation. Another proto-oncoprotein, c-Mos, is also
degraded by the ubiquitin system.
[0878] The human papilloma virus (HPV) derived E6 proteins
stimulate ATP and ubiquitin dependent conjugation and degradation
of p53, such a mechanism could explain the extremely low levels of
p53 observed in HPV-transformed cervical carcinoma lines and
propose a mechanism for the tumorigenicity of these
onco-proteins.
[0879] Several cell surface receptors, including the lymphocyte
homing receptor, growth hormone receptor, and growth factor
receptor (PDGF, steel factor) were also found to be modified by
ubiquitin.
[0880] This gene is expressed primarily in stromal cell and to a
lesser extent in retina, human atrophic endometrium, colon
carcinoma and myeloid progenitor cells.
[0881] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: defects of stromal cell
development, neuronal growth disorders and tumors. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and neural system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., retinal cells, endometrium, colon, and bone
marrow, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0882] The tissue distribution and homology to ubiquitin-activating
enzyme homolog indicate that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis or treatment of
some type of tumors, fucosidosis and neuronal growth disorders.
[0883] Once the polypeptides are being expressed intra-cellularly
via gene therapy, they may be employed to provide a signal for the
lymphocyte homing receptor thereby regulating lymphocyte
trafficking. The growth hormone receptor also utilizes ubiquitin to
signal ligands, and, therefore, the Ubiquitin Activating-like
polypeptides may be employed to regulate activation of the growth
receptor.
[0884] Ubiquitin Activating-like polypeptides may be employed to
overcome many viral infections by overcoming the suppressed
programmed cell death induced by these viruses, since programmed
cell death may be one of the primary antiviral defense mechanisms
of cells.
[0885] Ubiquitin Activating-like polypeptides may also be employed
to treat immuno-suppression related disorders, such as AIDS, by
targeting virus infected cells for cell death.
[0886] Ubiquitin Activating-like polypeptides may also be employed
to inhibit the cytotoxic properties of platelets and the production
of oxygen metabolites by platelets. These polypeptides may also be
employed to regulate immunological disorders in which platelets
seem to be involved, for example, hymenoptera venom
hypersensitivity and aspirin-sensitive asthma.
[0887] Ubiquitin Activating-like polypeptides may also be employed
to treat malignant transformation because proto-oncoproteins c-Mos
and v-Jun are degraded in a ubiquitin-dependent manner.
[0888] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 481 as residues: Lys-65 to Lys-72, Tyr-159 to
Thr-169, Arg-176 to Pro-182, Glu-200 to Glu-214, Ala-228 to
Asp-234, Ile-238 to Asp-250, Lys-271 to Pro-279, Val-287 to
Gly-306, Asp-345 to Ser-350, Asn-419 to Lys-426, Glu-494 to
Leu-506.
[0889] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:172 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1916 of SEQ ID NO:172, b is an
integer of 15 to 1930, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:172, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 163
[0890] This gene is expressed primarily in primary breast cancer
and hemangiopericytoma and to a lesser extent in adult brain and
cerebellum.
[0891] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: breast cancer, leukemia and
cerebellum disorders. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system and neural
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., mammary tissue, brain and other tissue of the nervous
system, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0892] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis or
treatment of various tumors and disease involved in neural
system.
[0893] This gene is believed to reside on chromosome 3.
Polynucleotides related to this gene are believed to be useful,
therefore, in linkage analysis as markers for chromosome 3.
[0894] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 482 as residues: Glu-29 to Tyr-42, Glu-44 to Glu-54,
Tyr-68 to Glu-73, Ala-145 to Leu-165, Gln-173 to Ser-183, Lys-215
to Cys-220.
[0895] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:173 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1495 of SEQ ID NO:173, b is an
integer of 15 to 1509, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:173, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 164
[0896] The translation product of this gene, "MAX.3", shares
sequence homology with proline rich proteins. Recently, another
group has also cloned this gene, calling it CD84 leukocyte antigen,
a new member of the Immunoglobulin superfamily. MAX.3 detects the
CD84 antigen, which is a member of the CD2 family of cell surface
molecules, and it is postulated that it is this interaction which
allows MAX.3 to function in intracellular signaling events. (See
Accession No. U82988, see also, Blood 90 (6), 2398-2405 (1997),
incorporated herein by reference.) The extracellular domain is
believed to comprise residues 23-220, the transmembrane domain
residues 221-245 and the intracellular domain residues 246-389, of
SEQ ID NO:483. Preferred polypeptides of the invention comprise the
extracellular domain alone, residues 23-220, or fused to the
intracellular domain; i.e., lacking the transmembrane domain.
[0897] This gene is expressed primarily in Weizmann olfactory
tissue and osteoclastoma and to a lesser extent in anergic
T-cells.
[0898] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: ostsis and immune disease.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., olfactory tissue, bone, and blood cells, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0899] The tissue distribution and homology to the Ig superfamily
CD84 antigen indicate that the protein product of this clone is
useful for treatment of osteoporosis, autoimmune disease, and other
immune disorders. Alternatively, the tissue distribution and
homology to a cell-surface antigen may indicate that MAX.3 plays a
role in the extracellular matrix remodelling and/or wound healing
immune responses in the body.
[0900] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 483 as residues: Ala-19 to Ser-24, Glu-102 to
Ile-111, Thr-113 to Tyr-124, Glu-158 to Asn-167, Thr-183 to
Leu-189, Asn-197 to Ser-207, Lys-250 to Thr-260, Arg-269 to
Ile-278, Pro-289 to Pro-294, Ala-311 to Ser-323.
[0901] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:174 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 3159 of SEQ ID NO:174, b is an
integer of 15 to 3173, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:174, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 166
[0902] This gene is expressed primarily in human primary breast
cancer and to a lesser extent in activated monocyte. This
polypeptide is a type Ia transmembrane protein. The extracellular
domain is believe to comprise residues from about 20-195, the
transmembrane domain residues from about 196 to about 212, and the
cytoplasmic tail residues from about 213 to about 217, of SEQ ID
NO: 485. Preferred polypeptide fragments comprise the amino acid
sequence: VTQPKHLSASMGGSVEIPFSFYYPWELAXXPXVRISWRRGHFHG
QSFYSTRPPSIHKDYVNRLFLNWTEGQESGFLRISNLRKEDQSVYFCRVELDTRRSG (SEQ ID
NO: 754), as well as N-terminal and C-terminal deletions. Also
preferred are polypeptides comprising the extracellular domain,
residues 20 to 195. Also provided are polynucleotide fragments
encoding these polypeptides.
[0903] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: breast cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., mammary tissue, and blood cells, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0904] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis of
breast cancer and other immune system disorders.
[0905] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:176 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1276 of SEQ ID NO:176, b is an
integer of 15 to 1290, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:176, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 167
[0906] This gene is expressed primarily in fetal tissues and to a
lesser extent in adult lung. This gene has also been mapped to
chromosomal location 9q34, and thus, can be used as a marker for
linkage analysis for chromosome 9.
[0907] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the embryo tissues, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., fetal tissues, and lung, and cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0908] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful as reagents for
differential identification of tissues or cell types present in a
biological sample.
[0909] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 486 as residues: Tyr-15 to Gly-23.
[0910] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:177 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2276 of SEQ ID NO:177, b is an
integer of 15 to 2290, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:177, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 168
[0911] The translation product of this gene shares sequence
homology with Ig Heavy Chain which is thought to be important in
immune response.
[0912] This gene is expressed primarily in prostate cancer tissue
specifically
[0913] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: prostate cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
prostate, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., prostate, tissue and cells of the immune system, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0914] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis of
prostate cancer and other immune system disorders.
[0915] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:178 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 535 of SEQ ID NO:178, b is an integer
of 15 to 549, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:178, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 169
[0916] The translation product of this gene shares sequence
homology with cytosolic acyl coenzyme-A hydrolase, which is thought
to be important in neuron-specific fatty acid metabolism. The gene
represented by this contig has since been published by Hajra and
colleagues (GenBank Accession No. U91316, incorporated herein by
reference).
[0917] This gene is expressed primarily in human pituitary gland
and to a lesser extent in colorectal cancer tissue. This gene has
also been observed in the LNCAP cell line.
[0918] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: hyperlipidemias of familial
and/or idiopathic origins. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly blood, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., pituitary and colon, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0919] The tissue distribution and homology to rat cytosolic acyl
coenzyme-A hydrolase indicate that polynucleotides and polypeptides
corresponding to this gene are useful for the detection or
treatment of hyperlipidemia disease states by virtue of the ability
of specific drugs to activate the enzyme.
[0920] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 488 as residues: Arg-48 to Glu-56.
[0921] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:179 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1495 of SEQ ID NO:179, b is an
integer of 15 to 1509, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:179, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 170
[0922] The translation product of this gene shares sequence
homology with a Caenorhabditis elegans gene which is thought to be
important in organism development. This gene is believed to be
important in embryogenesis and cancer.
[0923] Preferred polypeptide encoded by this gene comprise the
following amino acid sequence:
MATPAVPVSAPPATPTPVPAAAPASVPAPTPAPAAAPVPAAAPACILRPC
GSSGCNCGSWPDPGLSAXPAQTPAPALPGPALPGPFPGGRVVRLHPVILAIVDSY
ERRNEGAARVIGTLLGTVDKHSVEVTNCFSVPHNESEDEVAVDMEFAKNMYELHKKVSPNELILGWYAT
GHDITEHSVLIHEYYSREAPNPIHLTVDTSLQNGRMSIKAYVSTLMGVPGRTMGVMFTPLTVKYAYYDTE
RIGVDLIMKTCFSPNRVIGLSSDLQQVGGASARIQDALSTVLQYAEDVLSGKVSADNTVGRFLMSLVNQVP
KIVPDDFETMLNSNINDLLMVTYLANLTQSQIALNEKLVNL (SEQ ID NO: 755) and
MLNSNINDLLMVTYLANLTQSQIALNEKLVNL (SEQ ID NO: 756). Polynucleotides
encoding such polypeptides are also provided.
[0924] This gene is expressed primarily in human synovial sarcoma
tissue, bone marrow, and to a lesser extent in human brain.
[0925] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, bone, specifically synovial sarcoma. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the bone,
connective tissues and possibly immune system, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., synovial tissue,
bone marrow, brain and other tissue of the nervous system, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0926] The tissue distribution and homology to Caenorhabditis
elegans indicate that polynucleotides and polypeptides
corresponding to this gene are useful as a diagnostic and/or
therapeutic modality directed at the detection and/or treatment of
connective tissue sarcomas or other related bone diseases.
[0927] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:180 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1302 of SEQ ID NO:180, b is an
integer of 15 to 1316, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:180, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 171
[0928] The translation product of this gene shares sequence
homology with beta1-6GlcNAc transferase which is thought to be
important in the transfer and metabolism of beta1-6,
N-acetylglucosamine. This gene product has previously been shown to
suppress melanoma lung metastasis in both syngeneic and nude mice,
decreased invasiveness into the MATRIGEL.TM., and inhibition of
cell attachment to collagen and laminin without affecting cell
growth.
[0929] This gene is expressed primarily in human testes and
prostate tissues, and to a lesser extent in kidney, medulla, and
pancreas.
[0930] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancer particularly melanoma. Similarly, polypeptides
and antibodies directed to these polypeptides are useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
testes and other reproductive tissue, prostate, kidney, pancreas,
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0931] The tissue distribution and homology to beta1-6GlcNAc
transferase indicate that the protein product of this clone is
useful for the development of diagnostic and/or therapeutic
modalities directed at the detection and/or treatment of cancer,
the metastasis of malignant tissue or cells. Defects in this
potentially secreted enzyme may play a role in metastasis.
[0932] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:181 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 763 of SEQ ID NO:181, b is an integer
of 15 to 777, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:181, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 172
[0933] This gene is expressed primarily in fetal spleen and
liver.
[0934] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: immune disorders, Wilm's
tumor disease, hepatic disorders, and hematopoietic disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hematopoiesis and immune systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., spleen and liver, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0935] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the
treatment and identification of fetal defects along with correcting
diseases that affect hematopoiesis and the immune system.
[0936] This gene is believed to reside on chromosome 6. Therefore,
nucleic acids related to this gene are useful in linkage analysis
as markers for chromosome 6.
[0937] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:182 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 777 of SEQ ID NO:182, b is an integer
of 15 to 791, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:182, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 173
[0938] The translation product of this gene shares sequence
homology with ret II oncogene which is thought to be important in
Hirschsprung disease and many types of cancers.
[0939] This gene is expressed in multiple tissues including the
lymphatic system, brain, and thyroid.
[0940] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of
diseases and conditions: Hirschsprung disease and multiple cancers.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., lymphoid tissue, thyroid, and
brain and other tissue of the nervous system, and cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0941] The tissue distribution and homology to ret II oncogene
indicate that polynucleotides and polypeptides corresponding to
this gene are useful for diagnosis and treatment of various
cancers. It would also be useful for the diagnosis and treatment of
Hirschsprung disease. Preferred polypeptides of the invention
comprise the amino acid sequence:
MEAQQVNEAESAREQLQXLHDQIAGQKASKQELETELERLKQEFHYIEE
DLYRTKNTLQSRIKDRDEEIQKLRNQLTNKTLSNSSQSELENRLHQLTETLIQKQTMLESLSTEKNSLVFQL
ERLEQQMNSASGSSSNGSSINMSGIDNGEGTRLRNVPVLFNDTETNLAGMYGKVRKAASSIDQFSIRLGIFL
RRYPIARVFVIIYMALLHLWVMIVLLTYTPEMHHDQPYGK (SEQ ID NO: 757).
Polynucleotides encoding such polypeptides are also provided.
[0942] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:183 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1391 of SEQ ID NO:183, b is an
integer of 15 to 1405, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:183, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 174
[0943] The translation product of this gene shares sequence
homology with testis enhanced gene transcript which is thought to
be important in regulation of human development.
[0944] This gene is expressed primarily in infant brain and to a
lesser extent in a variety of other tissues and cell types,
including the prostate, testes, monocytes, macrophages, dendritic
cells, keratinocytes, and adipocytes.
[0945] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neurological, developmental,
immune and inflammation disorders. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the brain and immune systems,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
brain and other tissue of the nervous system, prostate, testes and
other reproductive tissue, blood cells, keratinocytes, and
adipocytes, and cancerous and wounded tissues) or bodily fluids
(e.g., serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0946] The tissue distribution and homology to testis enhanced gene
transcript indicate that the protein product of this clone is
useful for diagnosis and treatment of disorders involving the
developing brain and the immune system.
[0947] This gene is believed to reside on chromosome 10. Nucleic
acids related to this gene are useful therefore in linkage analysis
as markers for chromosome 10.
[0948] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 493 as residues: Ser-31 to Trp-37, Pro-41 to Thr-49,
Arg-54 to Lys-63, Tyr-119 to His-125, Pro-181 to Lys-189, Gly-340
to Lys-345.
[0949] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:184 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1582 of SEQ ID NO:184, b is an
integer of 15 to 1596, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:184, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 175
[0950] This gene is expressed primarily in prostate and to a lesser
extent in various other tissues, including placenta.
[0951] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, cancers, especially of the prostate. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
prostate, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., prostate and placenta, and cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0952] The tissue distribution indicates that the protein product
of this clone is useful for diagnosis and treatment of prostate
disorders and cancer. It may also be useful for the diagnosis and
treatment of endocrine disorders.
[0953] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 494 as residues: Lys-10 to Trp-19, Arg-49 to Leu-54,
Val-78 to Val-87, Pro-141 to Lys-148.
[0954] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:185 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2279 of SEQ ID NO:185, b is an
integer of 15 to 2293, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:185, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 176
[0955] The translation product of this gene shares sequence
homology with Sacchromyces cerevisiae YNT20 gene which is thought
to be important in mitochondrial function.
[0956] This gene is expressed at a particularly high level in
muscle tissue.
[0957] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases related to such tissues and cell types
including: muscle wasting diseases. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the neuromuscular system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
muscle and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0958] The tissue distribution and homology to the YNT20 gene
indicate that this protein is useful for treatment and detection of
neuromuscular diseases caused by loss of mitochondrial function.
For example this gene or its protein product could be used in
replacement therapy for such diseases.
[0959] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 495 as residues: Arg-91 to Pro-96, Val-106 to
Leu-113, Lys-180 to Lys-187, Asn-191 to Val-198.
[0960] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:186 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1198 of SEQ ID NO:186, b is an
integer of 15 to 1212, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:186, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 177
[0961] This gene is expressed primarily in the brain and to a
lesser extent in kidney, placenta, smooth muscle, heart and
lung.
[0962] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: neuromuscular diseases,
degenerative diseases of the central nervous system, and heart
disease. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the neuromuscular system, central nervous system, and heart,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
brain and other tissue of the nervous system, kidney, placenta,
muscle, heart and lung, and cancerous and wounded tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0963] This gene or its protein product could also be used for
replacement therapy for the above mentioned diseases.
[0964] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 496 as residues: Arg-12 to Glu-18, Asn-38 to Phe-47,
Arg-56 to Val-62.
[0965] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:187 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1591 of SEQ ID NO:187, b is an
integer of 15 to 1605, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:187, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 178
[0966] The translation product of this gene shares sequence
homology with caldesmon which is thought to be important in the
cellular response to changes in glucose levels.
[0967] This gene is expressed primarily in multiple tissues
including brain and retina.
[0968] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for identification of the tissue(s) or cell
type(s) present in a biological sample and for diagnosis of
diseases and conditions: central nervous system disorders and
retinopathy. Similarly, polypeptides and antibodies directed to
these polypeptides are useful in providing immunological probes for
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the CNS
disorders and retinopathy, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., brain and other tissue of the nervous system,
and retinal tissue, and cancerous and wounded tissues) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder.
[0969] The tissue distribution and homology to caldesmon indicate
that polynucleotides and polypeptides corresponding to this gene
are useful for treatment of retinopathies.
[0970] This gene is believe to be useful as a marker for
chromosomes 1 and 3 in linkage analysis studies.
[0971] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 497 as residues: Met-1 to Gly-7, Glu-17 to Glu-28,
Lys-39 to Asp-45, Ser-50 to Ser-63, Glu-82 to Phe-88, Pro-97 to
Lys-109, Gln-124 to Arg-129, Val-145 to Lys-157, Glu-163 to
Leu-168, Ile-186 to Glu-195, Glu-198 to Arg-230, Asn-240 to
Phe-252, Pro-256 to Glu-264.
[0972] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:188 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1502 of SEQ ID NO:188, b is an
integer of 15 to 1516, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:188, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 179
[0973] The translation product of this gene shares sequence
homology with mouse fibrosin protein which is thought to be
important in regulation of fibrinogenesis in certain chronic
inflammatory diseases.
[0974] This gene is expressed primarily in amniotic cells and
breast tissue.
[0975] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of breast cancer and abnormal embryo development.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the reproductive system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
and cell types (e.g., amniotic cells, and mammary tissue, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0976] The tissue distribution and homology to fibrosin indicate
that the protein product of this clone is useful for treatment of
breast cancer. This gene or its protein product could be used in
replacement therapy for breast cancer. In addition the protein
product of this gene is useful in the treatment of chronic
inflammatory diseases.
[0977] This gene is believed to reside on chromosome 15.
Polynucleotides related to this gene are believed to useful,
therefore, in linkage analysis as markers for chromosome 15.
[0978] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:189 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 667 of SEQ ID NO:189, b is an integer
of 15 to 681, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:189, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 180
[0979] This gene is expressed in several infant tissues including
brain and liver and various adult tissues including brain, lung,
liver, testes, and prostate.
[0980] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions which include, but are not
limited to, brain cancer, lung cancer, liver cancer and cancers of
the reproductive system. Similarly, polypeptides and antibodies
directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the central nervous system,
hepatic system, and reproductive system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., brain and other tissue of the
nervous system, lung, liver, testes and other reproductive tissue,
and prostate, and cancerous and wounded tissues) or bodily fluids
(e.g., serum, plasma, urine, synovial fluid or spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0981] The tissue distribution of this gene product indicates that
the protein product of this clone is involved in growth regulation
and could be used as a growth factor or growth blocker in a variety
of settings including treatment of cancers.
[0982] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 499 as residues: Pro-153 to Arg-158.
[0983] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:190 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1000 of SEQ ID NO:190, b is an
integer of 15 to 1014, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:190, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 181
[0984] This gene is expressed primarily in activated monocytes and
to a lesser extent in melanocytes and dendritic cells.
[0985] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of immune system diseases and cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues and cell types
(e.g., blood cells, melanocytes, and dendritic cells, and cancerous
and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0986] The tissue distribution indicates that the protein product
of this clone could be involved in growth regulation and could be
used as a growth factor or growth blocker in a variety of settings
including treatment of cancers.
[0987] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 500 as residues: Asp-35 to Asp-41, Ser-72 to Glu-77,
Lys-110 to His-115, Pro-160 to Gln-165.
[0988] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:191 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2765 of SEQ ID NO:191, b is an
integer of 15 to 2779, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:191, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 182
[0989] This gene is expressed primarily in placenta and several
tumors of various tissue origin and to a lesser extent in normal
tissues including liver, lung, brain, and skin,
[0990] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of cancers of all kinds. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the central nervous system,
respiratory system and skin, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., liver, lung, brain and other
tissues of the nervous system, and skin, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0991] The high expression of this gene in multiple tumors
indicates that the protein product of the clone may be involved in
cell growth control and therefore would be useful for treatment of
certain cancers. Likewise molecules developed to block the activity
of the protein product of this clone could be used to block its
potential role in tumor growth promotion.
[0992] This gene is believe to reside on chromosome 6.
Polynucleotides related to this gene are believed, therefore, to be
useful in linkage analysis for chromosome 6.
[0993] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 501 as residues: Gln-37 to Gln-43, Cys-51 to
Cys-65.
[0994] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:192 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1909 of SEQ ID NO:192, b is an
integer of 15 to 1923, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:192, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 183
[0995] The translation product of this gene shares sequence
homology with the mouse Ndr1 gene which is thought to be important
in cancer progression and atherogenesis (see J Biol Chem. 1996 Nov.
22; 271(47): 29659-29665), incorporated herein by reference in its
entirety.
[0996] This gene is expressed in multiple cell types and tissues
including brain, lung, kidney, bone marrow, liver, and spleen.
[0997] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of all types of cancers. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the nervous, immune, and
endocrine systems, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues and
cell types (e.g., brain and other tissue of the nervous system,
lung, kidney, bone marrow, liver and spleen, and cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0998] The tissue distribution and homology to Ndr1 gene, which is
thought to be involved in cancer progression, indicate that
polynucleotides and polypeptides corresponding to this gene are
useful for treatment of certain cancers. Likewise molecules
developed to block the activity of the protein product of this
clone could be used to block its potential role in tumor growth
promotion.
[0999] This gene is believed to reside on chromosome 8.
Polynucleotides derived from this gene are useful, therefore, in
linkage analysis as markers for chromosome 8.
[1000] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:193 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2332 of SEQ ID NO:193, b is an
integer of 15 to 2346, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:193, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 184
[1001] This gene is expressed primarily in early stage human brain
and liver and to a lesser extent in several other fetal
tissues.
[1002] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of diseases and conditions: brain and liver cancers.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system and immune system, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues and cell types (e.g., brain and other
tissue of the nervous system, liver, and fetal tissue, and
cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma, urine, synovial fluid or spinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[1003] The expression of this gene in embryonic tissues indicates
that the protein could be involved in growth regulation and could
be used as a growth factor or growth blocker in a variety of
settings including treatment of cancers.
[1004] This gene is believed to reside on chromosome 11.
Polynucleotides derived from this gene are useful, therefore, in
linkage analysis as markers for chromosome 11.
[1005] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 503 as residues: Pro-116 to Ser-127, Pro-136 to
Tyr-146.
[1006] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:194 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 3040 of SEQ ID NO:194, b is an
integer of 15 to 3054, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:194, and where
the b is greater than or equal to a +14.
Features of Protein Encoded by Gene No: 185
[1007] This gene is expressed primarily in infant and embryonic
brain.
[1008] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of degenerative nervous system disorders and brain
cancer. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues and cell types (e.g., embryonic tissue, brain and
other tissue of the nervous system, and cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[1009] The expression of this gene in embryonic tissues indicates
that the protein could be involved in growth regulation and could
be used as a growth factor or growth blocker in a variety of
settings including treatment of cancers.
[1010] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:195 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 893 of SEQ ID NO:195, b is an integer
of 15 to 907, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:195, and where the b is
greater than or equal to a +14.
Features of Protein Encoded by Gene No: 186
[1011] This gene is expressed primarily in multiple tissues
including placenta, fetal lung, fetal liver, and brain.
[1012] Therefore, polynucleotides and polypeptides of the invention
are useful as reagents for differential identification of the
tissue(s) or cell type(s) present in a biological sample and for
diagnosis of all types of cancers including liver, brain and lung.
Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system, pulmonary system, and hepatic system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues and cell types (e.g.,
placenta, lung, liver, and brain and other tissue of the nervous
system, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[1013] The expression of this gene in embryonic tissues indicates
that the protein could be involved in growth regulation and could
be used as a growth factor or growth blocker in a variety of
settings including treatment of cancers.
[1014] This gene is believed to reside on chromosome 10.
Polynucleotides derived from this gene are useful, therefore, in
linkage analysis as markers for chromosome 10.
[1015] Preferred epitopes include those comprising a sequence shown
in SEQ ID NO. 505 as residues: Pro-10 to Glu-17, Gly-42 to
Gln-51.
[1016] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:196 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1276 of SEQ ID NO:196, b is an
integer of 15 to 1290, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:196, and where
the b is greater than or equal to a +14.
TABLE-US-00002 TABLE 1 NT 5' 3' 5' NT AA SEQ NT NT of First SEQ
First Last First Last ATCC .TM. ID Total of of 5' NT AA of ID AA AA
AA of AA Gene cDNA Deposit Nr NO: NT Clone Clone of Start Signal
NO: of Sig of Sig Secreted of No. Clone ID and Date Vector X Seq.
Seq. Seq. Codon Pep Y Pep Pep Portion ORF 1 HTTEZ21 97897 Uni-ZAP
XR 11 1019 262 859 177 177 320 1 17 18 22 Feb. 26, 1997 209043 May
15, 1997 1 HTTEZ21 97897 Uni-ZAP XR 197 582 1 582 177 177 506 1 17
18 22 Feb. 26, 1997 209043 May 15, 1997 1 HTTEZ21 97897 Uni-ZAP XR
198 1020 296 830 442 442 507 1 18 19 22 Feb. 26, 1997 209043 May
15, 1997 2 HBGBW52 97897 Uni-ZAP XR 12 465 1 465 81 81 321 1 30 31
128 Feb. 26, 1997 209043 May 15, 1997 2 HBGBW52 97897 Uni-ZAP XR
199 524 229 343 196 508 1 20 21 33 Feb. 26, 1997 209043 May 15,
1997 3 HCUFM41 97897 ZAP Express 13 474 1 474 1 1 322 1 24 25 28
Feb. 26, 1997 209043 May 15, 1997 3 HCUFM41 97897 ZAP Express 200
332 1 319 35 35 509 1 24 25 28 Feb. 26, 1997 209043 May 15, 1997 4
HCUFQ22 97897 ZAP Express 14 314 1 298 122 122 323 1 34 35 64 Feb.
26, 1997 209043 May 15, 1997 5 HCUFV01 97897 ZAP Express 15 613 1
613 30 30 324 1 18 19 21 Feb. 26, 1997 209043 May 15, 1997 6
HCUGA50 97897 ZAP Express 16 356 1 356 239 239 325 1 22 23 39 Feb.
26, 1997 209043 May 15, 1997 7 HCUIM14 97897 ZAP Express 17 414 185
414 278 278 326 1 26 27 33 Feb. 26, 1997 209043 May 15, 1997 8
HLDOU93 97897 pCMVSport 3.0 18 469 1 469 77 77 327 1 44 45 88 Feb.
26, 1997 209043 May 15, 1997 9 HEIAX07 97897 Uni-ZAP XR 19 550 1
550 129 129 328 1 23 Feb. 26, 1997 209043 May 15, 1997 9 HEIAX07
97897 Uni-ZAP XR 201 376 9 376 1 510 1 8 9 15 Feb. 26, 1997 209043
May 15, 1997 10 HSAXR76 97897 Uni-ZAP XR 20 773 10 773 176 176 329
1 27 Feb. 26, 1997 209043 May 15, 1997 10 HSAXR76 97897 Uni-ZAP XR
202 741 55 741 190 190 511 1 27 Feb. 26, 1997 209043 May 15, 1997
11 HNGJJ68 97897 Uni-ZAP XR 21 991 1 991 62 62 330 1 30 31 63 Feb.
26, 1997 209043 May 15, 1997 11 HNGJJ68 97897 Uni-ZAP XR 203 1192
253 1137 409 512 1 19 Feb. 26, 1997 209043 May 15, 1997 12 HCFAW04
97897 pSport1 22 653 1 653 64 64 331 1 30 31 196 Feb. 26, 1997
209043 May 15, 1997 12 HCFAW04 97897 pSport1 204 589 1 513 109 109
513 1 29 Feb. 26, 1997 209043 May 15, 1997 13 HLMAV65 97897 Uni-ZAP
XR 23 1486 596 1418 102 102 332 1 54 55 251 Feb. 26, 1997 209043
May 15, 1997 13 HLMAV65 97897 Uni-ZAP XR 205 847 1 839 87 87 514 1
30 31 74 Feb. 26, 1997 209043 May 15, 1997 13 HLMAV65 97897 Uni-ZAP
XR 206 852 75 850 690 515 1 10 Feb. 26, 1997 209043 May 15, 1997 13
HTXEF04 209235 Uni-ZAP XR 207 1354 54 1354 100 100 516 1 33 34 206
Sep. 04, 1997 14 HPMFD84 97897 Uni-ZAP XR 24 2323 1017 2059 1242
1242 333 1 21 22 67 Feb. 26, 1997 209043 May 15, 1997 14 HPMFD84
97897 Uni-ZAP XR 208 1378 113 1226 303 303 517 1 25 26 36 Feb. 26,
1997 209043 May 15, 1997 15 HE6DB26 97897 Uni-ZAP XR 25 683 1 683
304 304 334 1 30 31 8 Feb. 26, 1997 209043 May 15, 1997 15 HE6DB26
97897 Uni-ZAP XR 209 1166 281 884 567 567 518 1 18 19 19 Feb. 26,
1997 209043 May 15, 1997 16 HHFFL33 97897 Uni-ZAP XR 26 2036 14
1959 214 214 335 1 20 21 36 Feb. 26, 1997 209043 May 15, 1997 17
HODBD33 97897 Uni-ZAP XR 27 717 1 717 70 70 336 1 30 31 62 Feb. 26,
1997 209043 May 15, 1997 17 HODBD33 97897 Uni-ZAP XR 210 697 2 697
33 33 519 1 31 32 32 Feb. 26, 1997 209043 May 15, 1997 18 HMDAE90
97897 Uni-ZAP XR 28 495 1 495 39 39 337 1 24 25 35 Feb. 26, 1997
209043 May 15, 1997 19 HOUAW01 97897 Uni-ZAP XR 29 556 1 556 116
116 338 1 19 20 23 Feb. 26, 1997 209043 May 15, 1997 20 HBJAE44
97897 Uni-ZAP XR 30 434 1 434 78 78 339 1 35 36 40 Feb. 26, 1997
209043 May 15, 1997 21 HCFME41 97897 pSport1 31 715 1 715 87 87 340
1 30 31 110 Feb. 26, 1997 209043 May 15, 1997 21 HCFME41 97897
pSport1 211 932 274 932 387 387 520 1 27 28 28 Feb. 26, 1997 209043
May 15, 1997 22 HOGCO71 97897 pCMVSport 2.0 32 486 1 486 137 137
341 1 21 22 106 Feb. 26, 1997 209043 May 15, 1997 23 HOSEX08 97897
Uni-ZAP XR 33 725 1 725 436 436 342 1 30 31 49 Feb. 26, 1997 209043
May 15, 1997 23 HOSEX08 97897 Uni-ZAP XR 212 661 1 647 81 81 521 1
25 26 26 Feb. 26, 1997 209043 May 15, 1997 24 HSKNJ72 97897
pBluescript .TM. 34 437 1 437 85 85 343 1 30 31 48 Feb. 26, 1997
209043 May 15, 1997 25 HEBEB69 97898 Uni-ZAP XR 35 943 1 943 196
196 344 1 30 31 40 Feb. 26, 1997 209044 May 15, 1997 25 HEBEB69
97898 Uni-ZAP XR 213 592 1 534 72 72 522 1 24 25 33 Feb. 26, 1997
209044 May 15, 1997 26 HE6EH18 97898 Uni-ZAP XR 36 604 1 604 375
375 345 1 20 21 76 Feb. 26, 1997 209044 May 15, 1997 26 HE6EH18
97898 Uni-ZAP XR 214 938 1 509 17 523 1 30 31 47 Feb. 26, 1997
209044 May 15, 1997 27 HSAUZ47 97898 Uni-ZAP XR 37 349 1 349 101
101 346 1 27 28 83 Feb. 26, 1997 209044 May 15, 1997 28 HSSDM73
97898 Uni-ZAP XR 38 672 1 672 22 22 347 1 38 39 42 Feb. 26, 1997
209044 May 15, 1997 29 HBMVK68 97898 Uni-ZAP XR 39 1908 135 1908
309 309 348 1 20 21 26 Feb. 26, 1997 209044 May 15, 1997 30 HMKDC66
97898 pSport1 40 458 93 458 147 147 349 1 24 25 26 Feb. 26, 1997
209044 May 15, 1997 31 HMKCU94 97898 pSport1 41 1153 500 1153 427
427 350 1 30 31 156 Feb. 26, 1997 209044 May 15, 1997 31 HMKCU94
97898 pSport1 215 1079 502 896 739 524 1 23 24 43 Feb. 26, 1997
209044 May 15, 1997 32 HRDEW41 97898 Uni-ZAP XR 42 1983 1092 1983
27 27 351 1 11 12 519 Feb. 26, 1997 209044 May 15, 1997 32 HRDEW41
97898 Uni-ZAP XR 216 3791 2757 3357 2030 525 1 3 Feb. 26, 1997
209044 May 15, 1997 33 HTOJN06 97898 Uni-ZAP XR 43 1406 1 695 19
352 1 19 20 39 Feb. 26, 1997 209044 May 15, 1997 34 HBGDA21 97898
Uni-ZAP XR 44 1391 851 1153 74 74 353 1 30 31 233 Feb. 26, 1997
209044 May 15, 1997 34 HBGDA21 97898 Uni-ZAP XR 217 1334 822 1036
638 526 1 18 19 174 Feb. 26, 1997 209044 May 15, 1997 35 HFGAK75
97898 Uni-ZAP XR 45 1569 768 1569 14 14 354 1 19 20 168 Feb. 26,
1997 209044 May 15, 1997 35 HFGAK75 97898 Uni-ZAP XR 218 1511 770
1404 844 844 527 1 32 33 43 Feb. 26, 1997 209044 May 15, 1997 36
HHPBD40 97898 Uni-ZAP XR 46 1924 1 1681 62 62 355 1 19 20 43 Feb.
26, 1997 209044 May 15, 1997 37 HOVCL83 97898 pSport1 47 475 252
396 141 141 356 1 37 38 78 Feb. 26, 1997 209044 May 15, 1997 38
HBCAY62 97898 Uni-ZAP XR 48 346 1 346 61 61 357 1 19 20 24 Feb. 26,
1997 209044 May 15, 1997 39 HBICM48 97898 Uni-ZAP XR 49 1366 882
1300 177 177 358 1 30 31 273
Feb. 26, 1997 209044 May 15, 1997 39 HBICM48 97898 Uni-ZAP XR 219
642 192 581 448 528 1 13 Feb. 26, 1997 209044 May 15, 1997 40
HLTCL35 97898 Uni-ZAP XR 50 1405 110 1404 61 61 359 1 30 31 46 Feb.
26, 1997 209044 May 15, 1997 40 HLTCL35 97898 Uni-ZAP XR 220 1241 1
1241 172 172 529 1 21 22 30 Feb. 26, 1997 209044 May 15, 1997 41
HLHCK50 97898 Uni-ZAP XR 51 2633 29 310 2064 2064 360 1 18 19 117
Feb. 26, 1997 209044 May 15, 1997 41 HLHCK50 97898 Uni-ZAP XR 221
504 207 485 222 222 530 1 3 Feb. 26, 1997 209044 May 15, 1997 42
HRSAN45 97899 ZAP Express 52 777 1 214 113 113 361 1 24 25 52 Feb.
26, 1997 209045 May 15, 1997 43 HSNBB14 97899 Uni-ZAP XR 53 602 1
419 41 41 362 1 59 60 131 Feb. 26, 1997 209045 May 15, 1997 43
HSNBB14 97899 Uni-ZAP XR 222 1080 186 686 399 399 531 1 26 27 47
Feb. 26, 1997 209045 May 15, 1997 44 HMABL38 97899 Uni-ZAP XR 54
1749 222 1749 166 166 363 1 30 31 203 Feb. 26, 1997 209045 May 15,
1997 44 HMABL38 97899 Uni-ZAP XR 223 1258 149 1190 254 254 532 1 18
19 26 Feb. 26, 1997 209045 May 15, 1997 45 HSKDK47 97899 Uni-ZAP XR
55 1896 596 1614 650 650 364 1 33 34 47 Feb. 26, 1997 209045 May
15, 1997 46 HOSFH03 97899 Uni-ZAP XR 56 1753 555 1753 414 414 365 1
18 19 72 Feb. 26, 1997 209045 May 15, 1997 46 HOSFH03 97899 Uni-ZAP
XR 224 1693 554 1693 526 533 1 25 26 58 Feb. 26, 1997 209045 May
15, 1997 47 HOGAV75 97899 pCMVSport 2.0 57 1220 690 1024 128 128
366 1 30 31 101 Feb. 26, 1997 209045 May 15, 1997 47 HOGAV75 97899
pCMVSport 2.0 225 1196 712 1163 1097 534 1 19 Feb. 26, 1997 209045
May 15, 1997 48 HFCAI74 97899 Uni-ZAP XR 58 1049 362 1049 335 335
367 1 33 34 48 Feb. 26, 1997 209045 May 15, 1997 49 HAGBI17 97899
Uni-ZAP XR 59 1776 854 1737 189 189 368 1 30 31 178 Feb. 26, 1997
209045 May 15, 1997 49 HAGBI17 97899 Uni-ZAP XR 226 1791 979 1791
1164 1164 535 1 18 19 40 Feb. 26, 1997 209045 May 15, 1997 50
HLFBC91 97899 pBluescript .TM. 60 443 1 443 164 164 369 1 21 22 25
Feb. 26, 1997 SK- 209045 May 15, 1997 51 HPRCA31 97899 Uni-ZAP XR
61 2888 1909 2888 90 90 370 1 30 31 223 Feb. 26, 1997 209045 May
15, 1997 51 HPRCA31 97899 Uni-ZAP XR 227 2517 1597 2517 1953 1953
536 1 18 19 57 Feb. 26, 1997 209045 May 15, 1997 52 HPRCE95 97899
Uni-ZAP XR 62 1851 1568 1736 139 139 371 1 30 31 348 Feb. 26, 1997
209045 May 15, 1997 52 HPRCE95 97899 Uni-ZAP XR 228 2424 299 2309
530 537 1 17 18 21 Feb. 26, 1997 209045 May 15, 1997 53 HHTLC66
97899 ZAP Express 63 3542 883 3492 964 964 372 1 25 26 467 Feb. 26,
1997 209045 May 15, 1997 54 HMADJ02 97899 Uni-ZAP XR 64 883 237 883
229 229 373 1 30 31 151 Feb. 26, 1997 209045 May 15, 1997 54
HMADJ02 97899 Uni-ZAP XR 229 1080 242 1033 436 436 538 1 24 25 39
Feb. 26, 1997 209045 May 15, 1997 55 HPRCU93 97899 Uni-ZAP XR 65
1541 1 1541 236 236 374 1 30 31 372 Feb. 26, 1997 209045 May 15,
1997 55 HPRCU93 97899 Uni-ZAP XR 230 1336 4 1336 946 946 539 1 25
26 128 Feb. 26, 1997 209045 May 15, 1997 56 HSAXS65 97899 Uni-ZAP
XR 66 732 41 698 163 163 375 1 18 19 82 Feb. 26, 1997 209045 May
15, 1997 56 HSAXS65 97899 Uni-ZAP XR 231 2043 1133 1756 1262 1262
540 1 20 21 82 Feb. 26, 1997 209045 May 15, 1997 57 HKTAG35 209011
Uni-ZAP XR 67 631 1 631 34 34 376 1 30 31 97 Apr. 28, 1997 57
HKTAG35 97899 Uni-ZAP XR 232 629 1 629 264 264 541 1 20 Feb. 26,
1997 209045 May 15, 1997 57 HMEFX42 97899 Lambda ZAP II 233 540 25
536 227 227 542 1 20 Feb. 26, 1997 209045 May 15, 1997 58 HHFHN61
97899 Uni-ZAP XR 68 1751 375 1751 95 95 377 1 19 20 227 Feb. 26,
1997 209045 May 15, 1997 59 HCWEF90 97899 ZAP Express 69 508 1 508
22 22 378 1 30 31 78 Feb. 26, 1997 209045 May 15, 1997 59 HCWEF90
97899 ZAP Express 234 448 9 448 1 543 1 22 23 75 Feb. 26, 1997
209045 May 15, 1997 60 HHGCM20 97899 Lambda ZAP II 70 245 1 245 93
93 379 1 28 29 50 Feb. 26, 1997 209045 May 15, 1997 61 HFRAU10
97900 Uni-ZAP XR 71 361 1 361 1 1 380 1 30 31 60 Feb. 26, 1997
209046 May 15, 1997 61 HFRAU10 97900 Uni-ZAP XR 235 407 1 407 210
210 544 1 17 18 60 Feb. 26, 1997 209046 May 15, 1997 62 HATDT67
97900 Uni-ZAP XR 72 713 8 713 169 169 381 1 30 31 39 Feb. 26, 1997
209046 May 15, 1997 62 HATDT67 97900 Uni-ZAP XR 236 830 190 580 329
329 545 1 28 29 39 Feb. 26, 1997 209046 May 15, 1997 63 HOUBG93
97900 Uni-ZAP XR 73 862 1 862 67 67 382 1 30 31 43 Feb. 26, 1997
209046 May 15, 1997 63 HOUBG93 97900 Uni-ZAP XR 237 932 138 905 287
287 546 1 2 Feb. 26, 1997 209046 May 15, 1997 64 HMWEX24 97900
Uni-Zap XR 74 4602 4162 4525 730 730 383 1 30 31 202 Feb. 26, 1997
209046 May 15, 1997 64 HMWEX24 97900 Uni-Zap XR 238 2786 2406 2739
2577 2577 547 1 22 23 36 Feb. 26, 1997 209046 May 15, 1997 65
HSGBA84 97900 Uni-ZAP XR 75 1255 1 1195 112 112 384 1 28 29 29 Feb.
26, 1997 209046 May 15, 1997 66 HTOCD52 97900 Uni-ZAP XR 76 475 1
475 13 13 385 1 30 31 135 Feb. 26, 1997 209046 May 15, 1997 66
HTOCD52 97900 Uni-ZAP XR 239 458 1 458 26 26 548 1 14 Feb. 26, 1997
209046 May 15, 1997 67 HTGCP16 97900 Uni-ZAP XR 77 465 25 299 74 74
386 1 33 34 41 Feb. 26, 1997 209046 May 15, 1997 68 HKIXR69 97900
pBluescript .TM. 78 1907 1627 1730 26 26 387 1 30 31 467 Feb. 26,
1997 209046 May 15, 1997 68 HKIXR69 97900 pBluescript .TM. 240 591
1 444 251 251 549 1 18 Feb. 26, 1997 209046 May 15, 1997 69 HETGJ09
97900 Uni-ZAP XR 79 1168 136 1168 267 267 388 1 20 21 29 Feb. 26,
1997 209046 May 15, 1997 70 HOBNC61 97900 Uni-ZAP XR 80 1285 132
1285 292 292 389 1 27 28 29 Feb. 26, 1997 209046 May 15, 1997 71
HFFAH94 97900 Lambda ZAP II 81 1290 768 1054 701 701 390 1 21 22
138 Feb. 26, 1997 209046 May 15, 1997 72 HBIAI95 97900 Uni-ZAP XR
82 684 1 684 119 119 391 1 30 31 74 Feb. 26, 1997 209046 May 15,
1997 73 HSQEL25 97900 Uni-ZAP XR 83 2024 1609 1953 200 200 392 1 30
31 520 Feb. 26, 1997 209046 May 15, 1997 73 HSQEL25 97900 Uni-ZAP
XR 241 2449 1511 2378 488 488 550 1 30 31 269 Feb. 26, 1997 209046
May 15, 1997 73 HSQEL25 97900 Uni-ZAP XR 242 1286 391 959 1204 551
1 9 10 10 Feb. 26, 1997 209046 May 15, 1997 74 HEBEG68 97900
Uni-ZAP XR 84 931 14 537 85 85 393 1 25 26 137 Feb. 26, 1997 209046
May 15, 1997 75 HBIAB39 97900 Uni-ZAP XR 85 825 59 802 66 66 394 1
30 31 185 Feb. 26, 1997 209046 May 15, 1997 75 HBIAB39 97900
Uni-ZAP XR 243 734 1 734 1 1 552 1 37 38 107 Feb. 26, 1997 209046
May 15, 1997 75 HBIAB39 97900 Uni-ZAP XR 244 809 80 794 294 553 1
15 16 106 Feb. 26, 1997 209046 May 15, 1997 76 HTXDU73 97900
Uni-ZAP XR 86 1238 36 918 17 17 395 1 1 Feb. 26, 1997
209046 May 15, 1997 77 HOEAS24 97900 Uni-ZAP XR 87 1460 9 1458 166
166 396 1 53 54 298 Feb. 26, 1997 209046 May 15, 1997 77 HOEAS24
97900 Uni-ZAP XR 245 2201 841 2080 507 507 554 1 43 44 135 Feb. 26,
1997 209046 May 15, 1997 77 HOEAS24 97900 Uni-ZAP XR 246 1661 311
1520 390 390 555 1 35 36 424 Feb. 26, 1997 209046 May 15, 1997 78
HTEIY30 97900 Uni-ZAP XR 88 1395 567 1395 639 639 397 1 36 37 49
Feb. 26, 1997 209046 May 15, 1997 79 HSKNE46 97900 pBluescript .TM.
89 1186 352 1186 540 540 398 1 49 50 60 Feb. 26, 1997 209046 May
15, 1997 79 HSKNE46 97900 pBluescript .TM. 247 1146 329 1146 564
564 556 1 21 22 39 Feb. 26, 1997 209046 May 15, 1997 80 HPMFL27
97900 Uni-ZAP XR 90 1821 1203 1614 1503 1503 399 1 30 31 79 Feb.
26, 1997 209046 May 15, 1997 81 HMWDN32 97900 Uni-Zap XR 91 862 253
862 359 359 400 1 32 33 36 Feb. 26, 1997 209046 May 15, 1997 82
HPRAX55 97900 Uni-ZAP XR 92 696 349 696 98 98 401 1 30 31 179 Feb.
26, 1997 209046 May 15, 1997 82 HPRAX55 97900 Uni-ZAP XR 248 1350
265 1230 348 348 557 1 32 33 58 Feb. 26, 1997 209046 May 15, 1997
83 HHFFW36 97900 Uni-ZAP XR 93 1886 1 1759 197 197 402 1 21 Feb.
26, 1997 209046 May 15, 1997 84 HE2PL77 97901 Uni-ZAP XR 94 1774
742 1772 785 785 403 1 21 22 60 Feb. 26, 1997 209047 May 15, 1997
85 HSDFV29 Uni-ZAP XR 95 1779 12 1662 219 219 404 1 36 37 185 85
HSDFV29 209076 Uni-ZAP XR 249 2503 1 1648 206 206 558 1 32 33 151
May 22, 1997 85 HCQAV53 97901 Lambda ZAP II 250 1529 72 911 191 191
559 1 20 21 33 Feb. 26, 1997 209047 May 15, 1997 86 HTPEG42 97901
Uni-ZAP XR 96 2801 418 2801 234 234 405 1 30 31 479 Feb. 26, 1997
209047 May 15, 1997 86 HTPEG42 97901 Uni-ZAP XR 251 1537 1 1537 125
125 560 1 21 22 367 Feb. 26, 1997 209047 May 15, 1997 87 HLHDR57
97901 Uni-ZAP XR 97 1631 916 1631 691 691 406 1 38 39 193 Feb. 26,
1997 209047 May 15, 1997 88 HAUAV32 97901 Uni-ZAP XR 98 504 26 504
197 197 407 1 23 24 77 Feb. 26, 1997 209047 May 15, 1997 88 HAUAV32
97901 Uni-ZAP XR 252 506 1 499 183 183 561 1 32 33 77 Feb. 26, 1997
209047 May 15, 1997 89 HNEBI60 97901 Uni-ZAP XR 99 1416 145 1416
456 456 408 1 18 19 73 Feb. 26, 1997 209047 May 15, 1997 89 HNEBI60
97901 Uni-ZAP XR 253 1348 84 1348 363 363 562 1 21 22 47 Feb. 26,
1997 209047 May 15, 1997 90 HSHCJ16 97901 Uni-ZAP XR 100 2847 1
2847 2 409 1 20 Feb. 26, 1997 209047 May 15, 1997 91 HTSEL31 97901
pBluescript .TM. 101 1394 608 1346 602 602 410 1 23 24 87 Feb. 26,
1997 209047 May 15, 1997 92 HAUBL57 97901 Uni-ZAP XR 102 794 1 794
518 518 411 1 30 31 92 Feb. 26, 1997 209047 May 15, 1997 92 HAUBL57
97901 Uni-ZAP XR 254 1766 42 1766 356 356 563 1 30 31 167 Feb. 26,
1997 209047 May 15, 1997 92 HAUBL57 97901 Uni-ZAP XR 255 2664 47
1708 147 564 1 18 19 124 Feb. 26, 1997 209047 May 15, 1997 93
HODAS59 97901 Uni-ZAP XR 103 1544 898 1531 975 975 412 1 21 Feb.
26, 1997 209047 May 15, 1997 94 HE6CT48 97901 Uni-ZAP XR 104 871
106 871 248 248 413 1 34 35 173 Feb. 26, 1997 209047 May 15, 1997
94 HE6CT48 97901 Uni-ZAP XR 256 865 97 865 258 258 565 1 19 20 177
Feb. 26, 1997 209047 May 15, 1997 95 HMDAA61 97901 Uni-ZAP XR 105
404 1 404 16 16 414 1 21 22 63 Feb. 26, 1997 209047 May 15, 1997 95
HMDAA61 97901 Uni-ZAP XR 257 2082 852 2074 829 829 566 1 22 23 72
Feb. 26, 1997 209047 May 15, 1997 96 HAQBK61 97901 Uni-ZAP XR 106
1542 506 1542 122 122 415 1 51 52 279 Feb. 26, 1997 209047 May 15,
1997 96 HAQBK61 97901 Uni-ZAP XR 258 1482 508 1482 633 567 1 15 16
45 Feb. 26, 1997 209047 May 15, 1997 96 HCUHB01 209215 ZAP Express
259 834 1 834 82 82 568 1 40 41 251 Aug. 21, 1997 97 HAQBF73 97901
Uni-ZAP XR 107 2327 1528 2327 465 465 416 1 30 31 283 Feb. 26, 1997
209047 May 15, 1997 97 HAQBF73 97901 Uni-ZAP XR 260 1508 885 1508
988 569 1 19 Feb. 26, 1997 209047 May 15, 1997 98 HAQBT94 97901
Uni-ZAP XR 108 1062 157 1062 172 172 417 1 28 29 187 Feb. 26, 1997
209047 May 15, 1997 99 HETHE07 97901 Uni-ZAP XR 109 2539 275 2501
903 903 418 1 30 31 236 Feb. 26, 1997 209047 May 15, 1997 99
HETHE07 97901 Uni-ZAP XR 261 2514 592 2431 176 176 570 1 30 31 216
Feb. 26, 1997 209047 May 15, 1997 99 HETHE07 97901 Uni-ZAP XR 262
2357 465 2288 1151 571 1 12 13 82 Feb. 26, 1997 209047 May 15, 1997
100 HLQAB52 97901 Lambda ZAP II 110 1751 969 1751 4 4 419 1 46 47
191 Feb. 26, 1997 209047 May 15, 1997 100 HLQAB52 97901 Lambda ZAP
II 263 689 218 655 314 314 572 1 18 19 95 Feb. 26, 1997 209047 May
15, 1997 100 HEONN58 209119 pSport1 264 2377 5 2377 25 25 573 1 28
29 53 Jun. 05, 1997 101 HCRAM28 97901 Uni-ZAP XR 111 1117 1 1117 1
420 1 19 20 21 Feb. 26, 1997 209047 May 15, 1997 101 HIBEK16 209627
Other 265 1193 69 1135 242 242 574 1 24 25 107 Feb. 12, 1998 102
HE2BG03 97901 Uni-ZAP XR 112 1313 128 1313 271 271 421 1 30 31 50
Feb. 26, 1997 209047 May 15, 1997 102 HE2BG03 97901 Uni-ZAP XR 266
1262 26 1262 35 35 575 1 35 36 50 Feb. 26, 1997 209047 May 15, 1997
103 HEBDJ82 97901 Uni-ZAP XR 113 1654 553 1654 709 709 422 1 32
Feb. 26, 1997 209047 May 15, 1997 104 HCUBC79 97901 ZAP Express 114
1171 540 1171 337 337 423 1 30 31 162 Feb. 26, 1997 209047 May 15,
1997 104 HCUBC79 97901 ZAP Express 267 1179 626 1161 335 335 576 1
30 31 252 Feb. 26, 1997 209047 May 15, 1997 104 HCUBC79 97901 ZAP
Express 268 1162 629 1131 942 942 577 1 18 Feb. 26, 1997 209047 May
15, 1997 105 HSVAF07 97901 Uni-ZAP XR 115 842 373 800 100 100 424 1
65 66 173 Feb. 26, 1997 209047 May 15, 1997 105 HSVAF07 97901
Uni-ZAP XR 269 735 290 735 25 25 578 1 65 66 80 Feb. 26, 1997
209047 May 15, 1997 105 HSVAF07 97901 Uni-ZAP XR 270 783 416 783
413 579 1 33 34 73 Feb. 26, 1997 209047 May 15, 1997 106 HT3AM65
97901 Uni-ZAP XR 116 1640 187 1470 581 581 425 1 30 31 49 Feb. 26,
1997 209047 May 15, 1997 106 HT3AM65 97901 Uni-ZAP XR 271 1638 301
1405 119 119 580 1 30 31 262 Feb. 26, 1997 209047 May 15, 1997 106
HT3AM65 97901 Uni-ZAP XR 272 1455 148 1188 438 438 581 1 24 25 70
Feb. 26, 1997 209047 May 15, 1997 107 HE6DK18 97901 Uni-ZAP XR 117
952 418 906 499 499 426 1 28 29 120 Feb. 26, 1997 209047 May 15,
1997 108 HEBEK93 97901 Uni-ZAP XR 118 1256 21 1079 301 301 427 1 30
31 158 Feb. 26, 1997 209047 May 15, 1997 108 HEBEK93 97901 Uni-ZAP
XR 273 1086 25 1050 227 227 582 1 23 24 34 Feb. 26, 1997 209047 May
15, 1997 109 HJPCM10 97901 Uni-ZAP XR 119 1143 171 1051 175 175 428
1 50 51 153 Feb. 26, 1997 209047 May 15, 1997 109 HJPCM10 97901
Uni-ZAP XR 274 1003 21 1003 115 115 583 1 34 35 103 Feb. 26, 1997
209047 May 15, 1997 109 HJPCM10 97901 Uni-ZAP XR 275 1234 174 1015
232 232 584 1 27 28 132 Feb. 26, 1997 209047 May 15, 1997 110
HSXBL78 97901 Uni-ZAP XR 120 1782 1 1720 138 138 429 1 32 33 204
Feb. 26, 1997
209047 May 15, 1997 111 HOEAW81 97901 Uni-ZAP XR 121 610 18 609 50
50 430 1 30 31 66 Feb. 26, 1997 209047 May 15, 1997 111 HOEAW81
97901 Uni-ZAP XR 276 574 1 566 337 337 585 1 27 28 32 Feb. 26, 1997
209047 May 15, 1997 112 HOEAP41 97901 Uni-ZAP XR 122 526 185 375
143 143 431 1 21 22 25 Feb. 26, 1997 209047 May 15, 1997 113
HEAAR60 97901 Uni-ZAP XR 123 2081 1179 1976 48 48 432 1 30 31 298
Feb. 26, 1997 209047 May 15, 1997 113 HEAAR60 97901 Uni-ZAP XR 277
1731 889 1626 886 886 586 1 18 19 28 Feb. 26, 1997 209047 May 15,
1997 114 HTXGS75 97902 Uni-ZAP XR 124 1717 764 1640 898 898 433 1
28 29 86 Feb. 26, 1997 209048 May 15, 1997 115 HOVBA03 97902
pSport1 125 804 1 804 145 145 434 1 15 16 197 Feb. 26, 1997 209048
May 15, 1997 115 HOVBA03 97902 pSport1 278 1320 77 637 280 280 587
1 22 23 40 Feb. 26, 1997 209048 May 15, 1997 116 HGBGK76 97902
Uni-ZAP XR 126 431 1 431 73 73 435 1 38 39 46 Feb. 26, 1997 209048
May 15, 1997 116 HGBGK76 97902 Uni-ZAP XR 279 515 1 515 43 43 588 1
20 21 30 Feb. 26, 1997 209048 May 15, 1997 117 HBMUW78 97902
Uni-ZAP XR 127 3752 3465 3752 748 748 436 1 30 31 369 Feb. 26, 1997
209048 May 15, 1997 117 HBMUW78 97902 Uni-ZAP XR 280 2995 2738 2995
2777 2777 589 1 18 19 29 Feb. 26, 1997 209048 May 15, 1997 118
HASAS24 97902 Uni-ZAP XR 128 1144 669 1144 896 896 437 1 30 Feb.
26, 1997 209048 May 15, 1997 119 HSIDN55 97902 Uni-ZAP XR 129 1830
1234 1830 1265 1265 438 1 24 Feb. 26, 1997 209048 May 15, 1997 120
HGBGZ64 97902 Uni-ZAP XR 130 1864 1505 1741 1578 1578 439 1 37 38
53 Feb. 26, 1997 209048 May 15, 1997 121 H6EBJ64 97902 Uni-ZAP XR
131 2041 1 1214 46 46 440 1 35 36 175 Feb. 26, 1997 209048 May 15,
1997 121 H6EBJ64 97902 Uni-ZAP XR 281 1990 8 1128 71 71 590 1 16 17
92 Feb. 26, 1997 209048 May 15, 1997 122 HOECP43 97902 Uni-ZAP XR
132 2012 853 1986 1127 1127 441 1 22 23 77 Feb. 26, 1997 209048 May
15, 1997 123 H2CBV31 97902 pBluescript .TM. 133 1669 670 1632 962
962 442 1 25 26 32 Feb. 26, 1997 SK- 209048 May 15, 1997 124
HPCAD23 97902 Uni-ZAP XR 134 1565 281 1565 274 274 443 1 25 26 30
Feb. 26, 1997 209048 May 15, 1997 125 HSPAG15 97902 pSport1 135
2007 1101 2007 1124 1124 444 1 39 40 69 Feb. 26, 1997 209048 May
15, 1997 126 HELGH31 97902 Uni-ZAP XR 136 1291 1 1180 911 911 445 1
58 59 63 Feb. 26, 1997 209048 May 15, 1997 127 HUSHH48 97902 Lambda
ZAP II 137 1906 1 1906 184 184 446 1 30 31 42 Feb. 26, 1997 209048
May 15, 1997 127 HUSHH48 97902 Lambda ZAP II 282 2436 572 2436 726
726 591 1 30 31 42 Feb. 26, 1997 209048 May 15, 1997 128 HLYAU95
97902 pSport1 138 1935 1044 1794 1183 1183 447 1 18 19 33 Feb. 26,
1997 209048 May 15, 1997 129 HHSCV65 97902 Uni-ZAP XR 139 1446 572
1347 585 585 448 1 25 26 53 Feb. 26, 1997 209048 May 15, 1997 130
HTTAD57 97902 Uni-ZAP XR 140 1109 639 1109 676 676 449 1 24 25 64
Feb. 26, 1997 209048 May 15, 1997 131 HEBGA37 97902 Uni-ZAP XR 141
497 9 497 95 95 450 1 34 Feb. 26, 1997 209048 May 15, 1997 132
HEBFU93 97902 Uni-ZAP XR 142 269 1 269 1 1 451 1 30 31 89 Feb. 26,
1997 209048 May 15, 1997 132 HEBFU93 97902 Uni-ZAP XR 283 782 408
781 571 592 1 31 32 70 Feb. 26, 1997 209048 May 15, 1997 133
HSGSC60 97902 Lambda ZAP II 143 1269 55 1262 55 55 452 1 25 26 350
Feb. 26, 1997 209048 May 15, 1997 134 HPMGD24 97902 Uni-ZAP XR 144
1944 97 1871 306 306 453 1 16 17 49 Feb. 26, 1997 209048 May 15,
1997 135 HPTVC60 97902 pBluescript .TM. 145 1021 526 1021 74 74 454
1 30 31 277 Feb. 26, 1997 209048 May 15, 1997 135 HPTVC60 97902
pBluescript .TM. 284 961 524 961 545 545 593 1 23 24 110 Feb. 26,
1997 209048 May 15, 1997 136 HSKNE18 97902 pBluescript .TM. 146
1285 5 1285 116 116 455 1 30 31 198 Feb. 26, 1997 209048 May 15,
1997 136 HSKNE18 97902 pBluescript .TM. 285 1228 9 1228 324 324 594
1 26 27 30 Feb. 26, 1997 209048 May 15, 1997 137 HMWIF35 97902
Uni-Zap XR 147 1386 169 1272 165 165 456 1 30 31 257 Feb. 26, 1997
209048 May 15, 1997 137 HMWIF35 97902 Uni-Zap XR 286 1327 169 1208
160 160 595 1 23 24 71 Feb. 26, 1997 209048 May 15, 1997 138
HMWGI25 97902 Uni-Zap XR 148 2098 721 2044 784 784 457 1 18 19 87
Feb. 26, 1997 209048 May 15, 1997 139 HSKGF03 97902 pBluescript
.TM. 149 1847 1689 1847 241 241 458 1 33 34 314 Feb. 26, 1997
209048 May 15, 1997 139 HSKGF03 97902 pBluescript .TM. 287 1847
1033 1847 243 243 596 1 30 31 123 Feb. 26, 1997 209048 May 15, 1997
139 HSKGF03 97902 pBluescript .TM. 288 799 1 799 243 597 1 12 13 47
Feb. 26, 1997 209048 May 15, 1997 140 HMSKE75 97902 Uni-ZAP XR 150
1569 113 1517 417 417 459 1 21 22 52 Feb. 26, 1997 209048 May 15,
1997 141 HCMSH30 97902 Uni-ZAP XR 151 1540 538 1540 48 48 460 1 30
31 382 Feb. 26, 1997 209048 May 15, 1997 141 HCMSH30 97902 Uni-ZAP
XR 289 2196 270 2196 294 294 598 1 32 33 39 Feb. 26, 1997 209048
May 15, 1997 142 HTWCB92 97902 pSport1 152 1719 690 1575 6 6 461 1
52 53 186 Feb. 26, 1997 209048 May 15, 1997 143 HBMDM46 97902
pBluescript .TM. 153 863 1 863 195 195 462 1 26 27 162 Feb. 26,
1997 209048 May 15, 1997 143 HBMDM46 97902 pBluescript .TM. 290
1185 277 1166 621 621 599 1 19 Feb. 26, 1997 209048 May 15, 1997
144 HFAMG13 97902 Uni-ZAP XR 154 1101 1 512 40 40 463 1 21 22 46
Feb. 26, 1997 209048 May 15, 1997 145 HFXHL79 97903 Lambda ZAP II
155 2031 669 2031 411 411 464 1 23 24 104 Feb. 26, 1997 209049 May
15, 1997 145 HFXHL79 97903 Lambda ZAP II 291 1634 615 1485 878 878
600 1 20 21 23 Feb. 26, 1997 209049 May 15, 1997 146 HSNAK17 97903
Uni-ZAP XR 156 1981 1458 1809 1592 1592 465 1 23 24 69 Feb. 26,
1997 209049 May 15, 1997 146 HSNAK17 97903 Uni-ZAP XR 292 1795 1458
1749 1562 1562 601 1 33 34 69 Feb. 26, 1997 209049 May 15, 1997 147
HCFBC03 97903 pSport1 157 915 45 912 22 22 466 1 22 23 154 Feb. 26,
1997 209049 May 15, 1997 147 HCFBC03 97903 pSport1 293 858 46 858
224 224 602 1 30 31 77 Feb. 26, 1997 209049 May 15, 1997 147
HSJAP03 209139 Uni-ZAP XR 294 915 1 915 22 22 603 1 22 23 154 Jul.
03, 1997 148 HSKGO26 97903 pBluescript .TM. 158 2117 51 1422 32 32
467 1 23 24 332 Feb. 26, 1997 209049 May 15, 1997 149 HCQAV96 97903
Lambda ZAP II 159 2395 14 887 722 722 468 1 22 23 48 Feb. 26, 1997
209049 May 15, 1997 150 HSHCC16 97903 Uni-ZAP XR 160 2120 1223 2108
317 317 469 1 51 52 548 Feb. 26, 1997 209049 May 15, 1997 151
HTLEF62 97903 Uni-ZAP XR 161 900 482 900 46 46 470 1 30 31 285 Feb.
26, 1997 209049 May 15, 1997 151 HTLEF62 97903 Uni-ZAP XR 295 1517
783 1517 1062 1062 604 1 24 Feb. 26, 1997 209049 May 15, 1997 152
HTLAD94 97903 Uni-ZAP XR 162 1003 1 1003 288 288 471 1 30 31 79
Feb. 26, 1997 209049 May 15, 1997 152 HTLAD94 97903 Uni-ZAP XR 296
3865 217 1195 281 281 605 1 16 17 38 Feb. 26, 1997 209049 May 15,
1997 153 HTSFQ12 97903 pBluescript .TM. 163 2196 1607 2180 1611
1611 472 1 30 31 47 Feb. 26, 1997 209049 May 15, 1997 154 HE6FL83
97903 Uni-ZAP XR 164 1945 271 1840 299 299 473 1 63 64 95 Feb. 26,
1997 209049 May 15, 1997 154 HE6FL83 97903 Uni-ZAP XR 297 1910 279
1818 355 355 606 1 39 40 69 Feb. 26, 1997 209049 May 15, 1997 155
HTXFJ55 97903 Uni-ZAP XR 165 2933 489 2871 258 258 474 1 30 31 398
Feb. 26, 1997 209049 May 15, 1997 155 HTXFJ55 97903 Uni-ZAP XR 298
3276 486 2838 525 607 1 45 46 308 Feb. 26, 1997 209049 May 15, 1997
156 HJPCJ76 97903 Uni-ZAP XR 166 2243 343 2221 1311 1311 475 1 20
21 45 Feb. 26, 1997 209049 May 15, 1997 157 HLTED27 97903 Uni-ZAP
XR 167 1816 1130 1816 284 284 476 1 31 32 272 Feb. 26, 1997 209049
May 15, 1997 157 HLTED27 97903 Uni-ZAP XR 299 1695 1098 1548 1306
1306 608 1 22 Feb. 26, 1997 209049 May 15, 1997 158 HMKBA64 97903
pSport1 168 945 1 787 208 208 477 1 18 19 192 Feb. 26, 1997 209049
May 15, 1997 159 HNFIP24 97903 pBluescript .TM. 169 902 46 816 19
19 478 1 26 27 234 Feb. 26, 1997 209049 May 15, 1997 160 HCELB21
97903 Uni-ZAP XR 170 1883 798 1869 1001 1001 479 1 45 46 104 Feb.
26, 1997 209049 May 15, 1997 160 HCELB21 97903 Uni-ZAP XR 300 1501
438 1501 510 510 609 1 24 Feb. 26, 1997 209049 May 15, 1997 161
HAWBA28 97903 pBluescript .TM. 171 2100 1642 2100 1722 1722 480 1
23 24 32 Feb. 26, 1997 SK- 209049 May 15, 1997 162 HSAAS44 97903
pBluescript .TM. 172 1930 187 1930 65 65 481 1 30 31 570 Feb. 26,
1997 SK- 209049 May 15, 1997 162 HSAAS44 97903 pBluescript .TM. 301
2683 183 2683 431 431 610 1 24 Feb. 26, 1997 SK- 209049 May 15,
1997 163 HAFAL73 97903 pBluescript .TM. 173 1509 962 1451 122 122
482 1 30 31 311 Feb. 26, 1997 SK- 209049 May 15, 1997 163 HAFAL73
97903 pBluescript .TM. 302 1454 961 1420 976 976 611 1 1 Feb. 26,
1997 SK- 209049 May 15, 1997 164 HSAWF26 97903 Uni-ZAP XR 174 3173
2197 2972 51 51 483 1 21 22 328 Feb. 26, 1997 209049 May 15, 1997
164 HSAWF26 97903 Uni-ZAP XR 303 828 52 828 305 305 612 1 8 Feb.
26, 1997 209049 May 15, 1997 165 HEAAL31 97903 Uni-ZAP XR 175 991
374 970 60 60 484 1 24 25 177 Feb. 26, 1997 209049 May 15, 1997 165
HEAAL31 97903 Uni-ZAP XR 304 2416 1387 2413 1473 1473 613 1 18 19
25 Feb. 26, 1997 209049 May 15, 1997 166 HFKFX55 97903 Uni-ZAP XR
176 1290 499 1290 130 130 485 1 19 20 238 Feb. 26, 1997 209049 May
15, 1997 167 H2LAO11 97903 pBluescript .TM. 177 2290 1 2290 173 173
486 1 22 23 62 Feb. 26, 1997 SK- 209049 May 15, 1997 168 HPFDZ95
97903 Uni-ZAP XR 178 549 1 549 11 11 487 1 21 22 27 Feb. 26, 1997
209049 May 15, 1997 168 HPFDZ95 97903 Uni-ZAP XR 305 545 1 545 17
17 614 1 21 22 27 Feb. 26, 1997 209049 May 15, 1997 169 HPTTU11
97904 Uni-ZAP XR 179 1509 294 1352 92 92 488 1 30 31 338 Feb. 26,
1997 209050 May 15, 1997 169 HPTTU11 97904 Uni-ZAP XR 306 1530 385
1530 562 562 615 1 23 24 61 Feb. 26, 1997 209050 May 15, 1997 170
HCFAE79 97904 pSport1 180 1316 985 1250 995 995 489 1 26 27 32 Feb.
26, 1997 209050 May 15, 1997 171 HTEDJ34 97904 Uni-ZAP XR 181 777 1
777 51 51 490 1 30 31 47 Feb. 26, 1997 209050 May 15, 1997 171
HTEDJ34 97904 Uni-ZAP XR 307 997 244 997 300 300 616 1 23 24 29
Feb. 26, 1997 209050 May 15, 1997 172 HODCW06 97904 Uni-ZAP XR 182
791 1 791 14 14 491 1 29 30 38 Feb. 26, 1997 209050 May 15, 1997
173 HFTAR26 97904 Uni-ZAP XR 183 1405 346 1405 575 575 492 1 20 21
61 Feb. 26, 1997 209050 May 15, 1997 174 H2MBF44 97904 pBluescript
.TM. 184 1596 75 1596 131 131 493 1 24 25 345 Feb. 26, 1997 SK-
209050 May 15, 1997 174 H2MBF44 97904 pBluescript .TM. 308 2345 75
2345 233 233 617 1 56 57 69 Feb. 26, 1997 SK- 209050 May 15, 1997
175 HE8BI92 97904 Uni-ZAP XR 185 2293 355 2288 67 67 494 1 30 31
236 Feb. 26, 1997 209050 May 15, 1997 175 HE8BI92 97904 Uni-ZAP XR
309 2369 2 1946 60 618 1 9 10 24 Feb. 26, 1997 209050 May 15, 1997
176 HFTBR48 97904 Uni-ZAP XR 186 1212 462 1180 257 257 495 1 30 31
199 Feb. 26, 1997 209050 May 15, 1997 176 HFTBR48 97904 Uni-ZAP XR
310 1181 424 1149 663 663 619 1 23 24 35 Feb. 26, 1997 209050 May
15, 1997 177 HE9CM64 97904 Uni-ZAP XR 187 1605 770 1554 166 166 496
1 30 31 350 Feb. 26, 1997 209050 May 15, 1997 177 HE9CM64 97904
Uni-ZAP XR 311 1537 719 1515 787 620 1 43 44 130 Feb. 26, 1997
209050 May 15, 1997 178 HATAV51 97904 Uni-ZAP XR 188 1516 960 1516
8 8 497 1 30 31 264 Feb. 26, 1997 209050 May 15, 1997 178 HATAV51
97904 Uni-ZAP XR 312 1493 1 1261 54 54 621 1 18 19 23 Feb. 26, 1997
209050 May 15, 1997 179 HAQAF27 97904 Uni-ZAP XR 189 681 287 681
401 498 1 25 Feb. 26, 1997 209050 May 15, 1997 180 HCEEK08 97904
Uni-ZAP XR 190 1014 703 1014 360 360 499 1 30 31 158 Feb. 26, 1997
209050 May 15, 1997 180 HCEEK08 97904 Uni-ZAP XR 313 577 1 577 175
622 1 6 Feb. 26, 1997 209050 May 15, 1997 181 HAFAU18 97904
pBluescript .TM. 191 2779 2207 2630 1153 1153 500 1 30 31 278 Feb.
26, 1997 SK- 209050 May 15, 1997 181 HAFAU18 97904 pBluescript .TM.
314 2860 163 2860 21 21 623 1 30 31 231 Feb. 26, 1997 SK- 209050
May 15, 1997 181 HAFAU18 97904 pBluescript .TM. 315 876 275 876 302
302 624 1 32 33 34 Feb. 26, 1997 SK- 209050 May 15, 1997 182
HETBY74 97904 Uni-ZAP XR 192 1923 30 1923 45 45 501 1 33 34 193
Feb. 26, 1997 209050 May 15, 1997 183 HTOAF35 97904 Uni-ZAP XR 193
2346 1160 2286 178 178 502 1 30 31 204 Feb. 26, 1997 209050 May 15,
1997 183 HTOAF35 97904 Uni-ZAP XR 316 2025 840 2025 971 971 625 1
18 19 21 Feb. 26, 1997 209050 May 15, 1997 184 HCRBX32 97904
Uni-ZAP XR 194 3054 2004 3054 434 434 503 1 11 12 146 Feb. 26, 1997
209050 May 15, 1997 184 HCRBX32 97904 Uni-ZAP XR 317 3026 1966 3026
2131 626 1 9 Feb. 26, 1997 209050 May 15, 1997 185 HEBGB80 97904
Uni-ZAP XR 195 907 152 907 297 297 504 1 30 31 63 Feb. 26, 1997
209050 May 15, 1997 185 HEBGB80 97904 Uni-ZAP XR 318 712 67 712 107
107 627 1 18 19 29 Feb. 26, 1997 209050 May 15, 1997 186 HFAMH74
97904 Uni-ZAP XR 196 1290 84 809 225 225 505 1 30 31 93 Feb. 26,
1997 209050 May 15, 1997 186 HFAMH74 97904 Uni-ZAP XR 319 1289 785
1289 927 927 628 1 28 29 30 Feb. 26, 1997 209050 May 15, 1997
[1017] Table 1 summarizes the information corresponding to each
"Gene No." described above. The nucleotide sequence identified as
"NT SEQ ID NO:X" was assembled from partially homologous
("overlapping") sequences obtained from the "cDNA clone ID"
identified in Table 1 and, in some cases, from additional related
DNA clones. The overlapping sequences were assembled into a single
contiguous sequence of high redundancy (usually three to five
overlapping sequences at each nucleotide position), resulting in a
final sequence identified as SEQ ID NO:X.
[1018] The cDNA Clone ID was deposited on the date and given the
corresponding deposit number listed in "ATCC.TM. Deposit No:Z and
Date." Some of the deposits contain multiple different clones
corresponding to the same gene. "Vector" refers to the type of
vector contained in the cDNA Clone ID.
[1019] "Total NT Seq." refers to the total number of nucleotides in
the contig identified by "Gene No." The deposited clone may contain
all or most of these sequences, reflected by the nucleotide
position indicated as "5' NT of Clone Seq." and the "3' NT of Clone
Seq." of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the
putative start codon (methionine) is identified as "5' NT of Start
Codon." Similarly, the nucleotide position of SEQ ID NO:X of the
predicted signal sequence is identified as "5' NT of First AA of
Signal Pep."
[1020] The translated amino acid sequence, beginning with the
methionine, is identified as "AA SEQ ID NO:Y," although other
reading frames can also be easily translated using known molecular
biology techniques. The polypeptides produced by these alternative
open reading frames are specifically contemplated by the present
invention.
[1021] The first and last amino acid position of SEQ ID NO:Y of the
predicted signal peptide is identified as "First AA of Sig Pep" and
"Last AA of Sig Pep." The predicted first amino acid position of
SEQ ID NO:Y of the secreted portion is identified as "Predicted
First AA of Secreted Portion." Finally, the amino acid position of
SEQ ID NO:Y of the last amino acid in the open reading frame is
identified as "Last AA of ORF."
[1022] SEQ ID NO:X (where X may be any of the polynucleotide
sequences disclosed in the sequence listing) and the translated SEQ
ID NO:Y (where Y may be any of the polypeptide sequences disclosed
in the sequence listing) are sufficiently accurate and otherwise
suitable for a variety of uses well known in the art and described
further below. For instance, SEQ ID NO:X is useful for designing
nucleic acid hybridization probes that will detect nucleic acid
sequences contained in SEQ ID NO:X or the cDNA contained in the
deposited clone. These probes will also hybridize to nucleic acid
molecules in biological samples, thereby enabling a variety of
forensic and diagnostic methods of the invention. Similarly,
polypeptides identified from SEQ ID NO:Y may be used, for example,
to generate antibodies which bind specifically to proteins
containing the polypeptides and the secreted proteins encoded by
the cDNA clones identified in Table 1.
[1023] Nevertheless, DNA sequences generated by sequencing
reactions can contain sequencing errors. The errors exist as
misidentified nucleotides, or as insertions or deletions of
nucleotides in the generated DNA sequence. The erroneously inserted
or deleted nucleotides cause frame shifts in the reading frames of
the predicted amino acid sequence. In these cases, the predicted
amino acid sequence diverges from the actual amino acid sequence,
even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base
insertion or deletion in an open reading frame of over 1000
bases).
[1024] Accordingly, for those applications requiring precision in
the nucleotide sequence or the amino acid sequence, the present
invention provides not only the generated nucleotide sequence
identified as SEQ ID NO:X and the predicted translated amino acid
sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA containing a human cDNA of the invention deposited with the
ATCC.TM., as set forth in Table 1. The nucleotide sequence of each
deposited clone can readily be determined by sequencing the
deposited clone in accordance with known methods. The predicted
amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a
particular clone can also be directly determined by peptide
sequencing or by expressing the protein in a suitable host cell
containing the deposited human cDNA, collecting the protein, and
determining its sequence.
[1025] The present invention also relates to the genes
corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone.
The corresponding gene can be isolated in accordance with known
methods using the sequence information disclosed herein. Such
methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from
appropriate sources of genomic material.
[1026] Also provided in the present invention are allelic variants,
orthologs, and/or species homologs. Procedures known in the art can
be used to obtain full-length genes, allelic variants, splice
variants, full-length coding portions, orthologs, and/or species
homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a
deposited clone, using information from the sequences disclosed
herein or the clones deposited with the ATCC.TM.. For example,
allelic variants and/or species homologs may be isolated and
identified by making suitable probes or primers from the sequences
provided herein and screening a suitable nucleic acid source for
allelic variants and/or the desired homologue.
[1027] The polypeptides of the invention can be prepared in any
suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[1028] The polypeptides may be in the form of the secreted protein,
including the mature form, or may be a part of a larger protein,
such as a fusion protein (see below). It is often advantageous to
include an additional amino acid sequence which contains secretory
or leader sequences, pro-sequences, sequences which aid in
purification, such as multiple histidine residues, or an additional
sequence for stability during recombinant production.
[1029] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of a polypeptide,
including the secreted polypeptide, can be substantially purified
using techniques described herein or otherwise known in the art,
such as, for example, by the one-step method described in Smith and
Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also
can be purified from natural, synthetic or recombinant sources
using techniques described herein or otherwise known in the art,
such as, for example, antibodies of the invention raised against
the secreted protein.
[1030] The present invention provides a polynucleotide comprising,
or alternatively consisting of, the nucleic acid sequence of SEQ ID
NO:X, and/or a cDNA contained in ATCC.TM. deposit Z. The present
invention also provides a polypeptide comprising, or alternatively,
consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide encoded by the cDNA contained in ATCC.TM. deposit Z.
Polynucleotides encoding a polypeptide comprising, or alternatively
consisting of the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide sequence encoded by the cDNA contained in ATCC.TM.
deposit Z are also encompassed by the invention.
Signal Sequences
[1031] The present invention also encompasses mature forms of the
polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or
the polypeptide sequence encoded by the cDNA in a deposited clone.
Polynucleotides encoding the mature forms (such as, for example,
the polynucleotide sequence in SEQ ID NO:X and/or the
polynucleotide sequence contained in the cDNA of a deposited clone)
are also encompassed by the invention. According to the signal
hypothesis, proteins secreted by mammalian cells have a signal or
secretary leader sequence which is cleaved from the mature protein
once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and
even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species of the protein. Further, it has long been known that
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide.
[1032] Methods for predicting whether a protein has a signal
sequence, as well as the cleavage point for that sequence, are
available. For instance, the method of McGeoch, Virus Res.
3:271-286 (1985), uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje, Nucleic Acids Res.
14:4683-4690 (1986) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2, where
+1 indicates the amino terminus of the secreted protein. The
accuracy of predicting the cleavage points of known mammalian
secretory proteins for each of these methods is in the range of
75-80%. (von Heinje, supra.) However, the two methods do not always
produce the same predicted cleavage point(s) for a given
protein.
[1033] In the present case, the deduced amino acid sequence of the
secreted polypeptide was analyzed by a computer program called
SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)),
which predicts the cellular location of a protein based on the
amino acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated. The analysis of the amino acid sequences of the
secreted proteins described herein by this program provided the
results shown in Table 1.
[1034] As one of ordinary skill would appreciate, however, cleavage
sites sometimes vary from organism to organism and cannot be
predicted with absolute certainty. Accordingly, the present
invention provides secreted polypeptides having a sequence shown in
SEQ ID NO:Y which have an N-terminus beginning within 5 residues
(i.e., + or -5 residues) of the predicted cleavage point.
Similarly, it is also recognized that in some cases, cleavage of
the signal sequence from a secreted protein is not entirely
uniform, resulting in more than one secreted species. These
polypeptides, and the polynucleotides encoding such polypeptides,
are contemplated by the present invention.
[1035] Moreover, the signal sequence identified by the above
analysis may not necessarily predict the naturally occurring signal
sequence. For example, the naturally occurring signal sequence may
be further upstream from the predicted signal sequence. However, it
is likely that the predicted signal sequence will be capable of
directing the secreted protein to the ER. Nonetheless, the present
invention provides the mature protein produced by expression of the
polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide
sequence contained in the cDNA of a deposited clone, in a mammalian
cell (e.g., COS cells, as described below). These polypeptides, and
the polynucleotides encoding such polypeptides, are contemplated by
the present invention.
Polynucleotide and Polypeptide Variants
[1036] The present invention is directed to variants of the
polynucleotide sequence disclosed in SEQ ID NO:X, the complementary
strand thereto, and/or the cDNA sequence contained in a deposited
clone.
[1037] The present invention also encompasses variants of the
polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a
deposited clone.
[1038] "Variant" refers to a polynucleotide or polypeptide
differing from the polynucleotide or polypeptide of the present
invention, but retaining essential properties thereof. Generally,
variants are overall closely similar, and, in many regions,
identical to the polynucleotide or polypeptide of the present
invention.
[1039] The present invention is also directed to nucleic acid
molecules which comprise, or alternatively consist of, a nucleotide
sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for example, the nucleotide coding sequence in SEQ ID
NO:X or the complementary strand thereto, the nucleotide coding
sequence contained in a deposited cDNA clone or the complementary
strand thereto, a nucleotide sequence encoding the polypeptide of
SEQ ID NO:Y, a nucleotide sequence encoding the polypeptide encoded
by the cDNA contained in a deposited clone, and/or polynucleotide
fragments of any of these nucleic acid molecules (e.g., those
fragments described herein). Polynucleotides which hybridize to
these nucleic acid molecules under stringent hybridization
conditions or lower stringency conditions are also encompassed by
the invention, as are polypeptides encoded by these
polynucleotides.
[1040] The present invention is also directed to polypeptides which
comprise, or alternatively consist of, an amino acid sequence which
is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to,
for example, the polypeptide sequence shown in SEQ ID NO:Y, the
polypeptide sequence encoded by the cDNA contained in a deposited
clone, and/or polypeptide fragments of any of these polypeptides
(e.g., those fragments described herein).
[1041] By a nucleic acid having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence of the
present invention, it is intended that the nucleotide sequence of
the nucleic acid is identical to the reference sequence except that
the nucleotide sequence may include up to five point mutations per
each 100 nucleotides of the reference nucleotide sequence encoding
the polypeptide. In other words, to obtain a nucleic acid having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence. The
query sequence may be an entire sequence shown in Table 1, the ORF
(open reading frame), or any fragment specified as described
herein.
[1042] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence of the presence
invention can be determined conventionally using known computer
programs. A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245 (1990)). In a sequence alignment the query and subject
sequences are both DNA sequences. An RNA sequence can be compared
by converting U's to T's. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
FASTDB alignment of DNA sequences to calculate percent identity
are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of
the subject nucleotide sequence, whichever is shorter.
[1043] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[1044] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[1045] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[1046] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, an amino acid sequences shown in Table 1 (SEQ ID NO:Y) or
to the amino acid sequence encoded by cDNA contained in a deposited
clone can be determined conventionally using known computer
programs. A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245 (1990)). In a sequence alignment the query and subject
sequences are either both nucleotide sequences or both amino acid
sequences. The result of said global sequence alignment is in
percent identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[1047] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[1048] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[1049] The variants may contain alterations in the coding regions,
non-coding regions, or both. Especially preferred are
polynucleotide variants containing alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Polynucleotide variants can be
produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human mRNA
to those preferred by a bacterial host such as E. coli).
[1050] Naturally occurring variants are called "allelic variants,"
and refer to one of several alternate forms of a gene occupying a
given locus on a chromosome of an organism. (Genes II, Lewin, B.,
ed., John Wiley & Sons, New York (1985).) These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[1051] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides of the present invention. For
instance, one or more amino acids can be deleted from the
N-terminus or C-terminus of the secreted protein without
substantial loss of biological function. The authors of Ron et al.,
J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins
having heparin binding activity even after deleting 3, 8, or 27
amino-terminal amino acid residues. Similarly, Interferon gamma
exhibited up to ten times higher activity after deleting 8-10 amino
acid residues from the carboxy terminus of this protein. (Dobeli et
al., J. Biotechnology 7:199-216 (1988).)
[1052] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem
268:22105-22111 (1993)) conducted extensive mutational analysis of
human cytokine IL-1a. They used random mutagenesis to generate over
3,500 individual IL-1a mutants that averaged 2.5 amino acid changes
per variant over the entire length of the molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]."
(See, Abstract.) In fact, only 23 unique amino acid sequences, out
of more than 3,500 nucleotide sequences examined, produced a
protein that significantly differed in activity from wild-type.
[1053] Furthermore, even if deleting one or more amino acids from
the N-terminus or C-terminus of a polypeptide results in
modification or loss of one or more biological functions, other
biological activities may still be retained. For example, the
ability of a deletion variant to induce and/or to bind antibodies
which recognize the secreted form will likely be retained when less
than the majority of the residues of the secreted form are removed
from the N-terminus or C-terminus. Whether a particular polypeptide
lacking N- or C-terminal residues of a protein retains such
immunogenic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[1054] Thus, the invention further includes polypeptide variants
which show substantial biological activity. Such variants include
deletions, insertions, inversions, repeats, and substitutions
selected according to general rules known in the art so as have
little effect on activity. For example, guidance concerning how to
make phenotypically silent amino acid substitutions is provided in
Bowie et al., Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main strategies for studying the
tolerance of an amino acid sequence to change.
[1055] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[1056] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The
resulting mutant molecules can then be tested for biological
activity.
[1057] As the authors state, these two strategies have revealed
that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Moreover, tolerated conservative amino acid
substitutions involve replacement of the aliphatic or hydrophobic
amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and
Glu; replacement of the amide residues Asn and Gln, replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized
amino acids Ala, Ser, Thr, Met, and Gly.
[1058] Besides conservative amino acid substitution, variants of
the present invention include (i) substitutions with one or more of
the non-conserved amino acid residues, where the substituted amino
acid residues may or may not be one encoded by the genetic code, or
(ii) substitution with one or more of amino acid residues having a
substituent group, or (iii) fusion of the mature polypeptide with
another compound, such as a compound to increase the stability
and/or solubility of the polypeptide (for example, polyethylene
glycol), or (iv) fusion of the polypeptide with additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or
leader or secretory sequence, or a sequence facilitating
purification or (v) fusion of the polypeptide with another
compound, such as albumin (including, but not limited to,
recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued
Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,
issued Jun. 16, 1998, herein incorporated by reference in their
entirety)). Such variant polypeptides are deemed to be within the
scope of those skilled in the art from the teachings herein.
[1059] For example, polypeptide variants containing amino acid
substitutions of charged amino acids with other charged or neutral
amino acids may produce proteins with improved characteristics,
such as less aggregation. Aggregation of pharmaceutical
formulations both reduces activity and increases clearance due to
the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).)
[1060] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of the present
invention having an amino acid sequence which contains at least one
amino acid substitution, but not more than 50 amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and still even more preferably, not more than 20
amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a peptide or polypeptide to
have an amino acid sequence which comprises the amino acid sequence
of the present invention, which contains at least one, but not more
than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In
specific embodiments, the number of additions, substitutions,
and/or deletions in the amino acid sequence of the present
invention or fragments thereof (e.g., the mature form and/or other
fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or
50-150, conservative amino acid substitutions are preferable.
[1061] Polynucleotide and Polypeptide Fragments
[1062] The present invention is also directed to polynucleotide
fragments of the polynucleotides of the invention.
[1063] In the present invention, a "polynucleotide fragment" refers
to a short polynucleotide having a nucleic acid sequence which: is
a portion of that contained in a deposited clone, or encoding the
polypeptide encoded by the cDNA in a deposited clone; is a portion
of that shown in SEQ ID NO:X or the complementary strand thereto,
or is a portion of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:Y. The nucleotide fragments of the
invention are preferably at least about 15 nt, and more preferably
at least about 20 nt, still more preferably at least about 30 nt,
and even more preferably, at least about 40 nt, at least about 50
nt, at least about 75 nt, or at least about 150 nt in length. A
fragment "at least 20 nt in length," for example, is intended to
include 20 or more contiguous bases from the cDNA sequence
contained in a deposited clone or the nucleotide sequence shown in
SEQ ID NO:X. In this context "about" includes the particularly
recited value, a value larger or smaller by several (5, 4, 3, 2, or
1) nucleotides, at either terminus or at both termini. These
nucleotide fragments have uses that include, but are not limited
to, as diagnostic probes and primers as discussed herein. Of
course, larger fragments (e.g., 50, 150, 500, 600, 2000
nucleotides) are preferred.
[1064] Moreover, representative examples of polynucleotide
fragments of the invention, include, for example, fragments
comprising, or alternatively consisting of, a sequence from about
nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,
301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700,
701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050,
1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,
1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,
1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,
1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary
strand thereto, or the cDNA contained in a deposited clone. In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini. Preferably, these fragments
encode a polypeptide which has biological activity. More
preferably, these polynucleotides can be used as probes or primers
as discussed herein. Polynucleotides which hybridize to these
nucleic acid molecules under stringent hybridization conditions or
lower stringency conditions are also encompassed by the invention,
as are polypeptides encoded by these polynucleotides.
[1065] In the present invention, a "polypeptide fragment" refers to
an amino acid sequence which is a portion of that contained in SEQ
ID NO:Y or encoded by the cDNA contained in a deposited clone.
Protein (polypeptide) fragments may be "free-standing," or
comprised within a larger polypeptide of which the fragment forms a
part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments comprising, or alternatively
consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding region. Moreover, polypeptide fragments can be about 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids
in length. In this context "about" includes the particularly
recited ranges or values, and ranges or values larger or smaller by
several (5, 4, 3, 2, or 1) amino acids, at either extreme or at
both extremes. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[1066] Preferred polypeptide fragments include the secreted protein
as well as the mature form. Further preferred polypeptide fragments
include the secreted protein or the mature form having a continuous
series of deleted residues from the amino or the carboxy terminus,
or both. For example, any number of amino acids, ranging from 1-60,
can be deleted from the amino terminus of either the secreted
polypeptide or the mature form. Similarly, any number of amino
acids, ranging from 1-30, can be deleted from the carboxy terminus
of the secreted protein or mature form. Furthermore, any
combination of the above amino and carboxy terminus deletions are
preferred. Similarly, polynucleotides encoding these polypeptide
fragments are also preferred.
[1067] Also preferred are polypeptide and polynucleotide fragments
characterized by structural or functional domains, such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Polypeptide fragments of SEQ ID NO:Y falling within conserved
domains are specifically contemplated by the present invention.
Moreover, polynucleotides encoding these domains are also
contemplated.
[1068] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
Polynucleotides encoding these polypeptide fragments are also
encompassed by the invention.
[1069] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a functional activity. By a
polypeptide demonstrating a "functional activity" is meant, a
polypeptide capable of displaying one or more known functional
activities associated with a full-length (complete) polypeptide of
invention protein. Such functional activities include, but are not
limited to, biological activity, antigenicity [ability to bind (or
compete with a polypeptide of the invention for binding) to an
antibody to the polypeptide of the invention], immunogenicity
(ability to generate antibody which binds to a polypeptide of the
invention), ability to form multimers with polypeptides of the
invention, and ability to bind to a receptor or ligand for a
polypeptide of the invention.
[1070] The functional activity of polypeptides of the invention,
and fragments, variants derivatives, and analogs thereof, can be
assayed by various methods.
[1071] For example, in one embodiment where one is assaying for the
ability to bind or compete with full-length polypeptide of the
invention for binding to an antibody of the polypeptide of the
invention, various immunoassays known in the art can be used,
including but not limited to, competitive and non-competitive assay
systems using techniques such as radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[1072] In another embodiment, where a ligand for a polypeptide of
the invention identified, or the ability of a polypeptide fragment,
variant or derivative of the invention to multimerize is being
evaluated, binding can be assayed, e.g., by means well-known in the
art, such as, for example, reducing and non-reducing gel
chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky, E., et al., 1995, Microbiol.
Rev. 59:94-123. In another embodiment, physiological correlates of
binding of a polypeptide of the invention to its substrates (signal
transduction) can be assayed.
[1073] In addition, assays described herein (see Examples) and
otherwise known in the art may routinely be applied to measure the
ability of polypeptides of the invention and fragments, variants
derivatives and analogs thereof to elicit related biological
activity related to that of the polypeptide of the invention
(either in vitro or in vivo). Other methods will be known to the
skilled artisan and are within the scope of the invention.
Epitopes and Antibodies
[1074] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:Y, or an epitope of the
polypeptide sequence encoded by a polynucleotide sequence contained
in ATCC.TM. deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:X or
contained in ATCC.TM. deposit No. Z under stringent hybridization
conditions or lower stringency hybridization conditions as defined
supra. The present invention further encompasses polynucleotide
sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID
NO:X), polynucleotide sequences of the complementary strand of a
polynucleotide sequence encoding an epitope of the invention, and
polynucleotide sequences which hybridize to the complementary
strand under stringent hybridization conditions or lower stringency
hybridization conditions defined supra.
[1075] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[1076] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[1077] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length. Additional
non-exclusive preferred antigenic epitopes include the antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[1078] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[1079] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[1080] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention (e.g., those
comprising an immunogenic or antigenic epitope) can be fused to
heterologous polypeptide sequences. For example, polypeptides of
the present invention (including fragments or variants thereof),
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof, resulting in chimeric polypeptides.
By way of another non-limiting example, polypeptides and/or
antibodies of the present invention (including fragments or
variants thereof) may be fused with albumin (including but not
limited to recombinant human serum albumin or fragments or variants
thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999,
EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16,
1998, herein incorporated by reference in their entirety)). In a
preferred embodiment, polypeptides and/or antibodies of the present
invention (including fragments or variants thereof) are fused with
the mature form of human serum albumin (i.e., amino acids 1-585 of
human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322
094) which is herein incorporated by reference in its entirety. In
another preferred embodiment, polypeptides and/or antibodies of the
present invention (including fragments or variants thereof) are
fused with polypeptide fragments comprising, or alternatively
consisting of, amino acid residues 1-z of human serum albumin,
where z is an integer from 369 to 419, as described in U.S. Pat.
No. 5,766,883 herein incorporated by reference in its entirety.
Polypeptides and/or antibodies of the present invention (including
fragments or variants thereof) may be fused to either the N- or
C-terminal end of the heterologous protein (e.g., immunoglobulin Fc
polypeptide or human serum albumin polypeptide). Polynucleotides
encoding fusion proteins of the invention are also encompassed by
the invention.
[1081] Such fusion proteins may facilitate purification and may
increase half-life in vivo. This has been shown for chimeric
proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. See, e.g., EP
394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced
delivery of an antigen across the epithelial barrier to the immune
system has been demonstrated for antigens (e.g., insulin)
conjugated to an FcRn binding partner such as IgG or Fc fragments
(see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG
Fusion proteins that have a disulfide-linked dimeric structure due
to the IgG portion desulfide bonds have also been found to be more
efficient in binding and neutralizing other molecules than
monomeric polypeptides or fragments thereof alone. See, e.g.,
Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic
acids encoding the above epitopes can also be recombined with a
gene of interest as an epitope tag (e.g., the hemagglutinin ("HA")
tag or flag tag) to aid in detection and purification of the
expressed polypeptide. For example, a system described by Janknecht
et al. allows for the ready purification of non-denatured fusion
proteins expressed in human cell lines (Janknecht et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene
of interest is subcloned into a vaccinia recombination plasmid such
that the open reading frame of the gene is translationally fused to
an amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix binding domain for the fusion protein. Extracts
from cells infected with the recombinant vaccinia virus are loaded
onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged
proteins can be selectively eluted with imidazole-containing
buffers.
[1082] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO:X and the polypeptides encoded by these polynucleotides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to generate variation in the polynucleotide sequence.
In another embodiment, polynucleotides of the invention, or the
encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. In another embodiment, one or
more components, motifs, sections, parts, domains, fragments, etc.,
of a polynucleotide encoding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
Antibodies
[1083] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y,
and/or an epitope, of the present invention (as determined by
immunoassays well known in the art for assaying specific
antibody-antigen binding). Antibodies of the invention include, but
are not limited to, polyclonal, monoclonal, multispecific, human,
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab') fragments, fragments produced by a Fab expression
library, anti-idiotypic (anti-Id) antibodies (including, e.g.,
anti-Id antibodies to antibodies of the invention), and
epitope-binding fragments of any of the above. The term "antibody,"
as used herein, refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds an antigen. The immunoglobulin molecules
of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA
and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule. In preferred embodiments, the
immunoglobulin molecules of the invention are IgG1. In other
preferred embodiments, the immunoglobulin molecules of the
invention are IgG4.
[1084] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[1085] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[1086] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[1087] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologs of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6M, 5.times.10.sup.-7 M, 10.sup.7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10 M,
10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, .sup.10-12M, 5.times.10.sup.-13 M, 10.sup.-13
M, 5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[1088] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[1089] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferrably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[1090] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[1091] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[1092] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[1093] The antibodies of the invention include derivatives that are
modified, i.e, by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[1094] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[1095] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[1096] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples (e.g., Example 16). In
a non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC.TM.. Hybridomas are selected
and cloned by limited dilution. The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide of the invention.
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[1097] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[1098] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[1099] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[1100] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[1101] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[1102] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[1103] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
ABGENIX.TM., Inc. (Freemont, Calif.) and Genpharm (San Jose,
Calif.) can be engaged to provide human antibodies directed against
a selected antigen using technology similar to that described
above.
[1104] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[1105] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[1106] Polynucleotides Encoding Antibodies
[1107] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:Y.
[1108] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[1109] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[1110] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[1111] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[1112] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[1113] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[1114] Methods of Producing Antibodies
[1115] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[1116] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[1117] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[1118] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[1119] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[1120] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[1121] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[1122] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[1123] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[1124] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[1125] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[1126] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[1127] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[1128] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452 (1991), which are incorporated by
reference in their entireties.
[1129] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341 (1992) (said references incorporated by
reference in their entireties).
[1130] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may
be fused or conjugated to the above antibody portions to increase
the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides corresponding to SEQ ID NO:Y may be fused or
conjugated to the above antibody portions to facilitate
purification. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[1131] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[1132] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or 99Tc.
[1133] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[1134] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[1135] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[1136] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[1137] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[1138] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[1139] Immunophenotyping
[1140] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[1141] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
[1142] Assays for Antibody Binding
[1143] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[1144] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[1145] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[1146] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[1147] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
[1148] Therapeutic Uses
[1149] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein. The treatment and/or prevention of
diseases, disorders, or conditions associated with aberrant
expression and/or activity of a polypeptide of the invention
includes, but is not limited to, alleviating symptoms associated
with those diseases, disorders or conditions. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[1150] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[1151] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[1152] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[1153] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, and
10.sup.-15 M.
[1154] Gene Therapy
[1155] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[1156] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[1157] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[1158] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both the heavy and light chains, or fragments thereof, of
the antibody.
[1159] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[1160] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; BIOLISTIC.TM.,
DUPONT.TM.), or coating with lipids or cell-surface receptors or
transfecting agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[1161] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[1162] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[1163] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[1164] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[1165] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[1166] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[1167] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[1168] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[1169] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[1170] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[1171] Demonstration of Therapeutic or Prophylactic Activity
[1172] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[1173] Therapeutic/Prophylactic Administration and Composition
[1174] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[1175] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[1176] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[1177] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[1178] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[1179] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[1180] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[1181] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; BIOLISTIC.TM., DUPONT.TM.), or
coating with lipids or cell-surface receptors or transfecting
agents, or by administering it in linkage to a homeobox-like
peptide which is known to enter the nucleus (see e.g., Joliot et
al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc.
Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, by homologous
recombination.
[1182] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[1183] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[1184] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[1185] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[1186] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[1187] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[1188] Diagnosis and Imaging
[1189] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases,
disorders, and/or conditions associated with the aberrant
expression and/or activity of a polypeptide of the invention. The
invention provides for the detection of aberrant expression of a
polypeptide of interest, comprising (a) assaying the expression of
the polypeptide of interest in cells or body fluid of an individual
using one or more antibodies specific to the polypeptide interest
and (b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[1190] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[1191] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[1192] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[1193] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99 mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[1194] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[1195] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[1196] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[1197] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
[1198] Kits
[1199] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[1200] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[1201] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[1202] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[1203] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (SIGMA.TM., St.
Louis, Mo.).
[1204] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[1205] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[1206] Fusion Proteins
[1207] Any polypeptide of the present invention can be used to
generate fusion proteins. For example, the polypeptide of the
present invention, when fused to a second protein, can be used as
an antigenic tag. Antibodies raised against the polypeptide of the
present invention can be used to indirectly detect the second
protein by binding to the polypeptide. Moreover, because secreted
proteins target cellular locations based on trafficking signals,
the polypeptides of the present invention can be used as targeting
molecules once fused to other proteins.
[1208] Examples of domains that can be fused to polypeptides of the
present invention include not only heterologous signal sequences,
but also other heterologous functional regions. The fusion does not
necessarily need to be direct, but may occur through linker
sequences.
[1209] Moreover, fusion proteins may also be engineered to improve
characteristics of the polypeptide of the present invention. For
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence during purification from the host
cell or subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to facilitate
handling of polypeptides are familiar and routine techniques in the
art.
[1210] Moreover, polypeptides of the present invention, including
fragments, and specifically epitopes, can be combined with parts of
the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or
portions thereof (CH1, CH2, CH3, and any combination thereof,
including both entire domains and portions thereof), resulting in
chimeric polypeptides. These fusion proteins facilitate
purification and show an increased half-life in vivo. One reported
example describes chimeric proteins consisting of the first two
domains of the human CD4-polypeptide and various domains of the
constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86
(1988).) Fusion proteins having disulfide-linked dimeric structures
(due to the IgG) can also be more efficient in binding and
neutralizing other molecules, than the monomeric secreted protein
or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 (1995).) Polynucleotides comprising or alternatively
consisting of nucleic acids which encode these fusion proteins are
also encompassed by the invention.
[1211] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. (See, D. Bennett et al.,
J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J.
Biol. Chem. 270:9459-9471 (1995).)
[1212] Moreover, the polypeptides of the present invention can be
fused to marker sequences, such as a peptide which facilitates
purification of the fused polypeptide. In preferred embodiments,
the marker amino acid sequence is a hexa-histidine peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. Another
peptide tag useful for purification, the "HA" tag, corresponds to
an epitope derived from the influenza hemagglutinin protein.
(Wilson et al., Cell 37:767 (1984).)
[1213] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
[1214] Vectors, Host Cells, and Protein Production
[1215] The present invention also relates to vectors containing the
polynucleotide of the present invention, host cells, and the
production of polypeptides by recombinant techniques. The vector
may be, for example, a phage, plasmid, viral, or retroviral vector.
Retroviral vectors may be replication competent or replication
defective. In the latter case, viral propagation generally will
occur only in complementing host cells.
[1216] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[1217] The polynucleotide insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, tip, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination, and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[1218] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae
or Pichia pastoris (ATCC.TM. Accession No. 201178)); insect cells
such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such
as CHO, COS, 293, and Bowes melanoma cells; and plant cells.
Appropriate culture mediums and conditions for the above-described
host cells are known in the art.
[1219] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript.TM.
vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from STRATAGENE.TM. Cloning Systems, Inc.; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 available from PHARMACIA.TM.
Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENE.TM.; and
pSVK3, pBPV, pMSG and pSVL available from PHARMACIA.TM.. Preferred
expression vectors for use in yeast systems include, but are not
limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and
PAO815 (all available from Invitrogen, Carlbad, Calif.). Other
suitable vectors will be readily apparent to the skilled
artisan.
[1220] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986). It is
specifically contemplated that the polypeptides of the present
invention may in fact be expressed by a host cell lacking a
recombinant vector.
[1221] A polypeptide of this invention can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification.
[1222] Polypeptides of the present invention, and preferably the
secreted form, can also be recovered from: products purified from
natural sources, including bodily fluids, tissues and cells,
whether directly isolated or cultured; products of chemical
synthetic procedures; and products produced by recombinant
techniques from a prokaryotic or eukaryotic host, including, for
example, bacterial, yeast, higher plant, insect, and mammalian
cells. Depending upon the host employed in a recombinant production
procedure, the polypeptides of the present invention may be
glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
Thus, it is well known in the art that the N-terminal methionine
encoded by the translation initiation codon generally is removed
with high efficiency from any protein after translation in all
eukaryotic cells. While the N-terminal methionine on most proteins
also is efficiently removed in most prokaryotes, for some proteins,
this prokaryotic removal process is inefficient, depending on the
nature of the amino acid to which the N-terminal methionine is
covalently linked.
[1223] In one embodiment, the yeast Pichia pastoris is used to
express the polypeptide of the present invention in a eukaryotic
system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol as its sole carbon source. A main step in the
methanol metabolization pathway is the oxidation of methanol to
formaldehyde using O.sub.2. This reaction is catalyzed by the
enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source, Pichia pastoris must generate high levels of alcohol
oxidase due, in part, to the relatively low affinity of alcohol
oxidase for O.sub.2. Consequently, in a growth medium depending on
methanol as a main carbon source, the promoter region of one of the
two alcohol oxidase genes (AOX1) is highly active. In the presence
of methanol, alcohol oxidase produced from the AOX1 gene comprises
up to approximately 30% of the total soluble protein in Pichia
pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21
(1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F.,
et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous
coding sequence, such as, for example, a polynucleotide of the
present invention, under the transcriptional regulation of all or
part of the AOX1 regulatory sequence is expressed at exceptionally
high levels in Pichia yeast grown in the presence of methanol.
[1224] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a polypeptide of the invention, as set forth herein,
in a Pichia yeast system essentially as described in "Pichia
Protocols: Methods in Molecular Biology," D. R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression
vector allows expression and secretion of a protein of the
invention by virtue of the strong AOX1 promoter linked to the
Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[1225] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[1226] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a
polynucleotide of the present invention, may be achieved by cloning
the heterologous polynucleotide of the invention into an expression
vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
[1227] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide sequences) that is operably associated with the
polynucleotides of the invention, and which activates, alters,
and/or amplifies endogenous polynucleotides. For example,
techniques known in the art may be used to operably associate
heterologous control regions (e.g., promoter and/or enhancer) and
endogenous polynucleotide sequences via homologous recombination,
resulting in the formation of a new transcription unit (see, e.g.,
U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.
5,733,761, issued Mar. 31, 1998; International Publication No. WO
96/29411, published Sep. 26, 1996; International Publication No. WO
94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), the disclosures of each of which are
incorporated by reference in their entireties).
[1228] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller et al., Nature, 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
a polypeptide sequence of the invention can be synthesized by use
of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[1229] The invention encompasses polypeptides which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[1230] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[1231] Also provided by the invention are chemically modified
derivatives of the polypeptides of the invention which may provide
additional advantages such as increased solubility, stability and
circulating time of the polypeptide, or decreased immunogenicity
(see U.S. Pat. No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[1232] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,
90,000, 95,000, or 100,000 kDa.
[1233] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
[1234] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[1235] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to a proteins via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[1236] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[1237] As indicated above, pegylation of the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol may be attached to the protein either directly
or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et
al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,
the disclosures of each of which are incorporated herein by
reference.
[1238] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1SO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[1239] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of
which is incorporated herein by reference. Pegylated protein
products produced using the reaction chemistries set out herein are
included within the scope of the invention.
[1240] The number of polyethylene glycol moieties attached to each
protein of the invention (i.e., the degree of substitution) may
also vary. For example, the pegylated proteins of the invention may
be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the
average degree of substitution within ranges such as 1-3, 2-4, 3-5,
4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule. Methods for determining the degree of
substitution are discussed, for example, in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[1241] The polypeptides of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to monomers and
multimers of the polypeptides of the invention, their preparation,
and compositions (preferably, Therapeutics) containing them. In
specific embodiments, the polypeptides of the invention are
monomers, dimers, trimers or tetramers. In additional embodiments,
the multimers of the invention are at least dimers, at least
trimers, or at least tetramers.
[1242] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only polypeptides corresponding to the amino acid
sequence of SEQ ID NO:Y or encoded by the cDNA contained in a
deposited clone (including fragments, variants, splice variants,
and fusion proteins, corresponding to these polypeptides as
described herein). These homomers may contain polypeptides having
identical or different amino acid sequences. In a specific
embodiment, a homomer of the invention is a multimer containing
only polypeptides having an identical amino acid sequence. In
another specific embodiment, a homomer of the invention is a
multimer containing polypeptides having different amino acid
sequences. In specific embodiments, the multimer of the invention
is a homodimer (e.g., containing polypeptides having identical or
different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid
sequences). In additional embodiments, the homomeric multimer of
the invention is at least a homodimer, at least a homotrimer, or at
least a homotetramer.
[1243] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the polypeptides
of the invention. In a specific embodiment, the multimer of the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In
additional embodiments, the heteromeric multimer of the invention
is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
[1244] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the polypeptides of the
invention. Such covalent associations may involve one or more amino
acid residues contained in the polypeptide sequence (e.g., that
recited in the sequence listing, or contained in the polypeptide
encoded by a deposited clone). In one instance, the covalent
associations are cross-linking between cysteine residues located
within the polypeptide sequences which interact in the native
(i.e., naturally occurring) polypeptide. In another instance, the
covalent associations are the consequence of chemical or
recombinant manipulation. Alternatively, such covalent associations
may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the
invention.
[1245] In one example, covalent associations are between the
heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent
associations of fusion proteins of the invention are between
heterologous polypeptide sequence from another protein that is
capable of forming covalently associated multimers, such as for
example, oseteoprotegerin (see, e.g., International Publication NO:
WO 98/49305, the contents of which are herein incorporated by
reference in its entirety). In another embodiment, two or more
polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising
multiple polypeptides of the invention separated by peptide linkers
may be produced using conventional recombinant DNA technology.
[1246] Another method for preparing multimer polypeptides of the
invention involves use of polypeptides of the invention fused to a
leucine zipper or isoleucine zipper polypeptide sequence. Leucine
zipper and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been
found in a variety of different proteins. Among the known leucine
zippers are naturally occurring peptides and derivatives thereof
that dimerize or trimerize. Examples of leucine zipper domains
suitable for producing soluble multimeric proteins of the invention
are those described in PCT application WO 94/10308, hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the invention fused to a polypeptide sequence that
dimerizes or trimerizes in solution are expressed in suitable host
cells, and the resulting soluble multimeric fusion protein is
recovered from the culture supernatant using techniques known in
the art.
[1247] Trimeric polypeptides of the invention may offer the
advantage of enhanced biological activity. Preferred leucine zipper
moieties and isoleucine moieties are those that preferentially form
trimers. One example is a leucine zipper derived from lung
surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric polypeptides of the invention.
[1248] In another example, proteins of the invention are associated
by interactions between Flag.RTM. polypeptide sequence contained in
fusion proteins of the invention containing Flag.RTM. polypeptide
sequence. In a further embodiment, associations proteins of the
invention are associated by interactions between heterologous
polypeptide sequence contained in Flag.RTM. fusion proteins of the
invention and anti-Flag.RTM. antibody.
[1249] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[1250] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hyrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety).
[1251] Uses of the Polynucleotides
[1252] Each of the polynucleotides identified herein can be used in
numerous ways as reagents. The following description should be
considered exemplary and utilizes known techniques.
[1253] The polynucleotides of the present invention are useful for
chromosome identification. There exists an ongoing need to identify
new chromosome markers, since few chromosome marking reagents,
based on actual sequence data (repeat polymorphisms), are presently
available. Each polynucleotide of the present invention can be used
as a chromosome marker.
[1254] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the sequences shown in SEQ
ID NO:X. Primers can be selected using computer analysis so that
primers do not span more than one predicted exon in the genomic
DNA. These primers are then used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the SEQ ID NO:X will
yield an amplified fragment.
[1255] Similarly, somatic hybrids provide a rapid method of PCR
mapping the polynucleotides to particular chromosomes. Three or
more clones can be assigned per day using a single thermal cycler.
Moreover, sublocalization of the polynucleotides can be achieved
with panels of specific chromosome fragments. Other gene mapping
strategies that can be used include in situ hybridization,
prescreening with labeled flow-sorted chromosomes, preselection by
hybridization to construct chromosome specific-cDNA libraries and
computer mapping techniques (See, e.g., Shuler, Trends Biotechnol
16:456-459 (1998) which is hereby incorporated by reference in its
entirety).
[1256] Precise chromosomal location of the polynucleotides can also
be achieved using fluorescence in situ hybridization (FISH) of a
metaphase chromosomal spread. This technique uses polynucleotides
as short as 500 or 600 bases; however, polynucleotides 2,000-4,000
bp are preferred. For a review of this technique, see Verma et al.,
"Human Chromosomes: a Manual of Basic Techniques," Pergamon Press,
New York (1988).
[1257] For chromosome mapping, the polynucleotides can be used
individually (to mark a single chromosome or a single site on that
chromosome) or in panels (for marking multiple sites and/or
multiple chromosomes).
[1258] The polynucleotides of the present invention would likewise
be useful for radiation hybrid mapping, HAPPY mapping, and long
range restriction mapping. For a review of these techniques and
others known in the art, see, e.g., Dear, "Genome Mapping: A
Practical Approach," IRL Press at Oxford University Press, London
(1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol.
Psychiatry. 3:483-492 (1998); Herrick et al., Chromosome Res.
7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280
(2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is
hereby incorporated by reference in its entirety.
[1259] Once a polynucleotide has been mapped to a precise
chromosomal location, the physical position of the polynucleotide
can be used in linkage analysis. Linkage analysis establishes
coinheritance between a chromosomal location and presentation of a
particular disease. (Disease mapping data are found, for example,
in V. McKusick, Mendelian Inheritance in Man (available on line
through Johns Hopkins University Welch Medical Library).) Assuming
1 megabase mapping resolution and one gene per 20 kb, a cDNA
precisely localized to a chromosomal region associated with the
disease could be one of 50-500 potential causative genes.
[1260] Thus, once coinheritance is established, differences in the
polynucleotide and the corresponding gene between affected and
unaffected individuals can be examined. First, visible structural
alterations in the chromosomes, such as deletions or
translocations, are examined in chromosome spreads or by PCR. If no
structural alterations exist, the presence of point mutations are
ascertained. Mutations observed in some or all affected
individuals, but not in normal individuals, indicates that the
mutation may cause the disease. However, complete sequencing of the
polypeptide and the corresponding gene from several normal
individuals is required to distinguish the mutation from a
polymorphism. If a new polymorphism is identified, this polymorphic
polypeptide can be used for further linkage analysis.
[1261] Furthermore, increased or decreased expression of the gene
in affected individuals as compared to unaffected individuals can
be assessed using polynucleotides of the present invention. Any of
these alterations (altered expression, chromosomal rearrangement,
or mutation) can be used as a diagnostic or prognostic marker.
[1262] Thus, the invention also provides a diagnostic method useful
during diagnosis of a disorder, involving measuring the expression
level of polynucleotides of the present invention in cells or body
fluid from an individual and comparing the measured gene expression
level with a standard level of polynucleotide expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a disorder.
[1263] In still another embodiment, the invention includes a kit
for analyzing samples for the presence of proliferative and/or
cancerous polynucleotides derived from a test subject. In a general
embodiment, the kit includes at least one polynucleotide probe
containing a nucleotide sequence that will specifically hybridize
with a polynucleotide of the present invention and a suitable
container. In a specific embodiment, the kit includes two
polynucleotide probes defining an internal region of the
polynucleotide of the present invention, where each probe has one
strand containing a 31' mer-end internal to the region. In a
further embodiment, the probes may be useful as primers for
polymerase chain reaction amplification.
[1264] Where a diagnosis of a disorder, has already been made
according to conventional methods, the present invention is useful
as a prognostic indicator, whereby patients exhibiting enhanced or
depressed polynucleotide of the present invention expression will
experience a worse clinical outcome relative to patients expressing
the gene at a level nearer the standard level.
[1265] By "measuring the expression level of polynucleotide of the
present invention" is intended qualitatively or quantitatively
measuring or estimating the level of the polypeptide of the present
invention or the level of the mRNA encoding the polypeptide in a
first biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the polypeptide level or mRNA level in a
second biological sample). Preferably, the polypeptide level or
mRNA level in the first biological sample is measured or estimated
and compared to a standard polypeptide level or mRNA level, the
standard being taken from a second biological sample obtained from
an individual not having the disorder or being determined by
averaging levels from a population of individuals not having a
disorder. As will be appreciated in the art, once a standard
polypeptide level or mRNA level is known, it can be used repeatedly
as a standard for comparison.
[1266] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains the polypeptide of the present
invention or mRNA. As indicated, biological samples include body
fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and spinal fluid) which contain the polypeptide of the present
invention, and other tissue sources found to express the
polypeptide of the present invention. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Where the biological sample is to include mRNA, a tissue biopsy is
the preferred source.
[1267] The method(s) provided above may preferrably be applied in a
diagnostic method and/or kits in which polynucleotides and/or
polypeptides are attached to a solid support. In one exemplary
method, the support may be a "gene chip" or a "biological chip" as
described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174.
Further, such a gene chip with polynucleotides of the present
invention attached may be used to identify polymorphisms between
the polynucleotide sequences, with polynucleotides isolated from a
test subject. The knowledge of such polymorphisms (i.e. their
location, as well as, their existence) would be beneficial in
identifying disease loci for many disorders, including cancerous
diseases and conditions. Such a method is described in U.S. Pat.
Nos. 5,858,659 and 5,856,104. The US patents referenced supra are
hereby incorporated by reference in their entirety herein.
[1268] The present invention encompasses polynucleotides of the
present invention that are chemically synthesized, or reproduced as
peptide nucleic acids (PNA), or according to other methods known in
the art. The use of PNAs would serve as the preferred form if the
polynucleotides are incorporated onto a solid support, or gene
chip. For the purposes of the present invention, a peptide nucleic
acid (PNA) is a polyamide type of DNA analog and the monomeric
units for adenine, guanine, thymine and cytosine are available
commercially (Perceptive Biosystems). Certain components of DNA,
such as phosphorus, phosphorus oxides, or deoxyribose derivatives,
are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm,
R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.
Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D.
A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen,
Nature 365, 666 (1993), PNAs bind specifically and tightly to
complementary DNA strands and are not degraded by nucleases. In
fact, PNA binds more strongly to DNA than DNA itself does. This is
probably because there is no electrostatic repulsion between the
two strands, and also the polyamide backbone is more flexible.
Because of this, PNA/DNA duplexes bind under a wider range of
stringency conditions than DNA/DNA duplexes, making it easier to
perform multiplex hybridization. Smaller probes can be used than
with DNA due to the strong binding. In addition, it is more likely
that single base mismatches can be determined with PNA/DNA
hybridization because a single mismatch in a PNA/DNA 15-mer lowers
the melting point (T.sub.m) by 8.degree.-20.degree. C., vs.
4.degree.-16.degree. C. for the DNA/DNA 15-mer duplex. Also, the
absence of charge groups in PNA means that hybridization can be
done at low ionic strengths and reduce possible interference by
salt during the analysis.
[1269] The present invention is useful for detecting cancer in
mammals. In particular the invention is useful during diagnosis of
pathological cell proliferative neoplasias which include, but are
not limited to: acute myelogenous leukemias including acute
monocytic leukemia, acute myeloblastic leukemia, acute
promyelocytic leukemia, acute myelomonocytic leukemia, acute
erythroleukemia, acute megakaryocytic leukemia, and acute
undifferentiated leukemia, etc.; and chronic myelogenous leukemias
including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs,
cows, pigs, horses, rabbits and humans. Particularly preferred are
humans.
[1270] Pathological cell proliferative diseases, disorders, and/or
conditions are often associated with inappropriate activation of
proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute
Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic
Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182
(1985)). Neoplasias are now believed to result from the qualitative
alteration of a normal cellular gene product, or from the
quantitative modification of gene expression by insertion into the
chromosome of a viral sequence, by chromosomal translocation of a
gene to a more actively transcribed region, or by some other
mechanism. (Gelmann et al., supra) It is likely that mutated or
altered expression of specific genes is involved in the
pathogenesis of some leukemias, among other tissues and cell types.
(Gelmann et al., supra) Indeed, the human counterparts of the
oncogenes involved in some animal neoplasias have been amplified or
translocated in some cases of human leukemia and carcinoma.
(Gelmann et al., supra)
[1271] For example, c-myc expression is highly amplified in the
non-lymphocytic leukemia cell line HL-60. When HL-60 cells are
chemically induced to stop proliferation, the level of c-myc is
found to be down-regulated. (International Publication Number WO
91/15580) However, it has been shown that exposure of HL-60 cells
to a DNA construct that is complementary to the 5' end of c-myc or
c-myb blocks translation of the corresponding mRNAs which
downregulates expression of the c-myc or c-myb proteins and causes
arrest of cell proliferation and differentiation of the treated
cells. (International Publication Number WO 91/15580; Wickstrom et
al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc.
Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan
would appreciate the present invention's usefulness would not be
limited to treatment of proliferative diseases, disorders, and/or
conditions of hematopoietic cells and tissues, in light of the
numerous cells and cell types of varying origins which are known to
exhibit proliferative phenotypes.
[1272] In addition to the foregoing, a polynucleotide can be used
to control gene expression through triple helix formation or
antisense DNA or RNA. Antisense techniques are discussed, for
example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance Lee et al., Nucleic Acids Research 6:
3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et
al., Science 251: 1360 (1991). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques,
preferred polynucleotides are usually oligonucleotides 20 to 40
bases in length and complementary to either the region of the gene
involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988).) Triple helix formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques are effective in model
systems, and the information disclosed herein can be used to design
antisense or triple helix polynucleotides in an effort to treat or
prevent disease.
[1273] Polynucleotides of the present invention are also useful in
gene therapy. One goal of gene therapy is to insert a normal gene
into an organism having a defective gene, in an effort to correct
the genetic defect. The polynucleotides disclosed in the present
invention offer a means of targeting such genetic defects in a
highly accurate manner. Another goal is to insert a new gene that
was not present in the host genome, thereby producing a new trait
in the host cell.
[1274] The polynucleotides are also useful for identifying
individuals from minute biological samples. The United States
military, for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identifying personnel. This
method does not suffer from the current limitations of "Dog Tags"
which can be lost, switched, or stolen, making positive
identification difficult. The polynucleotides of the present
invention can be used as additional DNA markers for RFLP.
[1275] The polynucleotides of the present invention can also be
used as an alternative to RFLP, by determining the actual
base-by-base DNA sequence of selected portions of an individual's
genome. These sequences can be used to prepare PCR primers for
amplifying and isolating such selected DNA, which can then be
sequenced. Using this technique, individuals can be identified
because each individual will have a unique set of DNA sequences.
Once an unique ID database is established for an individual,
positive identification of that individual, living or dead, can be
made from extremely small tissue samples.
[1276] Forensic biology also benefits from using DNA-based
identification techniques as disclosed herein. DNA sequences taken
from very small biological samples such as tissues, e.g., hair or
skin, or body fluids, e.g., blood, saliva, semen, synovial fluid,
amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,
urine, fecal matter, etc., can be amplified using PCR. In one prior
art technique, gene sequences amplified from polymorphic loci, such
as DQa class II HLA gene, are used in forensic biology to identify
individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).)
Once these specific polymorphic loci are amplified, they are
digested with one or more restriction enzymes, yielding an
identifying set of bands on a Southern blot probed with DNA
corresponding to the DQa class II HLA gene. Similarly,
polynucleotides of the present invention can be used as polymorphic
markers for forensic purposes.
[1277] There is also a need for reagents capable of identifying the
source of a particular tissue. Such need arises, for example, in
forensics when presented with tissue of unknown origin. Appropriate
reagents can comprise, for example, DNA probes or primers specific
to particular tissue prepared from the sequences of the present
invention. Panels of such reagents can identify tissue by species
and/or by organ type. In a similar fashion, these reagents can be
used to screen tissue cultures for contamination.
[1278] In the very least, the polynucleotides of the present
invention can be used as molecular weight markers on Southern gels,
as diagnostic probes for the presence of a specific mRNA in a
particular cell type, as a probe to "subtract-out" known sequences
in the process of discovering novel polynucleotides, for selecting
and making oligomers for attachment to a "gene chip" or other
support, to raise anti-DNA antibodies using DNA immunization
techniques, and as an antigen to elicit an immune response.
[1279] Uses of the Polypeptides
[1280] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[1281] A polypeptide of the present invention can be used to assay
protein levels in a biological sample using antibody-based
techniques. For example, protein expression in tissues can be
studied with classical immunohistological methods. (Jalkanen, M.,
et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J.
Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods
useful for detecting protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99 mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[1282] In addition to assaying secreted protein levels in a
biological sample, proteins can also be detected in vivo by
imaging. Antibody labels or markers for in vivo imaging of protein
include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma.
[1283] A protein-specific antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, 131I, 112In, 99 mTc), a radio-opaque
substance, or a material detectable by nuclear magnetic resonance,
is introduced (for example, parenterally, subcutaneously, or
intraperitoneally) into the mammal. It will be understood in the
art that the size of the subject and the imaging system used will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of 99 mTc. The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain the specific protein. In
vivo tumor imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982).)
[1284] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression of a
polypeptide of the present invention in cells or body fluid of an
individual; (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in
the assayed polypeptide gene expression level compared to the
standard expression level is indicative of a disorder. With respect
to cancer, the presence of a relatively high amount of transcript
in biopsied tissue from an individual may indicate a predisposition
for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[1285] Moreover, polypeptides of the present invention can be used
to treat, prevent, and/or diagnose disease. For example, patients
can be administered a polypeptide of the present invention in an
effort to replace absent or decreased levels of the polypeptide
(e.g., insulin), to supplement absent or decreased levels of a
different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD,
catalase, DNA repair proteins), to inhibit the activity of a
polypeptide (e.g., an oncogene or tumor supressor), to activate the
activity of a polypeptide (e.g., by binding to a receptor), to
reduce the activity of a membrane bound receptor by competing with
it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to bring about a desired response (e.g., blood
vessel growth inhibition, enhancement of the immune response to
proliferative cells or tissues).
[1286] Similarly, antibodies directed to a polypeptide of the
present invention can also be used to treat, prevent, and/or
diagnose disease. For example, administration of an antibody
directed to a polypeptide of the present invention can bind and
reduce overproduction of the polypeptide. Similarly, administration
of an antibody can activate the polypeptide, such as by binding to
a polypeptide bound to a membrane (receptor).
[1287] At the very least, the polypeptides of the present invention
can be used as molecular weight markers on SDS-PAGE gels or on
molecular sieve gel filtration columns using methods well known to
those of skill in the art. Polypeptides can also be used to raise
antibodies, which in turn are used to measure protein expression
from a recombinant cell, as a way of assessing transformation of
the host cell. Moreover, the polypeptides of the present invention
can be used to test the following biological activities.
[1288] Gene Therapy Methods
[1289] Another aspect of the present invention is to gene therapy
methods for treating or preventing disorders, diseases and
conditions. The gene therapy methods relate to the introduction of
nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an
animal to achieve expression of a polypeptide of the present
invention. This method requires a polynucleotide which codes for a
polypeptide of the invention that operatively linked to a promoter
and any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery
techniques are known in the art, see, for example, WO90/11092,
which is herein incorporated by reference.
[1290] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a polynucleotide of the invention ex vivo, with the
engineered cells then being provided to a patient to be treated
with the polypeptide. Such methods are well-known in the art. For
example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993);
Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T.,
et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer
Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene
Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy
4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38
(1996)), which are herein incorporated by reference. In one
embodiment, the cells which are engineered are arterial cells. The
arterial cells may be reintroduced into the patient through direct
injection to the artery, the tissues surrounding the artery, or
through catheter injection.
[1291] As discussed in more detail below, the polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[1292] In one embodiment, the polynucleotide of the invention is
delivered as a naked polynucleotide. The term "naked"
polynucleotide, DNA or RNA refers to sequences that are free from
any delivery vehicle that acts to assist, promote or facilitate
entry into the cell, including viral sequences, viral particles,
liposome formulations, LIPOFECTIN.TM. or precipitating agents and
the like. However, the polynucleotides of the invention can also be
delivered in liposome formulations and LIPOFECTIN.TM. formulations
and the like can be prepared by methods well known to those skilled
in the art. Such methods are described, for example, in U.S. Pat.
Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein
incorporated by reference.
[1293] The polynucleotide vector constructs of the invention used
in the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Appropriate vectors include pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from STRATAGENE.TM.; pSVK3, pBPV,
pMSG and pSVL available from PHARMACIA.TM.; and pEF1/V5, pcDNA3.1,
and pRc/CMV2 available from Invitrogen. Other suitable vectors will
be readily apparent to the skilled artisan.
[1294] Any strong promoter known to those skilled in the art can be
used for driving the expression of polynucleotide sequence of the
invention. Suitable promoters include adenoviral promoters, such as
the adenoviral major late promoter; or heterologous promoters, such
as the cytomegalovirus (CMV) promoter; the respiratory syncytial
virus (RSV) promoter; inducible promoters, such as the MMT
promoter, the metallothionein promoter; heat shock promoters; the
albumin promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for the polynucleotides of the invention.
[1295] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[1296] The polynucleotide construct of the invention can be
delivered to the interstitial space of tissues within the an
animal, including of muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus,
rectum, nervous system, eye, gland, and connective tissue.
Interstitial space of the tissues comprises the intercellular,
fluid, mucopolysaccharide matrix among the reticular fibers of
organ tissues, elastic fibers in the walls of vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of
bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery
to the interstitial space of muscle tissue is preferred for the
reasons discussed below. They may be conveniently delivered by
injection into the tissues comprising these cells. They are
preferably delivered to and expressed in persistent, non-dividing
cells which are differentiated, although delivery and expression
may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts. In vivo muscle cells are particularly competent
in their ability to take up and express polynucleotides.
[1297] For the naked nucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[1298] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
DNA constructs can be delivered to arteries during angioplasty by
the catheter used in the procedure.
[1299] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[1300] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
LIPOFECTIN.TM., precipitating agents, etc. Such methods of delivery
are known in the art.
[1301] In certain embodiments, the polynucleotide constructs of the
invention are complexed in a liposome preparation. Liposomal
preparations for use in the instant invention include cationic
(positively charged), anionic (negatively charged) and neutral
preparations. However, cationic liposomes are particularly
preferred because a tight charge complex can be formed between the
cationic liposome and the polyanionic nucleic acid. Cationic
liposomes have been shown to mediate intracellular delivery of
plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA,
84:7413-7416 (1987), which is herein incorporated by reference);
mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081
(1989), which is herein incorporated by reference); and purified
transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192
(1990), which is herein incorporated by reference), in functional
form.
[1302] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
LIPOFECTIN.TM., from GIBCO BRL, Grand Island, N.Y. (See, also,
Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987),
which is herein incorporated by reference). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[1303] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[1304] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[1305] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[1306] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology, 101:512-527
(1983), which is herein incorporated by reference. For example,
MLVs containing nucleic acid can be prepared by depositing a thin
film of phospholipid on the walls of a glass tube and subsequently
hydrating with a solution of the material to be encapsulated. SUVs
are prepared by extended sonication of MLVs to produce a
homogeneous population of unilamellar liposomes. The material to be
entrapped is added to a suspension of preformed MLVs and then
sonicated. When using liposomes containing cationic lipids, the
dried lipid film is resuspended in an appropriate solution such as
sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCl, sonicated, and then the preformed liposomes are mixed
directly with the DNA. The liposome and DNA form a very stable
complex due to binding of the positively charged liposomes to the
cationic DNA. SUVs find use with small nucleic acid fragments. LUVs
are prepared by a number of methods, well known in the art.
Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975);
Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al.,
Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem.
Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl.
Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al.,
Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase
evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980);
Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978);
Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein
incorporated by reference.
[1307] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[1308] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication NO: WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication NO: WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[1309] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding polypeptides of the invention. Retroviruses from
which the retroviral plasmid vectors may be derived include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[1310] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[1311] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding polypeptides
of the invention. Such retroviral vector particles then may be
employed, to transduce eukaryotic cells, either in vitro or in
vivo. The transduced eukaryotic cells will express polypeptides of
the invention.
[1312] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with polynucleotides of the invention contained in an
adenovirus vector. Adenovirus can be manipulated such that it
encodes and expresses polypeptides of the invention, and at the
same time is inactivated in terms of its ability to replicate in a
normal lytic viral life cycle. Adenovirus expression is achieved
without integration of the viral DNA into the host cell chromosome,
thereby alleviating concerns about insertional mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines
for many years with an excellent safety profile (Schwartz et al.,
Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus
mediated gene transfer has been demonstrated in a number of
instances including transfer of alpha-1-antitrypsin and CFTR to the
lungs of cotton rats (Rosenfeld et al., Science, 252:431-434
(1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore,
extensive studies to attempt to establish adenovirus as a causative
agent in human cancer were uniformly negative (Green et al. Proc.
Natl. Acad. Sci. USA, 76:6606 (1979)).
[1313] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155
(1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993);
Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are
herein incorporated by reference. For example, the adenovirus
vector Ad2 is useful and can be grown in human 293 cells. These
cells contain the E1 region of adenovirus and constitutively
express E1a and E1b, which complement the defective adenoviruses by
providing the products of the genes deleted from the vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and
Ad7) are also useful in the present invention.
[1314] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[1315] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, Curr. Topics in
Microbiol. Immunol., 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[1316] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The
polynucleotide construct containing polynucleotides of the
invention is inserted into the AAV vector using standard cloning
methods, such as those found in Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press (1989). The
recombinant AAV vector is then transfected into packaging cells
which are infected with a helper virus, using any standard
technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
polynucleotide construct of the invention. These viral particles
are then used to transduce eukaryotic cells, either ex vivo or in
vivo. The transduced cells will contain the polynucleotide
construct integrated into its genome, and will express the desired
gene product.
[1317] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding the polypeptide sequence of interest) via
homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; International Publication NO: WO 96/29411,
published Sep. 26, 1996; International Publication NO: WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438
(1989). This method involves the activation of a gene which is
present in the target cells, but which is not normally expressed in
the cells, or is expressed at a lower level than desired.
[1318] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the desired endogenous polynucleotide sequence
so the promoter will be operably linked to the endogenous sequence
upon homologous recombination.
[1319] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[1320] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[1321] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous sequence
is placed under the control of the promoter. The promoter then
drives the expression of the endogenous sequence.
[1322] The polynucleotides encoding polypeptides of the present
invention may be administered along with other polynucleotides
encoding other angiongenic proteins. Angiogenic proteins include,
but are not limited to, acidic and basic fibroblast growth factors,
VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor
alpha and beta, platelet-derived endothelial cell growth factor,
platelet-derived growth factor, tumor necrosis factor alpha,
hepatocyte growth factor, insulin like growth factor, colony
stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide
synthase.
[1323] Preferably, the polynucleotide encoding a polypeptide of the
invention contains a secretory signal sequence that facilitates
secretion of the protein. Typically, the signal sequence is
positioned in the coding region of the polynucleotide to be
expressed towards or at the 5' end of the coding region. The signal
sequence may be homologous or heterologous to the polynucleotide of
interest and may be homologous or heterologous to the cells to be
transfected. Additionally, the signal sequence may be chemically
synthesized using methods known in the art.
[1324] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, BIOLISTIC.TM.
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers.
(Kaneda et al., Science, 243:375 (1989)).
[1325] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[1326] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[1327] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[1328] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[1329] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian. Therapeutic compositions of
the present invention can be administered to any animal, preferably
to mammals and birds. Preferred mammals include humans, dogs, cats,
mice, rats, rabbits sheep, cattle, horses and pigs, with humans
being particularly
[1330] Biological Activities
[1331] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention can be used in assays to test
for one or more biological activities. If these polynucleotides and
polypeptides do exhibit activity in a particular assay, it is
likely that these molecules may be involved in the diseases
associated with the biological activity. Thus, the polynucleotides
or polypeptides, or agonists or antagonists could be used to treat
the associated disease.
[1332] Polynucleotides, translation products and antibodies
corresponding to this gene may be useful for the diagnosis,
prognosis, prevention, and/or treatment of diseases and/or
disorders associated with the following systems.
[1333] Immune Activity
[1334] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be useful in treating,
preventing, diagnosing and/or prognosing diseases, disorders,
and/or conditions of the immune system, by, for example, activating
or inhibiting the proliferation, differentiation, or mobilization
(chemotaxis) of immune cells. Immune cells develop through a
process called hematopoiesis, producing myeloid (platelets, red
blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells from pluripotent stem cells. The etiology of
these immune diseases, disorders, and/or conditions may be genetic,
somatic, such as cancer and some autoimmune diseases, acquired
(e.g., by chemotherapy or toxins), or infectious. Moreover,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention can be used as a marker or
detector of a particular immune system disease or disorder.
[1335] In another embodiment, a polypeptide of the invention, or
polynucleotides, antibodies, agonists, or antagonists corresponding
to that polypeptide, may be used to treat diseases and disorders of
the immune system and/or to inhibit or enhance an immune response
generated by cells associated with the tissue(s) in which the
polypeptide of the invention is expressed, including one, two,
three, four, five, or more tissues disclosed in Table 1, column 8
(Tissue Distribution Library Code).
[1336] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be useful in treating,
preventing, diagnosing, and/or prognosing immunodeficiencies,
including both congenital and acquired immunodeficiencies. Examples
of B cell immunodeficiencies in which immunoglobulin levels B cell
function and/or B cell numbers are decreased include: X-linked
agammaglobulinemia (Bruton's disease), X-linked infantile
agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non
X-linked immunodeficiency with hyper IgM, X-linked
lymphoproliferative syndrome (XLP), agammaglobulinemia including
congenital and acquired agammaglobulinemia, adult onset
agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, unspecified
hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type),
Selective IgM deficiency, selective IgA deficiency, selective IgG
subclass deficiencies, IgG subclass deficiency (with or without IgA
deficiency), Ig deficiency with increased IgM, IgG and IgA
deficiency with increased IgM, antibody deficiency with normal or
elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B
cell lymphoproliferative disorder (BLPD), common variable
immunodeficiency (CVID), common variable immunodeficiency (CVI)
(acquired), and transient hypogammaglobulinemia of infancy.
[1337] In specific embodiments, ataxia-telangiectasia or conditions
associated with ataxia-telangiectasia are treated, prevented,
diagnosed, and/or prognosing using the polypeptides or
polynucleotides of the invention, and/or agonists or antagonists
thereof.
[1338] Examples of congenital immunodeficiencies in which T cell
and/or B cell function and/or number is decreased include, but are
not limited to: DiGeorge anomaly, severe combined
immunodeficiencies (SCID) (including, but not limited to, X-linked
SCID, autosomal recessive SCID, adenosine deaminase deficiency,
purine nucleoside phosphorylase (PNP) deficiency, Class II MHC
deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome,
and ataxia telangiectasia), thymic hypoplasia, third and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous
candidiasis, natural killer cell deficiency (NK), idiopathic CD4+
T-lymphocytopenia, immunodeficiency with predominant T cell defect
(unspecified), and unspecified immunodeficiency of cell mediated
immunity.
[1339] In specific embodiments, DiGeorge anomaly or conditions
associated with DiGeorge anomaly are treated, prevented, diagnosed,
and/or prognosed using polypeptides or polynucleotides of the
invention, or antagonists or agonists thereof.
[1340] Other immunodeficiencies that may be treated, prevented,
diagnosed, and/or prognosed using polypeptides or polynucleotides
of the invention, and/or agonists or antagonists thereof, include,
but are not limited to, chronic granulomatous disease,
Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte
glucose-6-phosphate dehydrogenase deficiency, X-linked
lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency,
complement component deficiencies (including C1, C2, C3, C4, C5,
C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe
congenital leukopenia, dysplasia with immunodeficiency, neonatal
neutropenia, short limbed dwarfism, and Nezelof syndrome-combined
immunodeficiency with Igs.
[1341] In a preferred embodiment, the immunodeficiencies and/or
conditions associated with the immunodeficiencies recited above are
treated, prevented, diagnosed and/or prognosed using
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention.
[1342] In a preferred embodiment polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
could be used as an agent to boost immunoresponsiveness among
immunodeficient individuals. In specific embodiments,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention could be used as an agent to
boost immunoresponsiveness among B cell and/or T cell
immunodeficient individuals.
[1343] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
treating, preventing, diagnosing and/or prognosing autoimmune
disorders. Many autoimmune disorders result from inappropriate
recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to
the destruction of the host tissue. Therefore, the administration
of polynucleotides and polypeptides of the invention that can
inhibit an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing autoimmune disorders.
[1344] Autoimmune diseases or disorders that may be treated,
prevented, diagnosed and/or prognosed by polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention include, but are not limited to, one or more of
the following: systemic lupus erythematosus, rheumatoid arthritis,
ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis,
Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic
anemia, thrombocytopenia, autoimmune thrombocytopenia purpura,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, purpura (e.g., Henloch-Scoenlein purpura),
autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris,
myasthenia gravis, Grave's disease (hyperthyroidism), and
insulin-resistant diabetes mellitus.
[1345] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, and/or diagnosed with the
compositions of the invention include, but are not limited to, type
II collagen-induced arthritis, antiphospholipid syndrome,
dermatitis, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, neuritis, uveitis
ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man
Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre
Syndrome, insulin dependent diabetes mellitus, and autoimmune
inflammatory eye disorders.
[1346] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, diagnosed and/or
prognosed with the compositions of the invention include, but are
not limited to, scleroderma with anti-collagen antibodies (often
characterized, e.g., by nucleolar and other nuclear antibodies),
mixed connective tissue disease (often characterized, e.g., by
antibodies to extractable nuclear antigens (e.g.,
ribonucleoprotein)), polymyositis (often characterized, e.g., by
nonhistone ANA), pernicious anemia (often characterized, e.g., by
antiparietal cell, microsomes, and intrinsic factor antibodies),
idiopathic Addison's disease (often characterized, e.g., by humoral
and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g., by antispermatozoal antibodies),
glomerulonephritis (often characterized, e.g., by glomerular
basement membrane antibodies or immune complexes), bullous
pemphigoid (often characterized, e.g., by IgG and complement in
basement membrane), Sjogren's syndrome (often characterized, e.g.,
by multiple tissue antibodies, and/or a specific nonhistone ANA
(SS-B)), diabetes mellitus (often characterized, e.g., by
cell-mediated and humoral islet cell antibodies), and adrenergic
drug resistance (including adrenergic drug resistance with asthma
or cystic fibrosis) (often characterized, e.g., by beta-adrenergic
receptor antibodies).
[1347] Additional disorders that may have an autoimmune component
that may be treated, prevented, diagnosed and/or prognosed with the
compositions of the invention include, but are not limited to,
chronic active hepatitis (often characterized, e.g., by smooth
muscle antibodies), primary biliary cirrhosis (often characterized,
e.g., by mitochondria antibodies), other endocrine gland failure
(often characterized, e.g., by specific tissue antibodies in some
cases), vitiligo (often characterized, e.g., by melanocyte
antibodies), vasculitis (often characterized, e.g., by Ig and
complement in vessel walls and/or low serum complement), post-MI
(often characterized, e.g., by myocardial antibodies), cardiotomy
syndrome (often characterized, e.g., by myocardial antibodies),
urticaria (often characterized, e.g., by IgG and IgM antibodies to
IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM
antibodies to IgE), asthma (often characterized, e.g., by IgG and
IgM antibodies to IgE), and many other inflammatory, granulomatous,
degenerative, and atrophic disorders.
[1348] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using for example, antagonists or agonists, polypeptides
or polynucleotides, or antibodies of the present invention. In a
specific preferred embodiment, rheumatoid arthritis is treated,
prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present
invention.
[1349] In another specific preferred embodiment, systemic lupus
erythematosus is treated, prevented, and/or diagnosed using
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention. In another specific preferred
embodiment, idiopathic thrombocytopenia purpura is treated,
prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present
invention.
[1350] In another specific preferred embodiment IgA nephropathy is
treated, prevented, and/or diagnosed using polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention.
[1351] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention
[1352] In preferred embodiments, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a immunosuppressive agent(s).
[1353] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be useful in treating,
preventing, prognosing, and/or diagnosing diseases, disorders,
and/or conditions of hematopoietic cells. Polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention could be used to increase differentiation and
proliferation of hematopoietic cells, including the pluripotent
stem cells, in an effort to treat or prevent those diseases,
disorders, and/or conditions associated with a decrease in certain
(or many) types hematopoietic cells, including but not limited to,
leukopenia, neutropenia, anemia, and thrombocytopenia.
Alternatively, Polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention could be used to
increase differentiation and proliferation of hematopoietic cells,
including the pluripotent stem cells, in an effort to treat or
prevent those diseases, disorders, and/or conditions associated
with an increase in certain (or many) types of hematopoietic cells,
including but not limited to, histiocytosis.
[1354] Allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, diagnosed and/or prognosed using
polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists or antagonists thereof. Moreover, these molecules
can be used to treat, prevent, prognose, and/or diagnose
anaphylaxis, hypersensitivity to an antigenic molecule, or blood
group incompatibility.
[1355] Additionally, polypeptides or polynucleotides of the
invention, and/or agonists or antagonists thereof, may be used to
treat, prevent, diagnose and/or prognose IgE-mediated allergic
reactions. Such allergic reactions include, but are not limited to,
asthma, rhinitis, and eczema. In specific embodiments,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be used to modulate IgE
concentrations in vitro or in vivo.
[1356] Moreover, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention have uses in the
diagnosis, prognosis, prevention, and/or treatment of inflammatory
conditions. For example, since polypeptides, antibodies, or
polynucleotides of the invention, and/or agonists or antagonists of
the invention may inhibit the activation, proliferation and/or
differentiation of cells involved in an inflammatory response,
these molecules can be used to prevent and/or treat chronic and
acute inflammatory conditions. Such inflammatory conditions
include, but are not limited to, for example, inflammation
associated with infection (e.g., septic shock, sepsis, or systemic
inflammatory response syndrome), ischemia-reperfusion injury,
endotoxin lethality, complement-mediated hyperacute rejection,
nephritis, cytokine or chemokine induced lung injury, inflammatory
bowel disease, Crohn's disease, over production of cytokines (e.g.,
TNF or IL-1.), respiratory disorders (e.g., asthma and allergy);
gastrointestinal disorders (e.g., inflammatory bowel disease);
cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast);
CNS disorders (e.g., multiple sclerosis; ischemic brain injury
and/or stroke, traumatic brain injury, neurodegenerative disorders
(e.g., Parkinson's disease and Alzheimer's disease); AIDS-related
dementia; and prion disease); cardiovascular disorders (e.g.,
atherosclerosis, myocarditis, cardiovascular disease, and
cardiopulmonary bypass complications); as well as many additional
diseases, conditions, and disorders that are characterized by
inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma,
pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion
injury, Grave's disease, systemic lupus erythematosus, diabetes
mellitus, and allogenic transplant rejection).
[1357] Because inflammation is a fundamental defense mechanism,
inflammatory disorders can effect virtually any tissue of the body.
Accordingly, polynucleotides, polypeptides, and antibodies of the
invention, as well as agonists or antagonists thereof, have uses in
the treatment of tissue-specific inflammatory disorders, including,
but not limited to, adrenalitis, alveolitis, angiocholecystitis,
appendicitis, balanitis, blepharitis, bronchitis, bursitis,
carditis, cellulitis, cervicitis, cholecystitis, chorditis,
cochlitis, colitis, conjunctivitis, cystitis, dermatitis,
diverticulitis, encephalitis, endocarditis, esophagitis,
eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis,
gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis,
laryngitis, lymphangitis, mastitis, media otitis, meningitis,
metritis, mucitis, myocarditis, myosititis, myringitis, nephritis,
neuritis, orchitis, osteochondritis, otitis, pericarditis,
peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis,
prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis,
sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis,
stomatitis, synovitis, syringitis, tendonitis, tonsillitis,
urethritis, and vaginitis.
[1358] In specific embodiments, polypeptides, antibodies, or
polynucleotides of the invention, and/or agonists or antagonists
thereof, are useful to diagnose, prognose, prevent, and/or treat
organ transplant rejections and graft-versus-host disease. Organ
rejection occurs by host immune cell destruction of the
transplanted tissue through an immune response. Similarly, an
immune response is also involved in GVHD, but, in this case, the
foreign transplanted immune cells destroy the host tissues.
Polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists or antagonists thereof, that inhibit an immune
response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD. In specific
embodiments, polypeptides, antibodies, or polynucleotides of the
invention, and/or agonists or antagonists thereof, that inhibit an
immune response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing experimental allergic and hyperacute
xenograft rejection.
[1359] In other embodiments, polypeptides, antibodies, or
polynucleotides of the invention, and/or agonists or antagonists
thereof, are useful to diagnose, prognose, prevent, and/or treat
immune complex diseases, including, but not limited to, serum
sickness, post streptococcal glomerulonephritis, polyarteritis
nodosa, and immune complex-induced vasculitis.
[1360] Polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the invention can be used to treat, detect, and/or
prevent infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation activation
and/or differentiation of B and/or T cells, infectious diseases may
be treated, detected, and/or prevented. The immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may also directly inhibit the infectious agent
(refer to section of application listing infectious agents, etc),
without necessarily eliciting an immune response.
[1361] In another embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a vaccine adjuvant that enhances immune
responsiveness to an antigen. In a specific embodiment,
polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the present invention are used as an adjuvant to
enhance tumor-specific immune responses.
[1362] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an adjuvant to enhance anti-viral immune
responses. Anti-viral immune responses that may be enhanced using
the compositions of the invention as an adjuvant, include virus and
virus associated diseases or symptoms described herein or otherwise
known in the art. In specific embodiments, the compositions of the
invention are used as an adjuvant to enhance an immune response to
a virus, disease, or symptom selected from the group consisting of:
AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B).
In another specific embodiment, the compositions of the invention
are used as an adjuvant to enhance an immune response to a virus,
disease, or symptom selected from the group consisting of:
HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese
B encephalitis, influenza A and B, parainfluenza, measles,
cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever,
herpes simplex, and yellow fever.
[1363] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an adjuvant to enhance anti-bacterial or
anti-fungal immune responses. Anti-bacterial or anti-fungal immune
responses that may be enhanced using the compositions of the
invention as an adjuvant, include bacteria or fungus and bacteria
or fungus associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a bacteria or fungus, disease, or symptom
selected from the group consisting of: tetanus, Diphtheria,
botulism, and meningitis type B.
[1364] In another specific embodiment, the compositions of the
invention are used as an adjuvant to enhance an immune response to
a bacteria or fungus, disease, or symptom selected from the group
consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella
typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus
pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic
Escherichia coli, Enterohemorrhagic E. coli, and Borrelia
burgdorferi.
[1365] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an adjuvant to enhance anti-parasitic immune
responses. Anti-parasitic immune responses that may be enhanced
using the compositions of the invention as an adjuvant, include
parasite and parasite associated diseases or symptoms described
herein or otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to Plasmodium (malaria) or Leishmania.
[1366] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention may also be employed to treat infectious diseases
including silicosis, sarcoidosis, and idiopathic pulmonary
fibrosis; for example, by preventing the recruitment and activation
of mononuclear phagocytes.
[1367] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an antigen for the generation of antibodies
to inhibit or enhance immune mediated responses against
polypeptides of the invention.
[1368] In one embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are
administered to an animal (e.g., mouse, rat, rabbit, hamster,
guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow,
sheep, dog, cat, non-human primate, and human, most preferably
human) to boost the immune system to produce increased quantities
of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher affinity antibody production and immunoglobulin class
switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an
immune response.
[1369] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a stimulator of B cell responsiveness to
pathogens.
[1370] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an activator of T cells.
[1371] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent that elevates the immune status of
an individual prior to their receipt of immunosuppressive
therapies.
[1372] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to induce higher affinity
antibodies.
[1373] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to increase serum immunoglobulin
concentrations.
[1374] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to accelerate recovery of
immunocompromised individuals.
[1375] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to boost immunoresponsiveness among
aged populations and/or neonates.
[1376] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an immune system enhancer prior to, during,
or after bone marrow transplant and/or other transplants (e.g.,
allogeneic or xenogeneic organ transplantation). With respect to
transplantation, compositions of the invention may be administered
prior to, concomitant with, and/or after transplantation. In a
specific embodiment, compositions of the invention are administered
after transplantation, prior to the beginning of recovery of T-cell
populations. In another specific embodiment, compositions of the
invention are first administered after transplantation after the
beginning of recovery of T cell populations, but prior to full
recovery of B cell populations.
[1377] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to boost immunoresponsiveness among
individuals having an acquired loss of B cell function. Conditions
resulting in an acquired loss of B cell function that may be
ameliorated or treated by administering the polypeptides,
antibodies, polynucleotides and/or agonists or antagonists thereof,
include, but are not limited to, HIV Infection, AIDS, bone marrow
transplant, and B cell chronic lymphocytic leukemia (CLL).
[1378] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to boost immunoresponsiveness among
individuals having a temporary immune deficiency. Conditions
resulting in a temporary immune deficiency that may be ameliorated
or treated by administering the polypeptides, antibodies,
polynucleotides and/or agonists or antagonists thereof, include,
but are not limited to, recovery from viral infections (e.g.,
influenza), conditions associated with malnutrition, recovery from
infectious mononucleosis, or conditions associated with stress,
recovery from measles, recovery from blood transfusion, and
recovery from surgery.
[1379] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a regulator of antigen presentation by
monocytes, dendritic cells, and/or B-cells.
[1380] In one embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
enhance antigen presentation or antagonizes antigen presentation in
vitro or in vivo. Moreover, in related embodiments, said
enhancement or antagonism of antigen presentation may be useful as
an anti-tumor treatment or to modulate the immune system.
[1381] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as an agent to direct an individual's immune
system towards development of a humoral response (i.e. TH2) as
opposed to a TH1 cellular response.
[1382] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a means to induce tumor proliferation and
thus make it more susceptible to anti-neoplastic agents. For
example, multiple myeloma is a slowly dividing disease and is thus
refractory to virtually all anti-neoplastic regimens. If these
cells were forced to proliferate more rapidly their susceptibility
profile would likely change.
[1383] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a stimulator of B cell production in
pathologies such as AIDS, chronic lymphocyte disorder and/or Common
Variable Immunodificiency.
[1384] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a therapy for generation and/or regeneration
of lymphoid tissues following surgery, trauma or genetic defect. In
another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used in the pretreatment of bone marrow samples prior
to transplant.
[1385] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a gene-based therapy for genetically
inherited disorders resulting in
immuno-incompetence/immunodeficiency such as observed among SCID
patients.
[1386] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a means of activating monocytes/macrophages
to defend against parasitic diseases that effect monocytes such as
Leishmania.
[1387] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a means of regulating secreted cytokines that
are elicited by polypeptides of the invention.
[1388] In another embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used in one or more of the applications described
herein, as they may apply to veterinary medicine.
[1389] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a means of blocking various aspects of immune
responses to foreign agents or self. Examples of diseases or
conditions in which blocking of certain aspects of immune responses
may be desired include autoimmune disorders such as lupus, and
arthritis, as well as immunoresponsiveness to skin allergies,
inflammation, bowel disease, injury and diseases/disorders
associated with pathogens.
[1390] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a therapy for preventing the B cell
proliferation and Ig secretion associated with autoimmune diseases
such as idiopathic thrombocytopenic purpura, systemic lupus
erythematosus and multiple sclerosis.
[1391] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a inhibitor of B and/or T cell migration in
endothelial cells. This activity disrupts tissue architecture or
cognate responses and is useful, for example in disrupting immune
responses, and blocking sepsis.
[1392] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a therapy for chronic hypergammaglobulinemia
evident in such diseases as monoclonal gammopathy of undetermined
significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonal gammopathies, and plasmacytomas.
[1393] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention may be employed for instance to inhibit polypeptide
chemotaxis and activation of macrophages and their precursors, and
of neutrophils, basophils, B lymphocytes and some T-cell subsets,
e.g., activated and CD8 cytotoxic T cells and natural killer cells,
in certain autoimmune and chronic inflammatory and infective
diseases. Examples of autoimmune diseases are described herein and
include multiple sclerosis, and insulin-dependent diabetes.
[1394] The polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention may also be
employed to treat idiopathic hyper-eosinophilic syndrome by, for
example, preventing eosinophil production and migration.
[1395] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used to enhance or inhibit complement mediated cell
lysis.
[1396] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used to enhance or inhibit antibody dependent
cellular cytotoxicity.
[1397] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention may also be employed for treating atherosclerosis, for
example, by preventing monocyte infiltration in the artery
wall.
[1398] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention may be employed to treat adult respiratory distress
syndrome (ARDS).
[1399] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention may be useful for stimulating wound and tissue repair,
stimulating angiogenesis, and/or stimulating the repair of vascular
or lymphatic diseases or disorders. Additionally, agonists and
antagonists of the invention may be used to stimulate the
regeneration of mucosal surfaces.
[1400] In a specific embodiment, polynucleotides or polypeptides,
and/or agonists thereof are used to diagnose, prognose, treat,
and/or prevent a disorder characterized by primary or acquired
immunodeficiency, deficient serum immunoglobulin production,
recurrent infections, and/or immune system dysfunction. Moreover,
polynucleotides or polypeptides, and/or agonists thereof may be
used to treat or prevent infections of the joints, bones, skin,
and/or parotid glands, blood-borne infections (e.g., sepsis,
meningitis, septic arthritis, and/or osteomyelitis), autoimmune
diseases (e.g., those disclosed herein), inflammatory disorders,
and malignancies, and/or any disease or disorder or condition
associated with these infections, diseases, disorders and/or
malignancies) including, but not limited to, CVID, other primary
immune deficiencies, HIV disease, CLL, recurrent bronchitis,
sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis,
meningitis, herpes zoster (e.g., severe herpes zoster), and/or
pneumocystis carnii. Other diseases and disorders that may be
prevented, diagnosed, prognosed, and/or treated with
polynucleotides or polypeptides, and/or agonists of the present
invention include, but are not limited to, HIV infection, HTLV-BLV
infection, lymphopenia, phagocyte bactericidal dysfunction anemia,
thrombocytopenia, and hemoglobinuria.
[1401] In another embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
are used to treat, and/or diagnose an individual having common
variable immunodeficiency disease ("CVID"; also known as "acquired
agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease.
[1402] In a specific embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be used to diagnose, prognose, prevent, and/or treat cancers or
neoplasms including immune cell or immune tissue-related cancers or
neoplasms. Examples of cancers or neoplasms that may be prevented,
diagnosed, or treated by polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention include,
but are not limited to, acute myelogenous leukemia, chronic
myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma,
acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia,
plasmacytomas, multiple myeloma, Burkitt's lymphoma,
EBV-transformed diseases, and/or diseases and disorders described
in the section entitled "Hyperproliferative Disorders" elsewhere
herein.
[1403] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a therapy for decreasing cellular
proliferation of Large B-cell Lymphomas.
[1404] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are used as a means of decreasing the involvement of B
cells and Ig associated with Chronic Myelogenous Leukemia.
[1405] In specific embodiments, the compositions of the invention
are used as an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy.
[1406] Antagonists of the invention include, for example, binding
and/or inhibitory antibodies, antisense nucleic acids, ribozymes or
soluble forms of the polypeptides of the present invention (e.g.,
Fc fusion protein; see, e.g., Example 9). Agonists of the invention
include, for example, binding or stimulatory antibodies, and
soluble forms of the polypeptides (e.g., Fc fusion proteins; see,
e.g., Example 9). polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention may be employed in
a composition with a pharmaceutically acceptable carrier, e.g., as
described herein.
[1407] In another embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
invention are administered to an animal (including, but not limited
to, those listed above, and also including transgenic animals)
incapable of producing functional endogenous antibody molecules or
having an otherwise compromised endogenous immune system, but which
is capable of producing human immunoglobulin molecules by means of
a reconstituted or partially reconstituted immune system from
another animal (see, e.g., published PCT Application Nos.
WO98/24893, WO/9634096, WO/9633735, and WO/9110741). Administration
of polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the present invention to such animals is useful for
the generation of monoclonal antibodies against the polypeptides,
antibodies, polynucleotides and/or agonists or antagonists of the
present invention in an organ system listed above.
[1408] Blood-Related Disorders
[1409] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to
modulate hemostatic (the stopping of bleeding) or thrombolytic
(clot dissolving) activity. For example, by increasing hemostatic
or thrombolytic activity, polynucleotides or polypeptides, and/or
agonists or antagonists of the present invention could be used to
treat or prevent blood coagulation diseases, disorders, and/or
conditions (e.g., afibrinogenemia, factor deficiencies,
hemophilia), blood platelet diseases, disorders, and/or conditions
(e.g., thrombocytopenia), or wounds resulting from trauma, surgery,
or other causes. Alternatively, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
that can decrease hemostatic or thrombolytic activity could be used
to inhibit or dissolve clotting. These molecules could be important
in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[1410] In specific embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be used to prevent, diagnose, prognose, and/or treat
thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial
infarction, transient ischemic attack, unstable angina. In specific
embodiments, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used for
the prevention of occlusion of saphenous grafts, for reducing the
risk of periprocedural thrombosis as might accompany angioplasty
procedures, for reducing the risk of stroke in patients with atrial
fibrillation including nonrheumatic atrial fibrillation, for
reducing the risk of embolism associated with mechanical heart
valves and or mitral valves disease. Other uses for the
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention, include, but are not limited
to, the prevention of occlusions in extracorporeal devices (e.g.,
intravascular cannulas, vascular access shunts in hemodialysis
patients, hemodialysis machines, and cardiopulmonary bypass
machines).
[1411] In another embodiment, a polypeptide of the invention, or
polynucleotides, antibodies, agonists, or antagonists corresponding
to that polypeptide, may be used to prevent, diagnose, prognose,
and/or treat diseases and disorders of the blood and/or blood
forming organs associated with the tissue(s) in which the
polypeptide of the invention is expressed, including one, two,
three, four, five, or more tissues disclosed in Table 1, column 8
(Tissue Distribution Library Code).
[1412] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to
modulate hematopoietic activity (the formation of blood cells). For
example, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to
increase the quantity of all or subsets of blood cells, such as,
for example, erythrocytes, lymphocytes (B or T cells), myeloid
cells (e.g., basophils, eosinophils, neutrophils, mast cells,
macrophages) and platelets. The ability to decrease the quantity of
blood cells or subsets of blood cells may be useful in the
prevention, detection, diagnosis and/or treatment of anemias and
leukopenias described below. Alternatively, the polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may be used to decrease the quantity of all or
subsets of blood cells, such as, for example, erythrocytes,
lymphocytes (B or T cells), myeloid cells (e.g., basophils,
eosinophils, neutrophils, mast cells, macrophages) and platelets.
The ability to decrease the quantity of blood cells or subsets of
blood cells may be useful in the prevention, detection, diagnosis
and/or treatment of leukocytoses, such as, for example
eosinophilia.
[1413] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to
prevent, treat, or diagnose blood dyscrasia.
[1414] Anemias are conditions in which the number of red blood
cells or amount of hemoglobin (the protein that carries oxygen) in
them is below normal. Anemia may be caused by excessive bleeding,
decreased red blood cell production, or increased red blood cell
destruction (hemolysis). The polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in treating, preventing, and/or diagnosing anemias.
Anemias that may be treated prevented or diagnosed by the
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention include iron deficiency
anemia, hypochromic anemia, microcytic anemia, chlorosis,
hereditary sideroblastic anemia, idiopathic acquired sideroblastic
anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious
anemia, (vitamin B12 deficiency) and folic acid deficiency anemia),
aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic
anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal
hemoglobinuria). The polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be
useful in treating, preventing, and/or diagnosing anemias
associated with diseases including but not limited to, anemias
associated with systemic lupus erythematosus, cancers, lymphomas,
chronic renal disease, and enlarged spleens. The polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may be useful in treating, preventing, and/or
diagnosing anemias arising from drug treatments such as anemias
associated with methyldopa, dapsone, and/or sulfadrugs.
Additionally, rhe polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
treating, preventing, and/or diagnosing anemias associated with
abnormal red blood cell architecture including but not limited to,
hereditary spherocytosis, hereditary elliptocytosis,
glucose-6-phosphate dehydrogenase deficiency, and sickle cell
anemia.
[1415] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
treating, preventing, and/or diagnosing hemoglobin abnormalities,
(e.g., those associated with sickle cell anemia, hemoglobin C
disease, hemoglobin S--C disease, and hemoglobin E disease).
Additionally, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
diagnosing, prognosing, preventing, and/or treating thalassemias,
including, but not limited to major and minor forms of
alpha-thalassemia and beta-thalassemia.
[1416] In another embodiment, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in diagnosing, prognosing, preventing, and/or
treating bleeding disorders including, but not limited to,
thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and
thrombotic thrombocytopenic purpura), Von Willebrand's disease,
hereditary platelet disorders (e.g., storage pool disease such as
Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2
dysfunction, thromboasthenia, and Bernard-Soulier syndrome),
hemolytic-uremic syndrome, hemophelias such as hemophelia A or
Factor VII deficiency and Christmas disease or Factor IX
deficiency, Hereditary Hemorhhagic Telangiectsia, also known as
Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein
purpura) and disseminated intravascular coagulation.
[1417] The effect of the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention on the
clotting time of blood may be monitored using any of the clotting
tests known in the art including, but not limited to, whole blood
partial thromboplastin time (PTT), the activated partial
thromboplastin time (aPTT), the activated clotting time (ACT), the
recalcified activated clotting time, or the Lee-White Clotting
time.
[1418] Several diseases and a variety of drugs can cause platelet
dysfunction. Thus, in a specific embodiment, the polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may be useful in diagnosing, prognosing,
preventing, and/or treating acquired platelet dysfunction such as
platelet dysfunction accompanying kidney failure, leukemia,
multiple myeloma, cirrhosis of the liver, and systemic lupus
erythematosus as well as platelet dysfunction associated with drug
treatments, including treatment with aspirin, ticlopidine,
nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and
sprains), and penicillin in high doses.
[1419] In another embodiment, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in diagnosing, prognosing, preventing, and/or
treating diseases and disorders characterized by or associated with
increased or decreased numbers of white blood cells. Leukopenia
occurs when the number of white blood cells decreases below normal.
Leukopenias include, but are not limited to, neutropenia and
lymphocytopenia. An increase in the number of white blood cells
compared to normal is known as leukocytosis. The body generates
increased numbers of white blood cells during infection. Thus,
leukocytosis may simply be a normal physiological parameter that
reflects infection. Alternatively, leukocytosis may be an indicator
of injury or other disease such as cancer. Leokocytoses, include
but are not limited to, eosinophilia, and accumulations of
macrophages. In specific embodiments, the polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukopenia. In other specific
embodiments, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
diagnosing, prognosing, preventing, and/or treating
leukocytosis.
[1420] Leukopenia may be a generalized decreased in all types of
white blood cells, or may be a specific depletion of particular
types of white blood cells. Thus, in specific embodiments, the
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful in diagnosing,
prognosing, preventing, and/or treating decreases in neutrophil
numbers, known as neutropenia. Neutropenias that may be diagnosed,
prognosed, prevented, and/or treated by the polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention include, but are not limited to, infantile
genetic agranulocytosis, familial neutropenia, cyclic neutropenia,
neutropenias resulting from or associated with dietary deficiencies
(e.g., vitamin B 12 deficiency or folic acid deficiency),
neutropenias resulting from or associated with drug treatments
(e.g., antibiotic regimens such as penicillin treatment,
sulfonamide treatment, anticoagulant treatment, anticonvulsant
drugs, anti-thyroid drugs, and cancer chemotherapy), and
neutropenias resulting from increased neutrophil destruction that
may occur in association with some bacterial or viral infections,
allergic disorders, autoimmune diseases, conditions in which an
individual has an enlarged spleen (e.g., Felty syndrome, malaria
and sarcoidosis), and some drug treatment regimens.
[1421] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
diagnosing, prognosing, preventing, and/or treating
lymphocytopenias (decreased numbers of B and/or T lymphocytes),
including, but not limited lymphocytopenias resulting from or
associated with stress, drug treatments (e.g., drug treatment with
corticosteroids, cancer chemotherapies, and/or radiation
therapies), AIDS infection and/or other diseases such as, for
example, cancer, rheumatoid arthritis, systemic lupus
erythematosus, chronic infections, some viral infections and/or
hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich
Syndome, severe combined immunodeficiency, ataxia
telangiectsia).
[1422] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
diagnosing, prognosing, preventing, and/or treating diseases and
disorders associated with macrophage numbers and/or macrophage
function including, but not limited to, Gaucher's disease,
Niemann-Pick disease, Letterer-Siwe disease and
Hand-Schuller-Christian disease.
[1423] In another embodiment, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in diagnosing, prognosing, preventing, and/or
treating diseases and disorders associated with eosinophil numbers
and/or eosinophil function including, but not limited to,
idiopathic hypereosinophilic syndrome, eosinophilia-myalgia
syndrome, and Hand-Schuller-Christian disease.
[1424] In yet another embodiment, the polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukemias and lymphomas including, but
not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL),
acute myeloid (myelocytic, myelogenous, myeloblastic, or
myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B
cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell
leukemia), chronic myelocytic (myeloid, myelogenous, or
granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma,
Burkitt's lymphoma, and mycosis fungoides.
[1425] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in diagnosing, prognosing, preventing, and/or
treating diseases and disorders of plasma cells including, but not
limited to, plasma cell dyscrasias, monoclonal gammaopathies,
monoclonal gammopathies of undetermined significance, multiple
myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia,
cryoglobulinemia, and Raynaud's phenomenon.
[1426] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in treating, preventing, and/or diagnosing
myeloproliferative disorders, including but not limited to,
polycythemia vera, relative polycythemia, secondary polycythemia,
myelofibrosis, acute myelofibrosis, agnogenic myeloid metaplasia,
thrombocythemia, (including both primary and secondary
thrombocythemia) and chronic myelocytic leukemia.
[1427] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful as a treatment prior to surgery, to increase blood
cell production.
[1428] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful as an agent to enhance the migration, phagocytosis,
superoxide production, antibody dependent cellular cytotoxicity of
neutrophils, eosionophils and macrophages.
[1429] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful as an agent to increase the number of stem cells in
circulation prior to stem cells pheresis. In another specific
embodiment, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful as
an agent to increase the number of stem cells in circulation prior
to platelet pheresis.
[1430] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful as an agent to increase cytokine production.
[1431] In other embodiments, the polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be useful in preventing, diagnosing, and/or treating primary
hematopoietic disorders.
[1432] Hyperproliferative Disorders
[1433] In certain embodiments, polynucleotides or polypeptides, or
agonists or antagonists of the present invention can be used to
treat or detect hyperproliferative disorders, including neoplasms.
Polynucleotides or polypeptides, or agonists or antagonists of the
present invention may inhibit the proliferation of the disorder
through direct or indirect interactions. Alternatively,
Polynucleotides or polypeptides, or agonists or antagonists of the
present invention may proliferate other cells which can inhibit the
hyperproliferative disorder.
[1434] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[1435] Examples of hyperproliferative disorders that can be treated
or detected by polynucleotides or polypeptides, or agonists or
antagonists of the present invention include, but are not limited
to neoplasms located in the: colon, abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and
urogenital tract.
[1436] Similarly, other hyperproliferative disorders can also be
treated or detected by polynucleotides or polypeptides, or agonists
or antagonists of the present invention. Examples of such
hyperproliferative disorders include, but are not limited to: Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia,
Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical
Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary)
Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid
Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult
Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary
Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma,
AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct
Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain
Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter,
Central Nervous System (Primary) Lymphoma, Central Nervous System
Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical
Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood
(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,
Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma,
Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's
Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and
Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal
and Supratentorial Primitive Neuroectodermal Tumors, Childhood
Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft
Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma,
Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon
Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer,
Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine
Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ
Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric
Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors,
Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell
Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's
Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal
Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell
Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney
Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer,
Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[1437] In another preferred embodiment, polynucleotides or
polypeptides, or agonists or antagonists of the present invention
are used to diagnose, prognose, prevent, and/or treat premalignant
conditions and to prevent progression to a neoplastic or malignant
state, including but not limited to those disorders described
above. Such uses are indicated in conditions known or suspected of
preceding progression to neoplasia or cancer, in particular, where
non-neoplastic cell growth consisting of hyperplasia, metaplasia,
or most particularly, dysplasia has occurred (for review of such
abnormal growth conditions, see Robbins and Angell, 1976, Basic
Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp.
68-79.)
[1438] Hyperplasia is a form of controlled cell proliferation,
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or function. Hyperplastic
disorders which can be diagnosed, prognosed, prevented, and/or
treated with compositions of the invention (including
polynucleotides, polypeptides, agonists or antagonists) include,
but are not limited to, angiofollicular mediastinal lymph node
hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical
melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph
node hyperplasia, cementum hyperplasia, congenital adrenal
hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia,
cystic hyperplasia of the breast, denture hyperplasia, ductal
hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia,
focal epithelial hyperplasia, gingival hyperplasia, inflammatory
fibrous hyperplasia, inflammatory papillary hyperplasia,
intravascular papillary endothelial hyperplasia, nodular
hyperplasia of prostate, nodular regenerative hyperplasia,
pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia,
and verrucous hyperplasia.
[1439] Metaplasia is a form of controlled cell growth in which one
type of adult or fully differentiated cell substitutes for another
type of adult cell. Metaplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with compositions of the
invention (including polynucleotides, polypeptides, agonists or
antagonists) include, but are not limited to, agnogenic myeloid
metaplasia, apocrine metaplasia, atypical metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia,
epithelial metaplasia, intestinal metaplasia, metaplastic anemia,
metaplastic ossification, metaplastic polyps, myeloid metaplasia,
primary myeloid metaplasia, secondary myeloid metaplasia, squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid
metaplasia.
[1440] Dysplasia is frequently a forerunner of cancer, and is found
mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation.
Dysplastic disorders which can be diagnosed, prognosed, prevented,
and/or treated with compositions of the invention (including
polynucleotides, polypeptides, agonists or antagonists) include,
but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
ophthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[1441] Additional pre-neoplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with compositions of the
invention (including polynucleotides, polypeptides, agonists or
antagonists) include, but are not limited to, benign
dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue hypertrophy, intestinal polyps, colon polyps,
and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease,
Farmer's Skin, solar cheilitis, and solar keratosis.
[1442] In another embodiment, a polypeptide of the invention, or
polynucleotides, antibodies, agonists, or antagonists corresponding
to that polypeptide, may be used to diagnose and/or prognose
disorders associated with the tissue(s) in which the polypeptide of
the invention is expressed, including one, two, three, four, five,
or more tissues disclosed in Table 1, column 8 (Tissue Distribution
Library Code).
[1443] In another embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
conjugated to a toxin or a radioactive isotope, as described
herein, may be used to treat cancers and neoplasms, including, but
not limited to those described herein. In a further preferred
embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention conjugated to a
toxin or a radioactive isotope, as described herein, may be used to
treat acute myelogenous leukemia.
[1444] Additionally, polynucleotides, polypeptides, and/or agonists
or antagonists of the invention may affect apoptosis, and
therefore, would be useful in treating a number of diseases
associated with increased cell survival or the inhibition of
apoptosis. For example, diseases associated with increased cell
survival or the inhibition of apoptosis that could be diagnosed,
prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or agonists or antagonists of the invention,
include cancers (such as follicular lymphomas, carcinomas with p53
mutations, and hormone-dependent tumors, including, but not limited
to colon cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection.
[1445] In preferred embodiments, polynucleotides, polypeptides,
and/or agonists or antagonists of the invention are used to inhibit
growth, progression, and/or metastasis of cancers, in particular
those listed above.
[1446] Additional diseases or conditions associated with increased
cell survival that could be diagnosed, prognosed, prevented, and/or
treated by polynucleotides, polypeptides, and/or agonists or
antagonists of the invention, include, but are not limited to,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[1447] Diseases associated with increased apoptosis that could be
diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or agonists or antagonists of the invention,
include AIDS; neurodegenerative disorders (such as Alzheimer's
disease, Parkinson's disease, amyotrophic lateral sclerosis,
retinitis pigmentosa, cerebellar degeneration and brain tumor or
prior associated disease); autoimmune disorders (such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[1448] Hyperproliferative diseases and/or disorders that could be
diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or agonists or antagonists of the invention,
include, but are not limited to, neoplasms located in the liver,
abdomen, bone, breast, digestive system, pancreas, peritoneum,
endocrine glands (adrenal, parathyroid, pituitary, testicles,
ovary, thymus, thyroid), eye, head and neck, nervous system
(central and peripheral), lymphatic system, pelvis, skin, soft
tissue, spleen, thorax, and urogenital tract.
[1449] Similarly, other hyperproliferative disorders can also be
diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or agonists or antagonists of the invention.
Examples of such hyperproliferative disorders include, but are not
limited to: hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis,
and any other hyperproliferative disease, besides neoplasia,
located in an organ system listed above.
[1450] Another preferred embodiment utilizes polynucleotides of the
present invention to inhibit aberrant cellular division, by gene
therapy using the present invention, and/or protein fusions or
fragments thereof.
[1451] Thus, the present invention provides a method for treating
cell proliferative disorders by inserting into an abnormally
proliferating cell a polynucleotide of the present invention,
wherein said polynucleotide represses said expression.
[1452] Another embodiment of the present invention provides a
method of treating cell-proliferative disorders in individuals
comprising administration of one or more active gene copies of the
present invention to an abnormally proliferating cell or cells. In
a preferred embodiment, polynucleotides of the present invention is
a DNA construct comprising a recombinant expression vector
effective in expressing a DNA sequence encoding said
polynucleotides. In another preferred embodiment of the present
invention, the DNA construct encoding the polynucleotides of the
present invention is inserted into cells to be treated utilizing a
retrovirus, or more preferably an adenoviral vector (See G J.
Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated
by reference). In a most preferred embodiment, the viral vector is
defective and will not transform non-proliferating cells, only
proliferating cells. Moreover, in a preferred embodiment, the
polynucleotides of the present invention inserted into
proliferating cells either alone, or in combination with or fused
to other polynucleotides, can then be modulated via an external
stimulus (i.e. magnetic, specific small molecule, chemical, or drug
administration, etc.), which acts upon the promoter upstream of
said polynucleotides to induce expression of the encoded protein
product. As such the beneficial therapeutic affect of the present
invention may be expressly modulated (i.e. to increase, decrease,
or inhibit expression of the present invention) based upon said
external stimulus.
[1453] Polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes" is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[1454] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, LIPOFECTIN.TM., or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[1455] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. The polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[1456] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[1457] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[1458] The present invention is further directed to antibody-based
therapies which involve administering of anti-polypeptides and
anti-polynucleotide antibodies to a mammalian, preferably human,
patient for treating one or more of the described disorders.
Methods for producing anti-polypeptides and anti-polynucleotide
antibodies polyclonal and monoclonal antibodies are described in
detail elsewhere herein. Such antibodies may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[1459] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[1460] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating a subject having or
developing cell proliferative and/or differentiation disorders as
described herein. Such treatment comprises administering a single
or multiple doses of the antibody, or a fragment, derivative, or a
conjugate thereof.
[1461] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors,
for example., which serve to increase the number or activity of
effector cells which interact with the antibodies.
[1462] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides, including fragments thereof. Preferred binding
affinities include those with a dissociation constant or Kd less
than 5.times.10.sup.-6M, 10.sup.-6M, 5.times.10.sup.-7M,
10.sup.-7M, 5.times.10.sup.-8M, 10.sup.-8M, 5.times.10.sup.-9M,
10.sup.-9M, 5.times.10.sup.-10M, 10.sup.-10M, 5.times.10.sup.-11M,
10.sup.-11M, 5.times.10.sup.-12M, 10.sup.-12M, 5.times.10.sup.-13M,
10.sup.-13M, 5.times.10.sup.-14M, 10.sup.-14M, 5.times.10.sup.-15M,
and 10.sup.-15M.
[1463] Moreover, polypeptides of the present invention are useful
in inhibiting the angiogenesis of proliferative cells or tissues,
either alone, as a protein fusion, or in combination with other
polypeptides directly or indirectly, as described elsewhere herein.
In a most preferred embodiment, said anti-angiogenesis effect may
be achieved indirectly, for example, through the inhibition of
hematopoietic, tumor-specific cells, such as tumor-associated
macrophages (See Joseph I B, et al. J Natl Cancer Inst,
90(21):1648-53 (1998), which is hereby incorporated by reference).
Antibodies directed to polypeptides or polynucleotides of the
present invention may also result in inhibition of angiogenesis
directly, or indirectly (See Witte L, et al., Cancer Metastasis
Rev. 17(2):155-61 (1998), which is hereby incorporated by
reference)).
[1464] Polypeptides, including protein fusions, of the present
invention, or fragments thereof may be useful in inhibiting
proliferative cells or tissues through the induction of apoptosis.
Said polypeptides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as tumor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998),
which is hereby incorporated by reference). Moreover, in another
preferred embodiment of the present invention, said polypeptides
may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or
through stimulating the expression of said proteins, either alone
or in combination with small molecule drugs or adjuvants, such as
apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See
for example, Mutat Res 400(1-2):447-55 (1998), Med. Hypotheses.
50(5):423-33 (1998), Chem Biol Interact. Apr. 24; 111-112:23-34
(1998), J Mol. Med. 76(6):402-12 (1998), Int J Tissue React;
20(1):3-15 (1998), which are all hereby incorporated by
reference).
[1465] Polypeptides, including protein fusions to, or fragments
thereof, of the present invention are useful in inhibiting the
metastasis of proliferative cells or tissues Inhibition may occur
as a direct result of administering polypeptides, or antibodies
directed to said polypeptides as described elsewhere herein, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr
Top Microbiol Immunol 1998; 231:125-41, which is hereby
incorporated by reference). Such therapeutic affects of the present
invention may be achieved either alone, or in combination with
small molecule drugs or adjuvants.
[1466] In another embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing polypeptides or
polypeptide antibodies associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs) to targeted cells
expressing the polypeptide of the present invention. Polypeptides
or polypeptide antibodies of the invention may be associated with
heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent
interactions.
[1467] Polypeptides, protein fusions to, or fragments thereof, of
the present invention are useful in enhancing the immunogenicity
and/or antigenicity of proliferating cells or tissues, either
directly, such as would occur if the polypeptides of the present
invention `vaccinated` the immune response to respond to
proliferative antigens and immunogens, or indirectly, such as in
activating the expression of proteins known to enhance the immune
response (e.g. chemokines), to said antigens and immunogens.
[1468] Renal Disorders
[1469] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention, may be used to treat,
prevent, diagnose, and/or prognose disorders of the renal system.
Renal disorders which can be diagnosed, prognosed, prevented,
and/or treated with compositions of the invention include, but are
not limited to, kidney failure, nephritis, blood vessel disorders
of kidney, metabolic and congenital kidney disorders, urinary
disorders of the kidney, autoimmune disorders, sclerosis and
necrosis, electrolyte imbalance, and kidney cancers.
[1470] Kidney diseases which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention
include, but are not limited to, acute kidney failure, chronic
kidney failure, atheroembolic renal failure, end-stage renal
disease, inflammatory diseases of the kidney (e.g., acute
glomerulonephritis, postinfectious glomerulonephritis, rapidly
progressive glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis, familial nephrotic syndrome,
membranoproliferative glomerulonephritis I and II, mesangial
proliferative glomerulonephritis, chronic glomerulonephritis, acute
tubulointerstitial nephritis, chronic tubulointerstitial nephritis,
acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis,
lupus nephritis, chronic nephritis, interstitial nephritis, and
post-streptococcal glomerulonephritis), blood vessel disorders of
the kidneys (e.g., kidney infarction, atheroembolic kidney disease,
cortical necrosis, malignant nephrosclerosis, renal vein
thrombosis, renal underperfusion, renal retinopathy, renal
ischemia-reperfusion, renal artery embolism, and renal artery
stenosis), and kidney disorders resulting form urinary tract
disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal
lithiasis, nephrolithiasis), reflux nephropathy, urinary tract
infections, urinary retention, and acute or chronic unilateral
obstructive uropathy.)
[1471] In addition, compositions of the invention can be used to
diagnose, prognose, prevent, and/or treat metabolic and congenital
disorders of the kidney (e.g., uremia, renal amyloidosis, renal
osteodystrophy, renal tubular acidosis, renal glycosuria,
nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome,
renal fibrocystic osteosis (renal rickets), Hartnup disease,
Bartter's syndrome, Liddle's syndrome, polycystic kidney disease,
medullary cystic disease, medullary sponge kidney, Alport's
syndrome, nail-patella syndrome, congenital nephrotic syndrome,
CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic
diabetes insipidus, analgesic nephropathy, kidney stones, and
membranous nephropathy), and autoimmune disorders of the kidney
(e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome,
IgA nephropathy, and IgM mesangial proliferative
glomerulonephritis).
[1472] Compositions of the invention can also be used to diagnose,
prognose, prevent, and/or treat sclerotic or necrotic disorders of
the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal
segmental glomerulosclerosis (FSGS), necrotizing
glomerulonephritis, and renal papillary necrosis), cancers of the
kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell
cancer, transitional cell cancer, renal adenocarcinoma, squamous
cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g.,
nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,
hyponatremia, hypernatremia, hypokalemia, hyperkalemia,
hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
[1473] Polypeptides may be administered using any method known in
the art, including, but not limited to, direct needle injection at
the delivery site, intravenous injection, topical administration,
catheter infusion, BIOLISTIC.TM. injectors, particle accelerators,
gelfoam sponge depots, other commercially available depot
materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. Polypeptides may be administered as part of a Therapeutic,
described in more detail below. Methods of delivering
polynucleotides are described in more detail herein.
[1474] Cardiovascular Disorders
[1475] Polynucleotides or polypeptides, or agonists or antagonists
of the present invention, may be used to treat, prevent, diagnose,
and/or prognose cardiovascular disorders, including, but not
limited to, peripheral artery disease, such as limb ischemia.
[1476] Cardiovascular disorders include, but are not limited to,
cardiovascular abnormalities, such as arterio-arterial fistula,
arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart defects include, but are not limited to, aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross
heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of fallot, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[1477] Cardiovascular disorders also include, but are not limited
to, heart disease, such as arrhythmias, carcinoid heart disease,
high cardiac output, low cardiac output, cardiac tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest,
congestive heart failure, congestive cardiomyopathy, paroxysmal
dyspnea, cardiac edema, heart hypertrophy, congestive
cardiomyopathy, left ventricular hypertrophy, right ventricular
hypertrophy, post-infarction heart rupture, ventricular septal
rupture, heart valve diseases, myocardial diseases, myocardial
ischemia, pericardial effusion, pericarditis (including
constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic
heart disease, ventricular dysfunction, hyperemia, cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular
syphilis, and cardiovascular tuberculosis.
[1478] Arrhythmias include, but are not limited to, sinus
arrhythmia, atrial fibrillation, atrial flutter, bradycardia,
extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine
Syndrome, Mahaim-type pre-excitation syndrome,
Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and ventricular fibrillation. Tachycardias include paroxysmal
tachycardia, supraventricular tachycardia, accelerated
idioventricular rhythm, atrioventricular nodal reentry tachycardia,
ectopic atrial tachycardia, ectopic junctional tachycardia,
sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de Pointes, and ventricular tachycardia.
[1479] Heart valve diseases include, but are not limited to, aortic
valve insufficiency, aortic valve stenosis, hear murmurs, aortic
valve prolapse, mitral valve prolapse, tricuspid valve prolapse,
mitral valve insufficiency, mitral valve stenosis, pulmonary
atresia, pulmonary valve insufficiency, pulmonary valve stenosis,
tricuspid atresia, tricuspid valve insufficiency, and tricuspid
valve stenosis.
[1480] Myocardial diseases include, but are not limited to,
alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic
cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular
stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns
Syndrome, myocardial reperfusion injury, and myocarditis.
[1481] Myocardial ischemias include, but are not limited to,
coronary disease, such as angina pectoris, coronary aneurysm,
coronary arteriosclerosis, coronary thrombosis, coronary vasospasm,
myocardial infarction and myocardial stunning.
[1482] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, and venous insufficiency.
[1483] Aneurysms include, but are not limited to, dissecting
aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms,
aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart
aneurysms, and iliac aneurysms.
[1484] Arterial occlusive diseases include, but are not limited to,
arteriosclerosis, intermittent claudication, carotid stenosis,
fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis obliterans.
[1485] Cerebrovascular disorders include, but are not limited to,
carotid artery diseases, cerebral amyloid angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous malformation, cerebral artery diseases, cerebral
embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[1486] Embolisms include, but are not limited to, air embolisms,
amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome,
fat embolisms, pulmonary embolisms, and thromoboembolisms.
Thrombosis include, but are not limited to, coronary thrombosis,
hepatic vein thrombosis, retinal vein occlusion, carotid artery
thrombosis, sinus thrombosis, Wallenberg's syndrome, and
thrombophlebitis.
[1487] Ischemic disorders include, but are not limited to, cerebral
ischemia, ischemic colitis, compartment syndromes, anterior
compartment syndrome, myocardial ischemia, reperfusion injuries,
and peripheral limb ischemia. Vasculitis includes, but is not
limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis
obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
[1488] Polypeptides may be administered using any method known in
the art, including, but not limited to, direct needle injection at
the delivery site, intravenous injection, topical administration,
catheter infusion, BIOLISTIC.TM. injectors, particle accelerators,
gelfoam sponge depots, other commercially available depot
materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. Polypeptides may be administered as part of a Therapeutic,
described in more detail below. Methods of delivering
polynucleotides are described in more detail herein.
[1489] Respiratory Disorders
[1490] Polynucleotides or polypeptides, or agonists or antagonists
of the present invention may be used to treat, prevent, diagnose,
and/or prognose diseases and/or disorders of the respiratory
system.
[1491] Diseases and disorders of the respiratory system include,
but are not limited to, nasal vestibulitis, nonallergic rhinitis
(e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis,
vasomotor rhinitis), nasal polyps, and sinusitis, juvenile
angiofibromas, cancer of the nose and juvenile papillomas, vocal
cord polyps, nodules (singer's nodules), contact ulcers, vocal cord
paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial),
tonsillitis, tonsillar cellulitis, parapharyngeal abscess,
laryngitis, laryngoceles, and throat cancers (e.g., cancer of the
nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,
squamous cell carcinoma, small cell (oat cell) carcinoma, large
cell carcinoma, and adenocarcinoma), allergic disorders
(eosinophilic pneumonia, hypersensitivity pneumonitis (e.g.,
extrinsic allergic alveolitis, allergic interstitial pneumonitis,
organic dust pneumoconiosis, allergic bronchopulmonary
aspergillosis, asthma, Wegener's granulomatosis (granulomatous
vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial
pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal
pneumonia), Staphylococcus aureus (staphylococcal pneumonia),
Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and
Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus
influenzae pneumonia, Legionella pneumophila (Legionnaires'
disease), and Chlamydia psittaci (Psittacosis)), and viral
pneumonia (e.g., influenza, chickenpox (varicella). Additional
diseases and disorders of the respiratory system include, but are
not limited to bronchiolitis, polio (poliomyelitis), croup,
respiratory syncytial viral infection, mumps, erythema infectiosum
(fifth disease), roseola infantum, progressive rubella
panencephalitis, german measles, and subacute sclerosing
panencephalitis), fungal pneumonia (e.g., Histoplasmosis,
Coccidioidomycosis, Blastomycosis, fungal infections in people with
severely suppressed immune systems (e.g., cryptococcosis, caused by
Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.;
candidiasis, caused by Candida; and mucormycosis)), Pneumocystis
carinii (pneumocystis pneumonia), atypical pneumonias (e.g.,
Mycoplasma and Chlamydia spp.), opportunistic infection pneumonia,
nosocomial pneumonia, chemical pneumonitis, and aspiration
pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and
pneumothorax (e.g., simple spontaneous pneumothorax, complicated
spontaneous pneumothorax, tension pneumothorax)), obstructive
airway diseases (e.g., asthma, chronic obstructive pulmonary
disease (COPD), emphysema, chronic or acute bronchitis),
occupational lung diseases (e.g., silicosis, black lung (coal
workers' pneumoconiosis), asbestosis, berylliosis, occupational
asthma, byssinosis, and benign pneumoconioses), Infiltrative Lung
Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis,
usual interstitial pneumonia), idiopathic pulmonary fibrosis,
desquamative interstitial pneumonia, lymphoid interstitial
pneumonia, histiocytosis X (e.g., Letterer-Siwe disease,
Hand-Schuller-Christian disease, eosinophilic granuloma),
idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary
alveolar proteinosis), Acute respiratory distress syndrome (also
called, e.g., adult respiratory distress syndrome), edema,
pulmonary embolism, bronchitis (e.g., viral, bacterial),
bronchiectasis, atelectasis, lung abscess (caused by, e.g.,
Staphylococcus aureus or Legionella pneumophila), and cystic
fibrosis.
[1492] Anti-Angiogenesis Activity
[1493] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinej ad et al., Cell
56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulatory controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al.,
Biotech. 9:630-634 (1991); Folkman et al., N Engl. J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein
and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science
221:719-725 (1983). In a number of pathological conditions, the
process of angiogenesis contributes to the disease state. For
example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis. Folkman and
Klagsbrun, Science 235:442-447 (1987).
[1494] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
the polynucleotides and/or polypeptides of the invention, as well
as agonists or antagonists of the present invention. Malignant and
metastatic conditions which can be treated with the polynucleotides
and polypeptides, or agonists or antagonists of the invention
include, but are not limited to, malignancies, solid tumors, and
cancers described herein and otherwise known in the art (for a
review of such disorders, see Fishman et al., Medicine, 2d Ed., J.
B. Lippincott Co., Philadelphia (1985)). Thus, the present
invention provides a method of treating an angiogenesis-related
disease and/or disorder, comprising administering to an individual
in need thereof a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist of the
invention. For example, polynucleotides, polypeptides, antagonists
and/or agonists may be utilized in a variety of additional methods
in order to therapeutically treat a cancer or tumor. Cancers which
may be treated with polynucleotides, polypeptides, antagonists
and/or agonists include, but are not limited to solid tumors,
including prostate, lung, breast, ovarian, stomach, pancreas,
larynx, esophagus, testes, liver, parotid, biliary tract, colon,
rectum, cervix, uterus, endometrium, kidney, bladder, thyroid
cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer;
colorectal cancer; advanced malignancies; and blood born tumors
such as leukemias. For example, polynucleotides, polypeptides,
antagonists and/or agonists may be delivered topically, in order to
treat cancers such as skin cancer, head and neck tumors, breast
tumors, and Kaposi's sarcoma.
[1495] Within yet other aspects, polynucleotides, polypeptides,
antagonists and/or agonists may be utilized to treat superficial
forms of bladder cancer by, for example, intravesical
administration. Polynucleotides, polypeptides, antagonists and/or
agonists may be delivered directly into the tumor, or near the
tumor site, via injection or a catheter. Of course, as the artisan
of ordinary skill will appreciate, the appropriate mode of
administration will vary according to the cancer to be treated.
Other modes of delivery are discussed herein.
[1496] Polynucleotides, polypeptides, antagonists and/or agonists
may be useful in treating other disorders, besides cancers, which
involve angiogenesis. These disorders include, but are not limited
to: benign tumors, for example hemangiomas, acoustic neuromas,
neurofibromas, trachomas, and pyogenic granulomas; artheroscleric
plaques; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis;
psoriasis; delayed wound healing; endometriosis; vasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures;
scleroderma; trachoma; vascular adhesions; myocardial angiogenesis;
coronary collaterals; cerebral collaterals; arteriovenous
malformations; ischemic limb angiogenesis; Osler-Webber Syndrome;
plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's
disease; and atherosclerosis.
[1497] For example, within one aspect of the present invention
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering a polynucleotide, polypeptide,
antagonist and/or agonist of the invention to a hypertrophic scar
or keloid.
[1498] Within one embodiment of the present invention
polynucleotides, polypeptides, antagonists and/or agonists of the
invention are directly injected into a hypertrophic scar or keloid,
in order to prevent the progression of these lesions. This therapy
is of particular value in the prophylactic treatment of conditions
which are known to result in the development of hypertrophic scars
and keloids (e.g., burns), and is preferably initiated after the
proliferative phase has had time to progress (approximately 14 days
after the initial injury), but before hypertrophic scar or keloid
development. As noted above, the present invention also provides
methods for treating neovascular diseases of the eye, including for
example, corneal neovascularization, neovascular glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and
macular degeneration.
[1499] Moreover, Ocular disorders associated with
neovascularization which can be treated with the polynucleotides
and polypeptides of the present invention (including agonists
and/or antagonists) include, but are not limited to: neovascular
glaucoma, diabetic retinopathy, retinoblastoma, retrolental
fibroplasia, uveitis, retinopathy of prematurity macular
degeneration, corneal graft neovascularization, as well as other
eye inflammatory diseases, ocular tumors and diseases associated
with choroidal or iris neovascularization. See, e.g., reviews by
Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et
al., Surv. Ophthal. 22:291-312 (1978).
[1500] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a compound (as
described above) to the cornea, such that the formation of blood
vessels is inhibited. Briefly, the cornea is a tissue which
normally lacks blood vessels. In certain pathological conditions
however, capillaries may extend into the cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also becomes clouded, resulting in a decline in
the patient's visual acuity. Visual loss may become complete if the
cornea completely opacitates. A wide variety of disorders can
result in corneal neovascularization, including for example,
corneal infections (e.g., trachoma, herpes simplex keratitis,
leishmaniasis and onchocerciasis), immunological processes (e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns,
trauma, inflammation (of any cause), toxic and nutritional
deficiency states, and as a complication of wearing contact
lenses.
[1501] Within particularly preferred embodiments of the invention,
may be prepared for topical administration in saline (combined with
any of the preservatives and antimicrobial agents commonly used in
ocular preparations), and administered in eyedrop form. The
solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic
compositions, prepared as described above, may also be administered
directly to the cornea. Within preferred embodiments, the
anti-angiogenic composition is prepared with a muco-adhesive
polymer which binds to cornea. Within further embodiments, the
anti-angiogenic factors or anti-angiogenic compositions may be
utilized as an adjunct to conventional steroid therapy. Topical
therapy may also be useful prophylactically in corneal lesions
which are known to have a high probability of inducing an
angiogenic response (such as chemical burns). In these instances
the treatment, likely in combination with steroids, may be
instituted immediately to help prevent subsequent
complications.
[1502] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself.
[1503] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist to the eye,
such that the formation of blood vessels is inhibited. In one
embodiment, the compound may be administered topically to the eye
in order to treat early forms of neovascular glaucoma. Within other
embodiments, the compound may be implanted by injection into the
region of the anterior chamber angle. Within other embodiments, the
compound may also be placed in any location such that the compound
is continuously released into the aqueous humor. Within another
aspect of the present invention, methods are provided for treating
proliferative diabetic retinopathy, comprising the step of
administering to a patient a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist to the eyes,
such that the formation of blood vessels is inhibited.
[1504] Within particularly preferred embodiments of the invention,
proliferative diabetic retinopathy may be treated by injection into
the aqueous humor or the vitreous, in order to increase the local
concentration of the polynucleotide, polypeptide, antagonist and/or
agonist in the retina. Preferably, this treatment should be
initiated prior to the acquisition of severe disease requiring
photocoagulation.
[1505] Within another aspect of the present invention, methods are
provided for treating retrolental fibroplasia, comprising the step
of administering to a patient a therapeutically effective amount of
a polynucleotide, polypeptide, antagonist and/or agonist to the
eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous injection
and/or via intraocular implants.
[1506] Additionally, disorders which can be treated with the
polynucleotides, polypeptides, agonists and/or agonists include,
but are not limited to, hemangioma, arthritis, psoriasis,
angiofibroma, atherosclerotic plaques, delayed wound healing,
granulations, hemophilic joints, hypertrophic scars, nonunion
fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma,
trachoma, and vascular adhesions.
[1507] Moreover, disorders and/or states, which can be treated,
prevented, diagnosed, and/or prognosed with the polynucleotides,
polypeptides, agonists and/or agonists of the invention include,
but are not limited to, solid tumors, blood born tumors such as
leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for
example hemangiomas, acoustic neuromas, neurofibromas, trachomas,
and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and uvietis, delayed wound healing, endometriosis,
vascluogenesis, granulations, hypertrophic scars (keloids),
nonunion fractures, scleroderma, trachoma, vascular adhesions,
myocardial angiogenesis, coronary collaterals, cerebral
collaterals, arteriovenous malformations, ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia, hemophiliac joints, angiofibroma fibromuscular
dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control agent by preventing vascularization required for
embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary angiomatosis.
[1508] In one aspect of the birth control method, an amount of the
compound sufficient to block embryo implantation is administered
before or after intercourse and fertilization have occurred, thus
providing an effective method of birth control, possibly a "morning
after" method. Polynucleotides, polypeptides, agonists and/or
agonists may also be used in controlling menstruation or
administered as either a peritoneal lavage fluid or for peritoneal
implantation in the treatment of endometriosis.
[1509] Polynucleotides, polypeptides, agonists and/or agonists of
the present invention may be incorporated into surgical sutures in
order to prevent stitch granulomas.
[1510] Polynucleotides, polypeptides, agonists and/or agonists may
be utilized in a wide variety of surgical procedures. For example,
within one aspect of the present invention a compositions (in the
form of, for example, a spray or film) may be utilized to coat or
spray an area prior to removal of a tumor, in order to isolate
normal surrounding tissues from malignant tissue, and/or to prevent
the spread of disease to surrounding tissues. Within other aspects
of the present invention, compositions (e.g., in the form of a
spray) may be delivered via endoscopic procedures in order to coat
tumors, or inhibit angiogenesis in a desired locale. Within yet
other aspects of the present invention, surgical meshes which have
been coated with anti-angiogenic compositions of the present
invention may be utilized in any procedure wherein a surgical mesh
might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition
may be utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[1511] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering a polynucleotide, polypeptide, agonist and/or agonist
to the resection margins of a tumor subsequent to excision, such
that the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within one embodiment of the
invention, the anti-angiogenic compound is administered directly to
the tumor excision site (e.g., applied by swabbing, brushing or
otherwise coating the resection margins of the tumor with the
anti-angiogenic compound). Alternatively, the anti-angiogenic
compounds may be incorporated into known surgical pastes prior to
administration. Within particularly preferred embodiments of the
invention, the anti-angiogenic compounds are applied after hepatic
resections for malignancy, and after neurosurgical operations.
[1512] Within one aspect of the present invention, polynucleotides,
polypeptides, agonists and/or agonists may be administered to the
resection margin of a wide variety of tumors, including for
example, breast, colon, brain and hepatic tumors. For example,
within one embodiment of the invention, anti-angiogenic compounds
may be administered to the site of a neurological tumor subsequent
to excision, such that the formation of new blood vessels at the
site are inhibited.
[1513] The polynucleotides, polypeptides, agonists and/or agonists
of the present invention may also be administered along with other
anti-angiogenic factors. Representative examples of other
anti-angiogenic factors include: Anti-Invasive Factor, retinoic
acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor
of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator
Inhibitor-2, and various forms of the lighter "d group" transition
metals.
[1514] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[1515] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[1516] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[1517] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include platelet factor 4; protamine
sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated
Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this
compound may be enhanced by the presence of steroids such as
estrogen, and tamoxifen citrate); Staurosporine; modulators of
matrix metabolism, including for example, proline analogs,
cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,
alpha,alpha-dipyridyl, aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;
Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
[1518] Diseases at the Cellular Level
[1519] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated, prevented,
diagnosed, and/or prognosed using polynucleotides or polypeptides,
as well as antagonists or agonists of the present invention,
include cancers (such as follicular lymphomas, carcinomas with p53
mutations, and hormone-dependent tumors, including, but not limited
to colon cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection.
[1520] In preferred embodiments, polynucleotides, polypeptides,
and/or antagonists of the invention are used to inhibit growth,
progression, and/or metasis of cancers, in particular those listed
above.
[1521] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by polynucleotides
or polypeptides, or agonists or antagonists of the present
invention include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[1522] Diseases associated with increased apoptosis that could be
treated, prevented, diagnosed, and/or prognosed using
polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, include, but are not limited to, AIDS;
neurodegenerative disorders (such as Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis
pigmentosa, Cerebellar degeneration and brain tumor or prior
associated disease); autoimmune disorders (such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[1523] Wound Healing and Epithelial Cell Proliferation
[1524] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing
polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, for therapeutic purposes, for example, to
stimulate epithelial cell proliferation and basal keratinocytes for
the purpose of wound healing, and to stimulate hair follicle
production and healing of dermal wounds. Polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention, may be clinically useful in stimulating wound healing
including surgical wounds, excisional wounds, deep wounds involving
damage of the dermis and epidermis, eye tissue wounds, dental
tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,
cubitus ulcers, arterial ulcers, venous stasis ulcers, burns
resulting from heat exposure or chemicals, and other abnormal wound
healing conditions such as uremia, malnutrition, vitamin
deficiencies and complications associated with systemic treatment
with steroids, radiation therapy and antineoplastic drugs and
antimetabolites. Polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, could be used to
promote dermal reestablishment subsequent to dermal loss
[1525] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, could be used to increase the
adherence of skin grafts to a wound bed and to stimulate
re-epithelialization from the wound bed. The following are types of
grafts that polynucleotides or polypeptides, agonists or
antagonists of the present invention, could be used to increase
adherence to a wound bed: autografts, artificial skin, allografts,
autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown
grafts, bone graft, brephoplastic grafts, cutis graft, delayed
graft, dermic graft, epidermic graft, fascia graft, full thickness
graft, heterologous graft, xenograft, homologous graft,
hyperplastic graft, lamellar graft, mesh graft, mucosal graft,
Oller-Thiersch graft, omenpal graft, patch graft, pedicle graft,
penetrating graft, split skin graft, thick split graft.
Polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, can be used to promote skin strength and
to improve the appearance of aged skin.
[1526] It is believed that polynucleotides or polypeptides, as well
as agonists or antagonists of the present invention, will also
produce changes in hepatocyte proliferation, and epithelial cell
proliferation in the lung, breast, pancreas, stomach, small
intestine, and large intestine. Polynucleotides or polypeptides, as
well as agonists or antagonists of the present invention, could
promote proliferation of epithelial cells such as sebocytes, hair
follicles, hepatocytes, type II pneumocytes, mucin-producing goblet
cells, and other epithelial cells and their progenitors contained
within the skin, lung, liver, and gastrointestinal tract.
Polynucleotides or polypeptides, agonists or antagonists of the
present invention, may promote proliferation of endothelial cells,
keratinocytes, and basal keratinocytes.
[1527] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, could also be used to reduce
the side effects of gut toxicity that result from radiation,
chemotherapy treatments or viral infections. Polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention, may have a cytoprotective effect on the small intestine
mucosa. Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, may also stimulate healing of
mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
[1528] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, could further be used in full
regeneration of skin in full and partial thickness skin defects,
including burns, (i.e., repopulation of hair follicles, sweat
glands, and sebaceous glands), treatment of other skin defects such
as psoriasis. Polynucleotides or polypeptides, as well as agonists
or antagonists of the present invention, could be used to treat
epidermolysis bullosa, a defect in adherence of the epidermis to
the underlying dermis which results in frequent, open and painful
blisters by accelerating reepithelialization of these lesions.
Polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, could also be used to treat gastric and
doudenal ulcers and help heal by scar formation of the mucosal
lining and regeneration of glandular mucosa and duodenal mucosal
lining more rapidly. Inflammatory bowel diseases, such as Crohn's
disease and ulcerative colitis, are diseases which result in
destruction of the mucosal surface of the small or large intestine,
respectively. Thus, polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, could be used to
promote the resurfacing of the mucosal surface to aid more rapid
healing and to prevent progression of inflammatory bowel disease.
Treatment with polynucleotides or polypeptides, agonists or
antagonists of the present invention, is expected to have a
significant effect on the production of mucus throughout the
gastrointestinal tract and could be used to protect the intestinal
mucosa from injurious substances that are ingested or following
surgery. Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, could be used to treat
diseases associate with the under expression.
[1529] Moreover, polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, could be used to
prevent and heal damage to the lungs due to various pathological
states. Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, which could stimulate
proliferation and differentiation and promote the repair of alveoli
and brochiolar epithelium to prevent or treat acute or chronic lung
damage. For example, emphysema, which results in the progressive
loss of alveoli, and inhalation injuries, i.e., resulting from
smoke inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated using
polynucleotides or polypeptides, agonists or antagonists of the
present invention. Also, polynucleotides or polypeptides, as well
as agonists or antagonists of the present invention, could be used
to stimulate the proliferation of and differentiation of type II
pneumocytes, which may help treat or prevent disease such as
hyaline membrane diseases, such as infant respiratory distress
syndrome and bronchopulmonary displasia, in premature infants.
[1530] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, could stimulate the
proliferation and differentiation of hepatocytes and, thus, could
be used to alleviate or treat liver diseases and pathologies such
as fulminant liver failure caused by cirrhosis, liver damage caused
by viral hepatitis and toxic substances (i.e., acetaminophen,
carbon tetraholoride and other hepatotoxins known in the art).
[1531] In addition, polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, could be used
treat or prevent the onset of diabetes mellitus. In patients with
newly diagnosed Types I and II diabetes, where some islet cell
function remains, polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, could be used to
maintain the islet function so as to alleviate, delay or prevent
permanent manifestation of the disease. Also, polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention, could be used as an auxiliary in islet cell
transplantation to improve or promote islet cell function.
[1532] Neural Activity and Neurological Diseases
[1533] The polynucleotides, polypeptides and agonists or
antagonists of the invention may be used for the diagnosis and/or
treatment of diseases, disorders, damage or injury of the brain
and/or nervous system. Nervous system disorders that can be treated
with the compositions of the invention (e.g., polypeptides,
polynucleotides, and/or agonists or antagonists), include, but are
not limited to, nervous system injuries, and diseases or disorders
which result in either a disconnection of axons, a diminution or
degeneration of neurons, or demyelination. Nervous system lesions
which may be treated in a patient (including human and non-human
mammalian patients) according to the methods of the invention,
include but are not limited to, the following lesions of either the
central (including spinal cord, brain) or peripheral nervous
systems: (1) ischemic lesions, in which a lack of oxygen in a
portion of the nervous system results in neuronal injury or death,
including cerebral infarction or ischemia, or spinal cord
infarction or ischemia; (2) traumatic lesions, including lesions
caused by physical injury or associated with surgery, for example,
lesions which sever a portion of the nervous system, or compression
injuries; (3) malignant lesions, in which a portion of the nervous
system is destroyed or injured by malignant tissue which is either
a nervous system associated malignancy or a malignancy derived from
non-nervous system tissue; (4) infectious lesions, in which a
portion of the nervous system is destroyed or injured as a result
of infection, for example, by an abscess or associated with
infection by human immunodeficiency virus, herpes zoster, or herpes
simplex virus or with Lyme disease, tuberculosis, or syphilis; (5)
degenerative lesions, in which a portion of the nervous system is
destroyed or injured as a result of a degenerative process
including but not limited to, degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional diseases or disorders, in which a portion of the
nervous system is destroyed or injured by a nutritional disorder or
disorder of metabolism including, but not limited to, vitamin B12
deficiency, folic acid deficiency, Wernicke disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary
degeneration of the corpus callosum), and alcoholic cerebellar
degeneration; (7) neurological lesions associated with systemic
diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma,
or sarcoidosis; (8) lesions caused by toxic substances including
alcohol, lead, or particular neurotoxins; and (9) demyelinated
lesions in which a portion of the nervous system is destroyed or
injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated
myelopathy, transverse myelopathy or various etiologies,
progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[1534] In one embodiment, the polypeptides, polynucleotides, or
agonists or antagonists of the invention are used to protect neural
cells from the damaging effects of hypoxia. In a further preferred
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to protect neural cells from
the damaging effects of cerebral hypoxia. According to this
embodiment, the compositions of the invention are used to treat or
prevent neural cell injury associated with cerebral hypoxia. In one
non-exclusive aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention, are
used to treat or prevent neural cell injury associated with
cerebral ischemia. In another non-exclusive aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat or prevent neural
cell injury associated with cerebral infarction.
[1535] In another preferred embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat or prevent neural cell injury associated with a
stroke. In a specific embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat or prevent cerebral neural cell injury associated
with a stroke.
[1536] In another preferred embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat or prevent neural cell injury associated with a heart
attack. In a specific embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat or prevent cerebral neural cell injury associated
with a heart attack.
[1537] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture either in the
presence or absence of hypoxia or hypoxic conditions; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in
Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased
sprouting of neurons may be detected by methods known in the art,
such as, for example, the methods set forth in Pestronk et al.,
Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev.
Neurosci., 4:17-42 (1981); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[1538] In specific embodiments, motor neuron disorders that may be
treated according to the invention include, but are not limited to,
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including, but not limited to,
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[1539] Further, polypeptides or polynucleotides of the invention
may play a role in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Thus, compositions of the
invention (including polynucleotides, polypeptides, and agonists or
antagonists) may be used to diagnose and/or treat or prevent
diseases or disorders associated with these roles, including, but
not limited to, learning and/or cognition disorders. The
compositions of the invention may also be useful in the treatment
or prevention of neurodegenerative disease states and/or
behavioural disorders. Such neurodegenerative disease states and/or
behavioral disorders include, but are not limited to, Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease, Tourette
Syndrome, schizophrenia, mania, dementia, paranoia, obsessive
compulsive disorder, panic disorder, learning disabilities, ALS,
psychoses, autism, and altered behaviors, including disorders in
feeding, sleep patterns, balance, and perception. In addition,
compositions of the invention may also play a role in the
treatment, prevention and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders.
[1540] Additionally, polypeptides, polynucleotides and/or agonists
or antagonists of the invention, may be useful in protecting neural
cells from diseases, damage, disorders, or injury, associated with
cerebrovascular disorders including, but not limited to, carotid
artery diseases (e.g., carotid artery thrombosis, carotid stenosis,
or Moyamoya Disease), cerebral amyloid angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous malformations, cerebral artery diseases, cerebral
embolism and thrombosis (e.g., carotid artery thrombosis, sinus
thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g.,
epidural or subdural hematoma, or subarachnoid hemorrhage),
cerebral infarction, cerebral ischemia (e.g., transient cerebral
ischemia, Subclavian Steal Syndrome, or vertebrobasilar
insufficiency), vascular dementia (e.g., multi-infarct),
leukomalacia, periventricular, and vascular headache (e.g., cluster
headache or migraines).
[1541] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing
polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, for therapeutic purposes, for example, to
stimulate neurological cell proliferation and/or differentiation.
Therefore, polynucleotides, polypeptides, agonists and/or
antagonists of the invention may be used to treat and/or detect
neurologic diseases. Moreover, polynucleotides or polypeptides, or
agonists or antagonists of the invention, can be used as a marker
or detector of a particular nervous system disease or disorder.
[1542] Examples of neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include brain diseases, such
as metabolic brain diseases which includes phenylketonuria such as
maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate
dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain
edema, brain neoplasms such as cerebellar neoplasms which include
infratentorial neoplasms, cerebral ventricle neoplasms such as
choroid plexus neoplasms, hypothalamic neoplasms, supratentorial
neoplasms, canavan disease, cerebellar diseases such as cerebellar
ataxia which include spinocerebellar degeneration such as ataxia
telangiectasia, cerebellar dyssynergia, Friederich's Ataxia,
Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar
neoplasms such as infratentorial neoplasms, diffuse cerebral
sclerosis such as encephalitis periaxialis, globoid cell
leukodystrophy, metachromatic leukodystrophy and subacute
sclerosing panencephalitis.
[1543] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include cerebrovascular
disorders (such as carotid artery diseases which include carotid
artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral
amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral
arteriosclerosis, cerebral arteriovenous malformations, cerebral
artery diseases, cerebral embolism and thrombosis such as carotid
artery thrombosis, sinus thrombosis and Wallenberg's Syndrome,
cerebral hemorrhage such as epidural hematoma, subdural hematoma
and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia
such as transient cerebral ischemia, Subclavian Steal Syndrome and
vertebrobasilar insufficiency, vascular dementia such as
multi-infarct dementia, periventricular leukomalacia, vascular
headache such as cluster headache and migraine.
[1544] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include dementia such as AIDS
Dementia Complex, presenile dementia such as Alzheimer's Disease
and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's
Disease and progressive supranuclear palsy, vascular dementia such
as multi-infarct dementia, encephalitis which include encephalitis
periaxialis, viral encephalitis such as epidemic encephalitis,
Japanese Encephalitis, St. Louis Encephalitis, tick-borne
encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meningoencephalitis such as
uveomeningoencephalitic syndrome, Postencephalitic Parkinson
Disease and subacute sclerosing panencephalitis, encephalomalacia
such as periventricular leukomalacia, epilepsy such as generalized
epilepsy which includes infantile spasms, absence epilepsy,
myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic
epilepsy, partial epilepsy such as complex partial epilepsy,
frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua,
and Hallervorden-Spatz Syndrome.
[1545] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include hydrocephalus such as
Dandy-Walker Syndrome and normal pressure hydrocephalus,
hypothalamic diseases such as hypothalamic neoplasms, cerebral
malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis,
cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic
diseases, cerebral toxoplasmosis, intracranial tuberculoma and
Zellweger Syndrome, central nervous system infections such as AIDS
Dementia Complex, Brain Abscess, subdural empyema,
encephalomyelitis such as Equine Encephalomyelitis, Venezuelan
Equine Encephalomyelitis, Necrotizing Hemorrhagic
Encephalomyelitis, Visna, and cerebral malaria.
[1546] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include meningitis such as
arachnoiditis, aseptic meningtitis such as viral meningtitis which
includes lymphocytic choriomeningitis, Bacterial meningtitis which
includes Haemophilus Meningtitis, Listeria Meningtitis,
Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome,
Pneumococcal Meningtitis and meningeal tuberculosis, fungal
meningitis such as Cryptococcal Meningtitis, subdural effusion,
meningoencephalitis such as uvemeningoencephalitic syndrome,
myelitis such as transverse myelitis, neurosyphilis such as tabes
dorsalis, poliomyelitis which includes bulbar poliomyelitis and
postpoliomyelitis syndrome, prion diseases (such as
Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy,
Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral
toxoplasmosis.
[1547] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include central nervous system
neoplasms such as brain neoplasms that include cerebellar neoplasms
such as infratentorial neoplasms, cerebral ventricle neoplasms such
as choroid plexus neoplasms, hypothalamic neoplasms and
supratentorial neoplasms, meningeal neoplasms, spinal cord
neoplasms which include epidural neoplasms, demyelinating diseases
such as Canavan Diseases, diffuse cerebral sceloris which includes
adrenoleukodystrophy, encephalitis periaxialis, globoid cell
leukodystrophy, diffuse cerebral sclerosis such as metachromatic
leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic
encephalomyelitis, progressive multifocal leukoencephalopathy,
multiple sclerosis, central pontine myelinolysis, transverse
myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue
Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal
cord diseases such as amyotonia congenita, amyotrophic lateral
sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann
Disease, spinal cord compression, spinal cord neoplasms such as
epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man
Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat
Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such
as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease,
Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome,
Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such
as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal
syndrome, phenylketonuria such as maternal phenylketonuria,
Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome,
Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities
such as holoprosencephaly, neural tube defects such as anencephaly
which includes hydrangencephaly, Arnold-Chain Deformity,
encephalocele, meningocele, meningomyelocele, spinal dysraphism
such as spina bifida cystica and spina bifida occulta.
[1548] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include hereditary motor and
sensory neuropathies which include Charcot-Marie Disease,
Hereditary optic atrophy, Refsum's Disease, hereditary spastic
paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and
Autonomic Neuropathies such as Congenital Analgesia and Familial
Dysautonomia, Neurologic manifestations (such as agnosia that
include Gerstmann's Syndrome, Amnesia such as retrograde amnesia,
apraxia, neurogenic bladder, cataplexy, communicative disorders
such as hearing disorders that includes deafness, partial hearing
loss, loudness recruitment and tinnitus, language disorders such as
aphasia which include agraphia, anomia, broca aphasia, and Wernicke
Aphasia, Dyslexia such as Acquired Dyslexia, language development
disorders, speech disorders such as aphasia which includes anomia,
broca aphasia and Wernicke Aphasia, articulation disorders,
communicative disorders such as speech disorders which include
dysarthria, echolalia, mutism and stuttering, voice disorders such
as aphonia and hoarseness, decerebrate state, delirium,
fasciculation, hallucinations, meningism, movement disorders such
as angelman syndrome, ataxia, athetosis, chorea, dystonia,
hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and
tremor, muscle hypertonia such as muscle rigidity such as stiff-man
syndrome, muscle spasticity, paralysis such as facial paralysis
which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia,
ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's
Syndrome, Chronic progressive external ophthalmoplegia such as
Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis,
Paraplegia such as Brown-Sequard Syndrome, quadriplegia,
respiratory paralysis and vocal cord paralysis, paresis, phantom
limb, taste disorders such as ageusia and dysgeusia, vision
disorders such as amblyopia, blindness, color vision defects,
diplopia, hemianopsia, scotoma and subnormal vision, sleep
disorders such as hypersomnia which includes Kleine-Levin Syndrome,
insomnia, and somnambulism, spasm such as trismus, unconsciousness
such as coma, persistent vegetative state and syncope and vertigo,
neuromuscular diseases such as amyotonia congenita, amyotrophic
lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron
disease, muscular atrophy such as spinal muscular atrophy,
Charcot-Marie Disease and Werdnig-Hoffmann Disease,
Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis,
Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial
Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic
Paraparesis and Stiff-Man Syndrome, peripheral nervous system
diseases such as acrodynia, amyloid neuropathies, autonomic nervous
system diseases such as Adie's Syndrome, Barre-Lieou Syndrome,
Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic
Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as
Acoustic Nerve Diseases such as Acoustic Neuroma which includes
Neurofibromatosis 2, Facial Nerve Diseases such as Facial
Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders
which includes amblyopia, nystagmus, oculomotor nerve paralysis,
ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome,
Chronic Progressive External Ophthalmoplegia which includes Kearns
Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor
Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which
includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic
Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal
Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as
Neuromyelitis Optica and Swayback, and Diabetic neuropathies such
as diabetic foot.
[1549] Additional neurologic diseases which can be treated or
detected with polynucleotides, polypeptides, agonists, and/or
antagonists of the present invention include nerve compression
syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome,
thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve
compression syndrome, neuralgia such as causalgia, cervico-brachial
neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such
as experimental allergic neuritis, optic neuritis, polyneuritis,
polyradiculoneuritis and radiculities such as polyradiculitis,
hereditary motor and sensory neuropathies such as Charcot-Marie
Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary
Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory
and Autonomic Neuropathies which include Congenital Analgesia and
Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating
and Tetany).
[1550] Endocrine Disorders
[1551] Polynucleotides or polypeptides, or agonists or antagonists
of the present invention, may be used to treat, prevent, diagnose,
and/or prognose disorders and/or diseases related to hormone
imbalance, and/or disorders or diseases of the endocrine
system.
[1552] Hormones secreted by the glands of the endocrine system
control physical growth, sexual function, metabolism, and other
functions. Disorders may be classified in two ways: disturbances in
the production of hormones, and the inability of tissues to respond
to hormones. The etiology of these hormone imbalance or endocrine
system diseases, disorders or conditions may be genetic, somatic,
such as cancer and some autoimmune diseases, acquired (e.g., by
chemotherapy, injury or toxins), or infectious. Moreover,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention can be used as a marker or
detector of a particular disease or disorder related to the
endocrine system and/or hormone imbalance.
[1553] Endocrine system and/or hormone imbalance and/or diseases
encompass disorders of uterine motility including, but not limited
to: complications with pregnancy and labor (e.g., pre-term labor,
post-term pregnancy, spontaneous abortion, and slow or stopped
labor); and disorders and/or diseases of the menstrual cycle (e.g.,
dysmenorrhea and endometriosis).
[1554] Endocrine system and/or hormone imbalance disorders and/or
diseases include disorders and/or diseases of the pancreas, such
as, for example, diabetes mellitus, diabetes insipidus, congenital
pancreatic agenesis, pheochromocytoma--islet cell tumor syndrome;
disorders and/or diseases of the adrenal glands such as, for
example, Addison's Disease, corticosteroid deficiency, virilizing
disease, hirsutism, Cushing's Syndrome, hyperaldosteronism,
pheochromocytoma; disorders and/or diseases of the pituitary gland,
such as, for example, hyperpituitarism, hypopituitarism, pituitary
dwarfism, pituitary adenoma, panhypopituitarism, acromegaly,
gigantism; disorders and/or diseases of the thyroid, including but
not limited to, hyperthyroidism, hypothyroidism, Plummer's disease,
Graves' disease (toxic diffuse goiter), toxic nodular goiter,
thyroiditis (Hashimoto's thyroiditis, subacute granulomatous
thyroiditis, and silent lymphocytic thyroiditis), Pendred's
syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone
coupling defect, thymic aplasia, Hurthle cell tumours of the
thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid
carcinoma; disorders and/or diseases of the parathyroid, such as,
for example, hyperparathyroidism, hypoparathyroidism; disorders
and/or diseases of the hypothalamus.
[1555] In specific embodiments, the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists or
antagonists of those polypeptides (including antibodies) as well as
fragments and variants of those polynucleotides, polypeptides,
agonists and antagonists, may be used to diagnose, prognose, treat,
prevent, or ameliorate diseases and disorders associated with
aberrant glucose metabolism or glucose uptake into cells.
[1556] In a specific embodiment, the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists and/or
antagonists thereof may be used to diagnose, prognose, treat,
prevent, and/or ameliorate type I diabetes mellitus (insulin
dependent diabetes mellitus, IDDM).
[1557] In another embodiment, the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists and/or
antagonists thereof may be used to diagnose, prognose, treat,
prevent, and/or ameliorate type II diabetes mellitus (insulin
resistant diabetes mellitus).
[1558] Additionally, in other embodiments, the polynucleotides
and/or polypeptides corresponding to this gene and/or antagonists
thereof (especially neutralizing or antagonistic antibodies) may be
used to diagnose, prognose, treat, prevent, and/or ameliorate
conditions associated with (type I or type II) diabetes mellitus,
including, but not limited to, diabetic ketoacidosis, diabetic
coma, nonketotic hyperglycemic-hyperosmolar coma, seizures, mental
confusion, drowsiness, cardiovascular disease (e.g., heart disease,
atherosclerosis, microvascular disease, hypertension, stroke, and
other diseases and disorders as described in the "Cardiovascular
Disorders" section), dyslipidemia, kidney disease (e.g., renal
failure, nephropathy other diseases and disorders as described in
the "Renal Disorders" section), nerve damage, neuropathy, vision
impairment (e.g., diabetic retinopathy and blindness), ulcers and
impaired wound healing, infections (e.g., infectious diseases and
disorders as described in the "Infectious Diseases" section,
especially of the urinary tract and skin), carpal tunnel syndrome
and Dupuytren's contracture.
[1559] In other embodiments, the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists or
antagonists thereof are administered to an animal, preferably a
mammal, and most preferably a human, in order to regulate the
animal's weight. In specific embodiments the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists or
antagonists thereof are administered to an animal, preferably a
mammal, and most preferably a human, in order to control the
animal's weight by modulating a biochemical pathway involving
insulin. In still other embodiments the polynucleotides and/or
polypeptides corresponding to this gene and/or agonists or
antagonists thereof are administered to an animal, preferably a
mammal, and most preferably a human, in order to control the
animal's weight by modulating a biochemical pathway involving
insulin-like growth factor.
[1560] In addition, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases of the testes or ovaries, including cancer. Other
disorders and/or diseases of the testes or ovaries further include,
for example, ovarian cancer, polycystic ovary syndrome,
Klinefelter's syndrome, vanishing testes syndrome (bilateral
anorchia), congenital absence of Leydig's cells, cryptorchidism,
Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the
testis (benign), neoplasias of the testis and neo-testis.
[1561] Moreover, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases such as, for example, polyglandular deficiency syndromes,
pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and
disorders and/or cancers of endocrine tissues.
[1562] In another embodiment, a polypeptide of the invention, or
polynucleotides, antibodies, agonists, or antagonists corresponding
to that polypeptide, may be used to diagnose, prognose, prevent,
and/or treat endocrine diseases and/or disorders associated with
the tissue(s) in which the polypeptide of the invention is
expressed, including one, two, three, four, five, or more tissues
disclosed in Table 1, column 8 (Tissue Distribution Library
Code).
[1563] Reproductive System Disorders
[1564] The polynucleotides or polypeptides, or agonists or
antagonists of the invention may be used for the diagnosis,
treatment, or prevention of diseases and/or disorders of the
reproductive system. Reproductive system disorders that can be
treated by the compositions of the invention, include, but are not
limited to, reproductive system injuries, infections, neoplastic
disorders, congenital defects, and diseases or disorders which
result in infertility, complications with pregnancy, labor, or
parturition, and postpartum difficulties.
[1565] Reproductive system disorders and/or diseases include
diseases and/or disorders of the testes, including testicular
atrophy, testicular feminization, cryptorchism (unilateral and
bilateral), anorchia, ectopic testis, epididymitis and orchitis
(typically resulting from infections such as, for example,
gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion,
vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell
carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors,
and teratomas), stromal tumors (e.g., Leydig cell tumors),
hydrocele, hematocele, varicocele, spermatocele, inguinal hernia,
and disorders of sperm production (e.g., immotile cilia syndrome,
aspermia, asthenozoospermia, azoospermia, oligospermia, and
teratozoospermia).
[1566] Reproductive system disorders also include disorders of the
prostate gland, such as acute non-bacterial prostatitis, chronic
non-bacterial prostatitis, acute bacterial prostatitis, chronic
bacterial prostatitis, prostatodystonia, prostatosis, granulomatous
prostatitis, malacoplakia, benign prostatic hypertrophy or
hyperplasia, and prostate neoplastic disorders, including
adenocarcinomas, transitional cell carcinomas, ductal carcinomas,
and squamous cell carcinomas.
[1567] Additionally, the compositions of the invention may be
useful in the diagnosis, treatment, and/or prevention of disorders
or diseases of the penis and urethra, including inflammatory
disorders, such as balanoposthitis, balanitis xerotica obliterans,
phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea,
non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV,
AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and
pearly penile papules; urethral abnormalities, such as hypospadias,
epispadias, and phimosis; premalignant lesions, including
Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant
condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile
cancers, including squamous cell carcinomas, carcinoma in situ,
verrucous carcinoma, and disseminated penile carcinoma; urethral
neoplastic disorders, including penile urethral carcinoma,
bulbomembranous urethral carcinoma, and prostatic urethral
carcinoma; and erectile disorders, such as priapism, Peyronie's
disease, erectile dysfunction, and impotence.
[1568] Moreover, diseases and/or disorders of the vas deferens
include vasculititis and CBAVD (congenital bilateral absence of the
vas deferens); additionally, the polynucleotides, polypeptides, and
agonists or antagonists of the present invention may be used in the
diagnosis, treatment, and/or prevention of diseases and/or
disorders of the seminal vesicles, including hydatid disease,
congenital chloride diarrhea, and polycystic kidney disease.
[1569] Other disorders and/or diseases of the male reproductive
system include, for example, Klinefelter's syndrome, Young's
syndrome, premature ejaculation, diabetes mellitus, cystic
fibrosis, Kartagener's syndrome, high fever, multiple sclerosis,
and gynecomastia.
[1570] Further, the polynucleotides, polypeptides, and agonists or
antagonists of the present invention may be used in the diagnosis,
treatment, and/or prevention of diseases and/or disorders of the
vagina and vulva, including bacterial vaginosis, candida vaginitis,
herpes simplex virus, chancroid, granuloma inguinale,
lymphogranuloma venereum, scabies, human papillomavirus, vaginal
trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea,
trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum
contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus,
lichen planus, vulvodynia, toxic shock syndrome, vaginismus,
vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such
as squamous cell hyperplasia, clear cell carcinoma, basal cell
carcinoma, melanomas, cancer of Bartholin's gland, and vulvar
intraepithelial neoplasia.
[1571] Disorders and/or diseases of the uterus include
dysmenorrhea, retroverted uterus, endometriosis, fibroids,
adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome,
hydatidiform moles, Asherman's syndrome, premature menopause,
precocious puberty, uterine polyps, dysfunctional uterine bleeding
(e.g., due to aberrant hormonal signals), and neoplastic disorders,
such as adenocarcinomas, keiomyosarcomas, and sarcomas.
Additionally, the polypeptides, polynucleotides, or agonists or
antagonists of the invention may be useful as a marker or detector
of, as well as in the diagnosis, treatment, and/or prevention of
congenital uterine abnormalities, such as bicornuate uterus,
septate uterus, simple unicornuate uterus, unicornuate uterus with
a noncavitary rudimentary horn, unicornuate uterus with a
non-communicating cavitary rudimentary horn, unicornuate uterus
with a communicating cavitary horn, arcuate uterus, uterine
didelfus, and T-shaped uterus.
[1572] Ovarian diseases and/or disorders include anovulation,
polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian
cysts, ovarian hypofunction, ovarian insensitivity to
gonadotropins, ovarian overproduction of androgens, right ovarian
vein syndrome, amenorrhea, hirutism, and ovarian cancer (including,
but not limited to, primary and secondary cancerous growth,
Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian
papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma,
and Ovarian Krukenberg tumors).
[1573] Cervical diseases and/or disorders include cervicitis,
chronic cervicitis, mucopurulent cervicitis, cervical dysplasia,
cervical polyps, Nabothian cysts, cervical erosion, cervical
incompetence, and cervical neoplasms (including, for example,
cervical carcinoma, squamous metaplasia, squamous cell carcinoma,
adenosquamous cell neoplasia, and columnar cell neoplasia).
[1574] Additionally, diseases and/or disorders of the reproductive
system include disorders and/or diseases of pregnancy, including
miscarriage and stillbirth, such as early abortion, late abortion,
spontaneous abortion, induced abortion, therapeutic abortion,
threatened abortion, missed abortion, incomplete abortion, complete
abortion, habitual abortion, missed abortion, and septic abortion;
ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding
during pregnancy, gestational diabetes, intrauterine growth
retardation, polyhydramnios, HELLP syndrome, abruptio placentae,
placenta previa, hyperemesis, preeclampsia, eclampsia, herpes
gestationis, and urticaria of pregnancy. Additionally, the
polynucleotides, polypeptides, and agonists or antagonists of the
present invention may be used in the diagnosis, treatment, and/or
prevention of diseases that can complicate pregnancy, including
heart disease, heart failure, rheumatic heart disease, congenital
heart disease, mitral valve prolapse, high blood pressure, anemia,
kidney disease, infectious disease (e.g., rubella, cytomegalovirus,
toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and
genital herpes), diabetes mellitus, Graves' disease, thyroiditis,
hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis,
cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic
lupus eryematosis, rheumatoid arthritis, myasthenia gravis,
idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts,
gallbladder disorders, and obstruction of the intestine.
[1575] Complications associated with labor and parturition include
premature rupture of the membranes, pre-term labor, post-term
pregnancy, postmaturity, labor that progresses too slowly, fetal
distress (e.g., abnormal heart rate (fetal or maternal), breathing
problems, and abnormal fetal position), shoulder dystocia,
prolapsed umbilical cord, amniotic fluid embolism, and aberrant
uterine bleeding.
[1576] Further, diseases and/or disorders of the postdelivery
period, including endometritis, myometritis, parametritis,
peritonitis, pelvic thrombophlebitis, pulmonary embolism,
endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis,
cystitis, postpartum hemorrhage, and inverted uterus.
[1577] Other disorders and/or diseases of the female reproductive
system that may be diagnosed, treated, and/or prevented by the
polynucleotides, polypeptides, and agonists or antagonists of the
present invention include, for example, Turner's syndrome,
pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory
disease, pelvic congestion (vascular engorgement), frigidity,
anorgasmia, dyspareunia, ruptured fallopian tube, and
Mittelschmerz.
[1578] Infectious Disease
[1579] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention can be used to treat or detect
infectious agents. For example, by increasing the immune response,
particularly increasing the proliferation and differentiation of B
and/or T cells, infectious diseases may be treated. The immune
response may be increased by either enhancing an existing immune
response, or by initiating a new immune response. Alternatively,
polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention may also directly inhibit the infectious
agent, without necessarily eliciting an immune response.
[1580] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by a
polynucleotide or polypeptide and/or agonist or antagonist of the
present invention. Examples of viruses, include, but are not
limited to Examples of viruses, include, but are not limited to the
following DNA and RNA viruses and viral families: Arbovirus,
Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue,
EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B,
and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. polynucleotides or polypeptides, or agonists
or antagonists of the invention, can be used to treat or detect any
of these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat: meningitis, Dengue, EBV, and/or
hepatitis (e.g., hepatitis B). In an additional specific embodiment
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat patients nonresponsive to one or more
other commercially available hepatitis vaccines. In a further
specific embodiment polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat AIDS.
[1581] Similarly, bacterial and fungal agents that can cause
disease or symptoms and that can be treated or detected by a
polynucleotide or polypeptide and/or agonist or antagonist of the
present invention include, but not limited to, the following
Gram-Negative and Gram-positive bacteria, bacterial families, and
fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus
neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis),
Bacteroides (e.g., Bacteroides fragilis), Blastomycosis,
Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella,
Candidia, Campylobacter, Chlamydia, Clostridium (e.g., Clostridium
botulinum, Clostridium dificile, Clostridium perfringens,
Clostridium tetani), Coccidioides, Corynebacterium (e.g.,
Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli
(e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),
Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae
(Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella
enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella),
Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B),
Helicobacter, Legionella (e.g., Legionella pneumophila),
Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma,
Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium
tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g.,
Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea,
Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),
Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp.,
Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus
aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g.,
Streptococcus pneumoniae and Groups A, B, and C Streptococci), and
Ureaplasmas. These bacterial, parasitic, and fungal families can
cause diseases or symptoms, including, but not limited to:
antibiotic-resistant infections, bacteremia, endocarditis,
septicemia, eye infections (e.g., conjunctivitis), uveitis,
tuberculosis, gingivitis, bacterial diarrhea, opportunistic
infections (e.g., AIDS related infections), paronychia,
prosthesis-related infections, dental caries, Reiter's Disease,
respiratory tract infections, such as Whooping Cough or Empyema,
sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid
fever, food poisoning, Legionella disease, chronic and acute
inflammation, erythema, yeast infections, typhoid, pneumonia,
gonorrhea, meningitis (e.g., meningitis types A and B), chlamydia,
syphilis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax,
spontaneous abortions, birth defects, pneumonia, lung infections,
ear infections, deafness, blindness, lethargy, malaise, vomiting,
chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility,
pelvic inflammatory diseases, candidiasis, paratuberculosis,
tuberculosis, lupus, botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections, noscomial infections. Polynucleotides
or polypeptides, agonists or antagonists of the invention, can be
used to treat or detect any of these symptoms or diseases. In
specific embodiments, polynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat: tetanus,
diphtheria, botulism, and/or meningitis type B.
[1582] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
include, but not limited to, the following families or class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis,
Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis,
Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium
virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium
ovale). These parasites can cause a variety of diseases or
symptoms, including, but not limited to: Scabies, Trombiculiasis,
eye infections, intestinal disease (e.g., dysentery, giardiasis),
liver disease, lung disease, opportunistic infections (e.g., AIDS
related), malaria, pregnancy complications, and toxoplasmosis.
polynucleotides or polypeptides, or agonists or antagonists of the
invention, can be used to treat, prevent, and/or diagnose any of
these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
[1583] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention of the present invention could
either be by administering an effective amount of a polypeptide to
the patient, or by removing cells from the patient, supplying the
cells with a polynucleotide of the present invention, and returning
the engineered cells to the patient (ex vivo therapy). Moreover,
the polypeptide or polynucleotide of the present invention can be
used as an antigen in a vaccine to raise an immune response against
infectious disease.
[1584] Regeneration
[1585] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention can be used to differentiate,
proliferate, and attract cells, leading to the regeneration of
tissues. (See, Science 276:59-87 (1997)). The regeneration of
tissues could be used to repair, replace, or protect tissue damaged
by congenital defects, trauma (wounds, burns, incisions, or
ulcers), age, disease (e.g. osteoporosis, osteocarthritis,
periodontal disease, liver failure), surgery, including cosmetic
plastic surgery, fibrosis, reperfusion injury, or systemic cytokine
damage.
[1586] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[1587] Moreover, polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, may increase
regeneration of tissues difficult to heal. For example, increased
tendon/ligament regeneration would quicken recovery time after
damage. Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention could also be used
prophylactically in an effort to avoid damage. Specific diseases
that could be treated include of tendinitis, carpal tunnel
syndrome, and other tendon or ligament defects. A further example
of tissue regeneration of non-healing wounds includes pressure
ulcers, ulcers associated with vascular insufficiency, surgical,
and traumatic wounds.
[1588] Similarly, nerve and brain tissue could also be regenerated
by using polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, to proliferate and
differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention.
[1589] Gastrointestinal Disorders
[1590] Polynucleotides or polypeptides, or agonists or antagonists
of the present invention, may be used to treat, prevent, diagnose,
and/or prognose gastrointestinal disorders, including inflammatory
diseases and/or conditions, infections, cancers (e.g., intestinal
neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's
lymphoma of the small intestine, small bowl lymphoma)), and ulcers,
such as peptic ulcers.
[1591] Gastrointestinal disorders include dysphagia, odynophagia,
inflammation of the esophagus, peptic esophagitis, gastric reflux,
submucosal fibrosis and stricturing, Mallory-Weiss lesions,
leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric
retention disorders, gastroenteritis, gastric atrophy,
gastric/stomach cancers, polyps of the stomach, autoimmune
disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral, eosinophilic, stress-induced, chronic erosive,
atrophic, plasma cell, and Menetrier's), and peritoneal diseases
(e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis,
neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess).
[1592] Gastrointestinal disorders also include disorders associated
with the small intestine, such as malabsorption syndromes,
distension, irritable bowel syndrome, sugar intolerance, celiac
disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's
disease, intestinal lymphangiectasia, Crohn's disease,
appendicitis, obstructions of the ileum, Meckel's diverticulum,
multiple diverticula, failure of complete rotation of the small and
large intestine, lymphoma, and bacterial and parasitic diseases
(such as Traveler's diarrhea, typhoid and paratyphoid, cholera,
infection by Roundworms (Ascariasis lumbricoides), Hookworms
(Ancylostoma duodenale), Threadworms (Enterobius vermicularis),
Tapeworms (Taenia saginata, Echinococcus granulosus,
Diphyllobothrium spp., and T. solium).
[1593] Liver diseases and/or disorders include intrahepatic
cholestasis (alagille syndrome, biliary liver cirrhosis), fatty
liver (alcoholic fatty liver, reye syndrome), hepatic vein
thrombosis, hepatolentricular degeneration, hepatomegaly,
hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension
(esophageal and gastric varices), liver abscess (amebic liver
abscess), liver cirrhosis (alcoholic, biliary and experimental),
alcoholic liver diseases (fatty liver, hepatitis, cirrhosis),
parasitic (hepatic echinococcosis, fascioliasis, amebic liver
abscess), jaundice (hemolytic, hepatocellular, and cholestatic),
cholestasis, portal hypertension, liver enlargement, ascites,
hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis
(autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced),
toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B,
hepatitis C, hepatitis D, hepatitis E), Wilson's disease,
granulomatous hepatitis, secondary biliary cirrhosis, hepatic
encephalopathy, portal hypertension, varices, hepatic
encephalopathy, primary biliary cirrhosis, primary sclerosing
cholangitis, hepatocellular adenoma, hemangiomas, bile stones,
liver failure (hepatic encephalopathy, acute liver failure), and
liver neoplasms (angiomyolipoma, calcified liver metastases, cystic
liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma,
focal nodular hyperplasia, hepatic adenoma, hepatobiliary
cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma,
liver cancer, liver hemangioendothelioma, mesenchymal hamartoma,
mesenchymal tumors of liver, nodular regenerative hyperplasia,
benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver
disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal
tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma,
Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor,
Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct
hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular
hyperplasia, Nodular regenerative hyperplasia)], malignant liver
tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma,
cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors
of blood vessels, angiosarcoma, Karposi's sarcoma,
hemangioendothelioma, other tumors, embryonal sarcoma,
fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,
teratoma, carcinoid, squamous carcinoma, primary lymphoma]),
peliosis hepatis, erythrohepatic porphyria, hepatic porphyria
(acute intermittent porphyria, porphyria cutanea tarda), Zellweger
syndrome).
[1594] Pancreatic diseases and/or disorders include acute
pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis,
alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas,
cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic
neoplasms, islet-cell tumors, pancreoblastoma), and other
pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic
pseudocyst, pancreatic fistula, insufficiency)).
[1595] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis), postcholecystectomy syndrome, diverticulosis
of the gallbladder, acute cholecystitis, chronic cholecystitis,
bile duct tumors, and mucocele.
[1596] Diseases and/or disorders of the large intestine include
antibiotic-associated colitis, diverticulitis, ulcerative colitis,
acquired megacolon, abscesses, fungal and bacterial infections,
anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases
(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps
(e.g., villous adenoma), colon carcinoma, colorectal cancer],
colonic diverticulitis, colonic diverticulosis, megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases
[proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease,
diarrhea (infantile diarrhea, dysentery), duodenal diseases
(duodenal neoplasms, duodenal obstruction, duodenal ulcer,
duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal
diseases (ileal neoplasms, ileitis), immunoproliferative small
intestinal disease, inflammatory bowel disease (ulcerative colitis,
Crohn's disease), intestinal atresia, parasitic diseases
(anisakiasis, balantidiasis, blastocystis infections,
cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis),
intestinal fistula (rectal fistula), intestinal neoplasms (cecal
neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms,
intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal
obstruction (afferent loop syndrome, duodenal obstruction, impacted
feces, intestinal pseudo-obstruction [cecal volvulus],
intussusception), intestinal perforation, intestinal polyps
(colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal
diseases (jejunal neoplasms), malabsorption syndromes (blind loop
syndrome, celiac disease, lactose intolerance, short bowl syndrome,
tropical sprue, whipple's disease), mesenteric vascular occlusion,
pneumatosis cystoides intestinalis, protein-losing enteropathies
(intestinal lymphagiectasis), rectal diseases (anus diseases, fecal
incontinence, hemorrhoids, proctitis, rectal fistula, rectal
prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic
esophagitis, hemorrhage, perforation, stomach ulcer,
Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping
syndrome), stomach diseases (e.g., achlorhydria, duodenogastric
reflux (bile reflux), gastric antral vascular ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or
hypertrophic), gastroparesis, stomach dilatation, stomach
diverticulum, stomach neoplasms (gastric cancer, gastric polyps,
gastric adenocarcinoma, hyperplastic gastric polyp), stomach
rupture, stomach ulcer, stomach volvulus), tuberculosis,
visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,
postoperative nausea and vomiting) and hemorrhagic colitis.
[1597] Further diseases and/or disorders of the gastrointestinal
system include biliary tract diseases, such as, gastroschisis,
fistula (e.g., biliary fistula, esophageal fistula, gastric
fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g.,
biliary tract neoplasms, esophageal neoplasms, such as
adenocarcinoma of the esophagus, esophageal squamous cell
carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such
as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the
pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and
peritoneal neoplasms), esophageal disease (e.g., bullous diseases,
candidiasis, glycogenic acanthosis, ulceration, barrett esophagus
varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum),
fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g., CREST syndrome, deglutition disorders, achalasia, spasm,
gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave
syndrome, Mallory-Weiss syndrome), stenosis, esophagitis,
diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal
diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk
virus infection), hemorrhage (e.g., hematemesis, melena, peptic
ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric
polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g.,
congenital diaphragmatic hernia, femoral hernia, inguinal hernia,
obturator hernia, umbilical hernia, ventral hernia), and intestinal
diseases (e.g., cecal diseases (appendicitis, cecal
neoplasms)).
[1598] Chemotaxis
[1599] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention may have chemotaxis activity.
A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or endothelial cells) to a particular site in the
body, such as inflammation, infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or
heal the particular trauma or abnormality.
[1600] Polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention may increase chemotaxic
activity of particular cells. These chemotactic molecules can then
be used to treat inflammation, infection, hyperproliferative
disorders, or any immune system disorder by increasing the number
of cells targeted to a particular location in the body. For
example, chemotaxic molecules can be used to treat wounds and other
trauma to tissues by attracting immune cells to the injured
location. Chemotactic molecules of the present invention can also
attract fibroblasts, which can be used to treat wounds.
[1601] It is also contemplated that polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention may inhibit chemotactic activity. These molecules could
also be used to treat disorders. Thus, polynucleotides or
polypeptides, as well as agonists or antagonists of the present
invention could be used as an inhibitor of chemotaxis.
[1602] Binding Activity
[1603] A polypeptide of the present invention may be used to screen
for molecules that bind to the polypeptide or for molecules to
which the polypeptide binds. The binding of the polypeptide and the
molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the polypeptide or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides,
proteins (e.g., receptors), or small molecules.
[1604] Preferably, the molecule is closely related to the natural
ligand of the polypeptide, e.g., a fragment of the ligand, or a
natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2):Chapter
5 (1991)). Similarly, the molecule can be closely related to the
natural receptor to which the polypeptide binds, or at least, a
fragment of the receptor capable of being bound by the polypeptide
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[1605] Preferably, the screening for these molecules involves
producing appropriate cells which express the polypeptide.
Preferred cells include cells from mammals, yeast, Drosophila, or
E. coli. Cells expressing the polypeptide (or cell membrane
containing the expressed polypeptide) are then preferably contacted
with a test compound potentially containing the molecule to observe
binding, stimulation, or inhibition of activity of either the
polypeptide or the molecule.
[1606] The assay may simply test binding of a candidate compound to
the polypeptide, wherein binding is detected by a label, or in an
assay involving competition with a labeled competitor. Further, the
assay may test whether the candidate compound results in a signal
generated by binding to the polypeptide.
[1607] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing a polypeptide, measuring polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule
activity or binding to a standard.
[1608] Preferably, an ELISA assay can measure polypeptide level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure polypeptide level
or activity by either binding, directly or indirectly, to the
polypeptide or by competing with the polypeptide for a
substrate.
[1609] Additionally, the receptor to which the polypeptide of the
present invention binds can be identified by numerous methods known
to those of skill in the art, for example, ligand panning and FACS
sorting (Coligan, et al., Current Protocols in Immun., 1(2),
Chapter 5, (1991)). For example, expression cloning is employed
wherein polyadenylated RNA is prepared from a cell responsive to
the polypeptides, for example, NIH3T3 cells which are known to
contain multiple receptors for the FGF family proteins, and SC-3
cells, and a cDNA library created from this RNA is divided into
pools and used to transfect COS cells or other cells that are not
responsive to the polypeptides. Transfected cells which are grown
on glass slides are exposed to the polypeptide of the present
invention, after they have been labeled. The polypeptides can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase.
[1610] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[1611] As an alternative approach for receptor identification, the
labeled polypeptides can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE analysis and exposed to
X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and
subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the genes encoding the putative receptors.
[1612] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of the
polypeptide of the present invention thereby effectively generating
agonists and antagonists of the polypeptide of the present
invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238,
5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al.,
Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends
Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.
Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.
Biotechniques 24(2):308-13 (1998); each of these patents and
publications are hereby incorporated by reference). In one
embodiment, alteration of polynucleotides and corresponding
polypeptides may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments into a desired
molecule by homologous, or site-specific, recombination. In another
embodiment, polynucleotides and corresponding polypeptides may be
altered by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to
recombination. In another embodiment, one or more components,
motifs, sections, parts, domains, fragments, etc., of the
polypeptide of the present invention may be recombined with one or
more components, motifs, sections, parts, domains, fragments, etc.
of one or more heterologous molecules. In preferred embodiments,
the heterologous molecules are family members. In further preferred
embodiments, the heterologous molecule is a growth factor such as,
for example, platelet-derived growth factor (PDGF), insulin-like
growth factor (IGF-I), transforming growth factor (TGF)-alpha,
epidermal growth factor (EGF), fibroblast growth factor (FGF),
TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6,
BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin,
growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived
neurotrophic factor (GDNF).
[1613] Other preferred fragments are biologically active fragments
of the polypeptide of the present invention. Biologically active
fragments are those exhibiting activity similar, but not
necessarily identical, to an activity of the polypeptide of the
present invention. The biological activity of the fragments may
include an improved desired activity, or a decreased undesirable
activity.
[1614] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of the
polypeptide of the present invention. An example of such an assay
comprises combining a mammalian fibroblast cell, a the polypeptide
of the present invention, the compound to be screened and .sup.3[H]
thymidine under cell culture conditions where the fibroblast cell
would normally proliferate. A control assay may be performed in the
absence of the compound to be screened and compared to the amount
of fibroblast proliferation in the presence of the compound to
determine if the compound stimulates proliferation by determining
the uptake of .sup.3[H] thymidine in each case. The amount of
fibroblast cell proliferation is measured by liquid scintillation
chromatography which measures the incorporation of .sup.3[H]
thymidine. Both agonist and antagonist compounds may be identified
by this procedure.
[1615] In another method, a mammalian cell or membrane preparation
expressing a receptor for a polypeptide of the present invention is
incubated with a labeled polypeptide of the present invention in
the presence of the compound. The ability of the compound to
enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system
following interaction of a compound to be screened and the receptor
is measured and the ability of the compound to bind to the receptor
and elicit a second messenger response is measured to determine if
the compound is a potential agonist or antagonist. Such second
messenger systems include but are not limited to, cAMP guanylate
cyclase, ion channels or phosphoinositide hydrolysis.
[1616] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat disease or to bring about a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
polypeptide/molecule. Moreover, the assays can discover agents
which may inhibit or enhance the production of the polypeptides of
the invention from suitably manipulated cells or tissues.
[1617] Therefore, the invention includes a method of identifying
compounds which bind to a polypeptide of the invention comprising
the steps of: (a) incubating a candidate binding compound with a
polypeptide of the present invention; and (b) determining if
binding has occurred. Moreover, the invention includes a method of
identifying agonists/antagonists comprising the steps of: (a)
incubating a candidate compound with a polypeptide of the present
invention, (b) assaying a biological activity, and (b) determining
if a biological activity of the polypeptide has been altered.
[1618] Targeted Delivery
[1619] In another embodiment, the invention provides a method of
delivering compositions to targeted cells expressing a receptor for
a polypeptide of the invention, or cells expressing a cell bound
form of a polypeptide of the invention.
[1620] As discussed herein, polypeptides or antibodies of the
invention may be associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic,
hydrophilic, ionic and/or covalent interactions. In one embodiment,
the invention provides a method for the specific delivery of
compositions of the invention to cells by administering
polypeptides of the invention (including antibodies) that are
associated with heterologous polypeptides or nucleic acids. In one
example, the invention provides a method for delivering a
therapeutic protein into the targeted cell. In another example, the
invention provides a method for delivering a single stranded
nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic acid (e.g., DNA that can integrate into the cell's genome
or replicate episomally and that can be transcribed) into the
targeted cell.
[1621] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention (e.g.,
polypeptides of the invention or antibodies of the invention) in
association with toxins or cytotoxic prodrugs.
[1622] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, or any molecules or
enzymes not normally present in or on the surface of a cell that
under defined conditions cause the cell's death. Toxins that may be
used according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a
non-toxic compound that is converted by an enzyme, normally present
in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may
be used according to the methods of the invention include, but are
not limited to, glutamyl derivatives of benzoic acid mustard
alkylating agent, phosphate derivatives of etoposide or mitomycin
C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives of doxorubicin.
[1623] Drug Screening
[1624] Further contemplated is the use of the polypeptides of the
present invention, or the polynucleotides encoding these
polypeptides, to screen for molecules which modify the activities
of the polypeptides of the present invention. Such a method would
include contacting the polypeptide of the present invention with a
selected compound(s) suspected of having antagonist or agonist
activity, and assaying the activity of these polypeptides following
binding.
[1625] This invention is particularly useful for screening
therapeutic compounds by using the polypeptides of the present
invention, or binding fragments thereof, in any of a variety of
drug screening techniques. The polypeptide or fragment employed in
such a test may be affixed to a solid support, expressed on a cell
surface, free in solution, or located intracellularly. One method
of drug screening utilizes eukaryotic or prokaryotic host cells
which are stably transformed with recombinant nucleic acids
expressing the polypeptide or fragment. Drugs are screened against
such transformed cells in competitive binding assays. One may
measure, for example, the formulation of complexes between the
agent being tested and a polypeptide of the present invention.
[1626] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the polypeptides of the present invention. These methods comprise
contacting such an agent with a polypeptide of the present
invention or a fragment thereof and assaying for the presence of a
complex between the agent and the polypeptide or a fragment
thereof, by methods well known in the art. In such a competitive
binding assay, the agents to screen are typically labeled.
Following incubation, free agent is separated from that present in
bound form, and the amount of free or uncomplexed label is a
measure of the ability of a particular agent to bind to the
polypeptides of the present invention.
[1627] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the polypeptides of the present invention, and is described in
great detail in European Patent Application 84/03564, published on
Sep. 13, 1984, which is incorporated herein by reference herein.
Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds are reacted
with polypeptides of the present invention and washed. Bound
polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in
the aforementioned drug screening techniques. In addition,
non-neutralizing antibodies may be used to capture the peptide and
immobilize it on the solid support.
[1628] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding polypeptides of the present invention specifically compete
with a test compound for binding to the polypeptides or fragments
thereof. In this manner, the antibodies are used to detect the
presence of any peptide which shares one or more antigenic epitopes
with a polypeptide of the invention.
[1629] Polypeptides of the Invention Binding Peptides and Other
Molecules
[1630] The invention also encompasses screening methods for
identifying polypeptides and nonpolypeptides that bind polypeptides
of the invention, and the polypeptide of the invention binding
molecules identified thereby. These binding molecules are useful,
for example, as agonists and antagonists of the polypeptides of the
invention. Such agonists and antagonists can be used, in accordance
with the invention, in the therapeutic embodiments described in
detail, below.
[1631] This method comprises the steps of: contacting a polypeptide
of the invention with a plurality of molecules; and identifying a
molecule that binds the polypeptide of the invention.
[1632] The step of contacting the polypeptide of the invention with
the plurality of molecules may be effected in a number of ways. For
example, one may contemplate immobilizing the polypeptide of the
invention on a solid support and bringing a solution of the
plurality of molecules in contact with the immobilized polypeptide
of the invention. Such a procedure would be akin to an affinity
chromatographic process, with the affinity matrix being comprised
of the immobilized polypeptide of the invention. The molecules
having a selective affinity for the polypeptide of the invention
can then be purified by affinity selection. The nature of the solid
support, process for attachment of the polypeptide of the invention
to the solid support, solvent, and conditions of the affinity
isolation or selection are largely conventional and well known to
those of ordinary skill in the art.
[1633] Alternatively, one may also separate a plurality of
polypeptides into substantially separate fractions comprising a
subset of or individual polypeptides. For instance, one can
separate the plurality of polypeptides by gel electrophoresis,
column chromatography, or like method known to those of ordinary
skill for the separation of polypeptides. The individual
polypeptides can also be produced by a transformed host cell in
such a way as to be expressed on or about its outer surface (e.g.,
a recombinant phage). Individual isolates can then be "probed" by
the polypeptide of the invention, optionally in the presence of an
inducer should one be required for expression, to determine if any
selective affinity interaction takes place between the polypeptide
of the invention and the individual clone. Prior to contacting the
polypeptide of the invention with each fraction comprising
individual polypeptides, the polypeptides could first be
transferred to a solid support for additional convenience. Such a
solid support may simply be a piece of filter membrane, such as one
made of nitrocellulose or nylon. In this manner, positive clones
could be identified from a collection of transformed host cells of
an expression library, which harbor a DNA construct encoding a
polypeptide having a selective affinity for a polypeptide of the
invention. Furthermore, the amino acid sequence of the polypeptide
having a selective affinity for the polypeptide of the invention
can be determined directly by conventional means or the coding
sequence of the DNA encoding the polypeptide can frequently be
determined more conveniently. The primary sequence can then be
deduced from the corresponding DNA sequence. If the amino acid
sequence is to be determined from the polypeptide itself, one may
use microsequencing techniques. The sequencing technique may
include mass spectroscopy.
[1634] In certain situations, it may be desirable to wash away any
unbound polypeptide of the invention, or alternatively, unbound
polypeptides, from a mixture of the polypeptide of the invention
and the plurality of polypeptides prior to attempting to determine
or to detect the presence of a selective affinity interaction. Such
a wash step may be particularly desirable when the polypeptide of
the invention or the plurality of polypeptides is bound to a solid
support.
[1635] The plurality of molecules provided according to this method
may be provided by way of diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries which can be screened
for molecules that specifically bind to a polypeptide of the
invention. Many libraries are known in the art that can be used,
e.g., chemically synthesized libraries, recombinant (e.g., phage
display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized libraries are described in Fodor
et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature
354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994,
Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal
Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad.
Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci.
USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618;
Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT
Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc.
Natl. Acad. Sci. USA 89:5381-5383.
[1636] Examples of phage display libraries are described in Scott
and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science,
249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol.
227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et
al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318
dated Aug. 18, 1994.
[1637] In vitro translation-based libraries include but are not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci.
USA 91:9022-9026.
[1638] By way of examples of nonpeptide libraries, a benzodiazepine
library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA
91:4708-4712) can be adapted for use. Peptoid libraries (Simon et
al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be
used. Another example of a library that can be used, in which the
amide functionalities in peptides have been permethylated to
generate a chemically transformed combinatorial library, is
described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA
91:11138-11142).
[1639] The variety of non-peptide libraries that are useful in the
present invention is great. For example, Ecker and Crooke, 1995,
Bio/Technology 13:351-360 list benzodiazepines, hydantoins,
piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,
arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,
aminimides, and oxazolones as among the chemical species that form
the basis of various libraries.
[1640] Non-peptide libraries can be classified broadly into two
types: decorated monomers and oligomers. Decorated monomer
libraries employ a relatively simple scaffold structure upon which
a variety functional groups is added. Often the scaffold will be a
molecule with a known useful pharmacological activity. For example,
the scaffold might be the benzodiazepine structure.
[1641] Non-peptide oligomer libraries utilize a large number of
monomers that are assembled together in ways that create new shapes
that depend on the order of the monomers. Among the monomer units
that have been used are carbamates, pyrrolinones, and morpholinos.
Peptoids, peptide-like oligomers in which the side chain is
attached to the alpha amino group rather than the alpha carbon,
form the basis of another version of non-peptide oligomer
libraries. The first non-peptide oligomer libraries utilized a
single type of monomer and thus contained a repeating backbone.
Recent libraries have utilized more than one monomer, giving the
libraries added flexibility.
[1642] Screening the libraries can be accomplished by any of a
variety of commonly known methods. See, e.g., the following
references, which disclose screening of peptide libraries: Parmley
and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith,
1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques
13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA
89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al.,
1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;
Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.
5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346,
all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673;
and CT Publication No. WO 94/18318.
[1643] In a specific embodiment, screening to identify a molecule
that binds a polypeptide of the invention can be carried out by
contacting the library members with a polypeptide of the invention
immobilized on a solid phase and harvesting those library members
that bind to the polypeptide of the invention. Examples of such
screening methods, termed "panning" techniques are described by way
of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et
al., 1992, BioTechniques 13:422-427; PCT Publication No. WO
94/18318; and in references cited herein.
[1644] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, 1989, Nature
340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically
bind to a polypeptide of the invention.
[1645] Where the polypeptide of the invention binding molecule is a
polypeptide, the polypeptide can be conveniently selected from any
peptide library, including random peptide libraries, combinatorial
peptide libraries, or biased peptide libraries. The term "biased"
is used herein to mean that the method of generating the library is
manipulated so as to restrict one or more parameters that govern
the diversity of the resulting collection of molecules, in this
case peptides.
[1646] Thus, a truly random peptide library would generate a
collection of peptides in which the probability of finding a
particular amino acid at a given position of the peptide is the
same for all 20 amino acids. A bias can be introduced into the
library, however, by specifying, for example, that a lysine occur
every fifth amino acid or that positions 4, 8, and 9 of a
decapeptide library be fixed to include only arginine. Clearly,
many types of biases can be contemplated, and the present invention
is not restricted to any particular bias. Furthermore, the present
invention contemplates specific types of peptide libraries, such as
phage displayed peptide libraries and those that utilize a DNA
construct comprising a lambda phage vector with a DNA insert.
[1647] As mentioned above, in the case of a polypeptide of the
invention binding molecule that is a polypeptide, the polypeptide
may have about 6 to less than about 60 amino acid residues,
preferably about 6 to about 10 amino acid residues, and most
preferably, about 6 to about 22 amino acids. In another embodiment,
a polypeptide of the invention binding polypeptide has in the range
of 15-100 amino acids, or 20-50 amino acids.
[1648] The selected polypeptide of the invention binding
polypeptide can be obtained by chemical synthesis or recombinant
expression.
[1649] Antisense and Ribozyme (Antagonists)
[1650] In specific embodiments, antagonists according to the
present invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:X, or the complementary strand thereof,
and/or to nucleotide sequences contained a deposited clone. In one
embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is
separately administered (see, for example, O'Connor, Neurochem.,
56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense
technology can be used to control gene expression through antisense
DNA or RNA, or through triple-helix formation. Antisense techniques
are discussed for example, in Okano, Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance, Lee et al., Nucleic Acids Research,
6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan
et al., Science, 251:1300 (1991). The methods are based on binding
of a polynucleotide to a complementary DNA or RNA.
[1651] For example, the use of c-myc and c-myb antisense RNA
constructs to inhibit the growth of the non-lymphocytic leukemia
cell line HL-60 and other cell lines was previously described.
(Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments
were performed in vitro by incubating cells with the
oligoribonucleotide. A similar procedure for in vivo use is
described in WO 91/15580. Briefly, a pair of oligonucleotides for a
given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is
flanked by an EcoR1 site on the 5 end and a HindIII site on the 3
end. Next, the pair of oligonucleotides is heated at 90.degree. C.
for one minute and then annealed in 2.times. ligation buffer (20 mM
TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM
ATP) and then ligated to the EcoR1/Hind III site of the retroviral
vector PMV7 (WO 91/15580).
[1652] For example, the 5' coding portion of a polynucleotide that
encodes the mature polypeptide of the present invention may be used
to design an antisense RNA oligonucleotide of from about 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[1653] In one embodiment, the antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the
antisense nucleic acid of the invention. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in vertebrate cells. Expression
of the sequence encoding a polypeptide of the invention, or
fragments thereof, can be by any promoter known in the art to act
in vertebrate, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include, but are not
limited to, the SV40 early promoter region (Bernoist and Chambon,
Nature, 29:304-310 (1981), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,
22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,
Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory
sequences of the metallothionein gene (Brinster et al., Nature,
296:39-42 (1982)), etc.
[1654] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a gene of interest. However, absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA," referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double stranded antisense
nucleic acids of the invention, a single strand of the duplex DNA
may thus be tested, or triplex formation may be assayed. The
ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base
mismatches with a RNA sequence of the invention it may contain and
still form a stable duplex (or triplex as the case may be). One
skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.
[1655] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of a
polynucleotide sequence of the invention could be used in an
antisense approach to inhibit translation of endogenous mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of mRNA, antisense nucleic acids should
be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[1656] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987);
PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[1657] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[1658] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[1659] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[1660] In yet another embodiment, the antisense oligonucleotide is
an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual b-units, the strands run parallel to each
other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The
oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl.
Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue
(Inoue et al., FEBS Lett. 215:327-330 (1987)).
[1661] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(Nucl. Acids Res., 16:3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.,
85:7448-7451 (1988)), etc.
[1662] While antisense nucleotides complementary to the coding
region sequence of the invention could be used, those complementary
to the transcribed untranslated region are most preferred.
[1663] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al,
Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy mRNAs
corresponding to the polynucleotides of the invention, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Haseloff
and Gerlach, Nature, 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within each nucleotide
sequence disclosed in the sequence listing. Preferably, the
ribozyme is engineered so that the cleavage recognition site is
located near the 5' end of the mRNA corresponding to the
polynucleotides of the invention; i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[1664] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express the polynucleotides of the invention in vivo. DNA
constructs encoding the ribozyme may be introduced into the cell in
the same manner as described above for the introduction of
antisense encoding DNA. A preferred method of delivery involves
using a DNA construct "encoding" the ribozyme under the control of
a strong constitutive promoter, such as, for example, pol III or
pol II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous messages and
inhibit translation. Since ribozymes unlike antisense molecules,
are catalytic, a lower intracellular concentration is required for
efficiency.
[1665] Antagonist/agonist compounds may be employed to inhibit the
cell growth and proliferation effects of the polypeptides of the
present invention on neoplastic cells and tissues, i.e. stimulation
of angiogenesis of tumors, and, therefore, retard or prevent
abnormal cellular growth and proliferation, for example, in tumor
formation or growth.
[1666] The antagonist/agonist may also be employed to prevent
hyper-vascular diseases, and prevent the proliferation of
epithelial lens cells after extracapsular cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the
present invention may also be desirous in cases such as restenosis
after balloon angioplasty.
[1667] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[1668] The antagonist/agonist may also be employed to treat,
prevent, and/or diagnose the diseases described herein.
[1669] Thus, the invention provides a method of treating or
preventing diseases, disorders, and/or conditions, including but
not limited to the diseases, disorders, and/or conditions listed
throughout this application, associated with overexpression of a
polynucleotide of the present invention by administering to a
patient (a) an antisense molecule directed to the polynucleotide of
the present invention, and/or (b) a ribozyme directed to the
polynucleotide of the present invention.
[1670] Other Activities
[1671] The polypeptide of the present invention, as a result of the
ability to stimulate vascular endothelial cell growth, may be
employed in treatment for stimulating re-vascularization of
ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions.
These polypeptide may also be employed to stimulate angiogenesis
and limb regeneration, as discussed above.
[1672] The polypeptide may also be employed for treating wounds due
to injuries, burns, post-operative tissue repair, and ulcers since
they are mitogenic to various cells of different origins, such as
fibroblast cells and skeletal muscle cells, and therefore,
facilitate the repair or replacement of damaged or diseased
tissue.
[1673] The polypeptide of the present invention may also be
employed stimulate neuronal growth and to treat, prevent, and/or
diagnose neuronal damage which occurs in certain neuronal disorders
or neuro-degenerative conditions such as Alzheimer's disease,
Parkinson's disease, and AIDS-related complex. The polypeptide of
the invention may have the ability to stimulate chondrocyte growth,
therefore, they may be employed to enhance bone and periodontal
regeneration and aid in tissue transplants or bone grafts.
[1674] The polypeptide of the present invention may be also be
employed to prevent skin aging due to sunburn by stimulating
keratinocyte growth.
[1675] The polypeptide of the invention may also be employed for
preventing hair loss, since FGF family members activate
hair-forming cells and promotes melanocyte growth. Along the same
lines, the polypeptides of the present invention may be employed to
stimulate growth and differentiation of hematopoietic cells and
bone marrow cells when used in combination with other
cytokines.
[1676] The polypeptide of the invention may also be employed to
maintain organs before transplantation or for supporting cell
culture of primary tissues.
[1677] The polypeptide of the present invention may also be
employed for inducing tissue of mesodermal origin to differentiate
in early embryos.
[1678] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also increase or decrease
the differentiation or proliferation of embryonic stem cells,
besides, as discussed above, hematopoietic lineage.
[1679] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also be used to modulate
mammalian characteristics, such as body height, weight, hair color,
eye color, skin, percentage of adipose tissue, pigmentation, size,
and shape (e.g., cosmetic surgery). Similarly, polypeptides or
polynucleotides and/or agonist or antagonists of the present
invention may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of
energy.
[1680] A polypeptide, polynucleotide, agonist, or antagonist of the
present invention may be used to treat weight disorders, including
but not limited to, obesity, cachexia, wasting disease, anorexia,
and bulimia.
[1681] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may be used to change a mammal's mental
state or physical state by influencing biorhythms, caricadic
rhythms, depression (including depressive diseases, disorders,
and/or conditions), tendency for violence, tolerance for pain,
reproductive capabilities (preferably by Activin or Inhibin-like
activity), hormonal or endocrine levels, appetite, libido, memory,
stress, or other cognitive qualities.
[1682] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used as a food additive or
preservative, such as to increase or decrease storage capabilities,
fat content, lipid, protein, carbohydrate, vitamins, minerals,
cofactors or other nutritional components.
Other Preferred Embodiments
[1683] Other preferred embodiments of the claimed invention include
an isolated nucleic acid molecule comprising a nucleotide sequence
which is at least 95% identical to a sequence of at least about 50
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table 1.
[1684] Also preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
Clone Sequence and ending with the nucleotide at about the position
of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID
NO:X in Table 1.
[1685] Also preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
Start Codon and ending with the nucleotide at about the position of
the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X
in Table 1.
[1686] Similarly preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
First Amino Acid of the Signal Peptide and ending with the
nucleotide at about the position of the 3' Nucleotide of the Clone
Sequence as defined for SEQ ID NO:X in Table 1.
[1687] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least about 150 contiguous nucleotides in the
nucleotide sequence of SEQ ID NO:X.
[1688] Further preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least about 500 contiguous nucleotides in the
nucleotide sequence of SEQ ID NO:X.
[1689] A further preferred embodiment is a nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
the nucleotide sequence of SEQ ID NO:X beginning with the
nucleotide at about the position of the 5' Nucleotide of the First
Amino Acid of the Signal Peptide and ending with the nucleotide at
about the position of the 3' Nucleotide of the Clone Sequence as
defined for SEQ ID NO:X in Table 1.
[1690] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to the complete nucleotide sequence of SEQ ID NO:X.
[1691] Also preferred is an isolated nucleic acid molecule which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule, wherein said nucleic acid molecule which hybridizes
does not hybridize under stringent hybridization conditions to a
nucleic acid molecule having a nucleotide sequence consisting of
only A residues or of only T residues.
[1692] Also preferred is a composition of matter comprising a DNA
molecule which comprises a human cDNA clone identified by a cDNA
Clone Identifier in Table 1, which DNA molecule is contained in the
material deposited with the American Type Culture Collection and
given the ATCC.TM. Deposit Number shown in Table 1 for said cDNA
Clone Identifier.
[1693] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least 50 contiguous nucleotides in the nucleotide
sequence of a human cDNA clone identified by a cDNA Clone
Identifier in Table 1, which DNA molecule is contained in the
deposit given the ATCC.TM. Deposit Number shown in Table 1.
[1694] Also preferred is an isolated nucleic acid molecule, wherein
said sequence of at least 50 contiguous nucleotides is included in
the nucleotide sequence of the complete open reading frame sequence
encoded by said human cDNA clone.
[1695] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
sequence of at least 150 contiguous nucleotides in the nucleotide
sequence encoded by said human cDNA clone.
[1696] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to sequence of at least 500 contiguous nucleotides in the
nucleotide sequence encoded by said human cDNA clone.
[1697] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to the complete nucleotide sequence encoded by said human
cDNA clone.
[1698] A further preferred embodiment is a method for detecting in
a biological sample a nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least
50 contiguous nucleotides in a sequence selected from the group
consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is
any integer as defined in Table 1; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC.TM. Deposit
Number shown for said cDNA clone in Table 1; which method comprises
a step of comparing a nucleotide sequence of at least one nucleic
acid molecule in said sample with a sequence selected from said
group and determining whether the sequence of said nucleic acid
molecule in said sample is at least 95% identical to said selected
sequence.
[1699] Also preferred is the above method wherein said step of
comparing sequences comprises determining the extent of nucleic
acid hybridization between nucleic acid molecules in said sample
and a nucleic acid molecule comprising said sequence selected from
said group. Similarly, also preferred is the above method wherein
said step of comparing sequences is performed by comparing the
nucleotide sequence determined from a nucleic acid molecule in said
sample with said sequence selected from said group. The nucleic
acid molecules can comprise DNA molecules or RNA molecules.
[1700] A further preferred embodiment is a method for identifying
the species, tissue or cell type of a biological sample which
method comprises a step of detecting nucleic acid molecules in said
sample, if any, comprising a nucleotide sequence that is at least
95% identical to a sequence of at least 50 contiguous nucleotides
in a sequence selected from the group consisting of: a nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in
Table 1; and a nucleotide sequence encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC.TM. Deposit Number shown for said cDNA
clone in Table 1.
[1701] The method for identifying the species, tissue or cell type
of a biological sample can comprise a step of detecting nucleic
acid molecules comprising a nucleotide sequence in a panel of at
least two nucleotide sequences, wherein at least one sequence in
said panel is at least 95% identical to a sequence of at least 50
contiguous nucleotides in a sequence selected from said group.
[1702] Also preferred is a method for diagnosing in a subject a
pathological condition associated with abnormal structure or
expression of a gene encoding a secreted protein identified in
Table 1, which method comprises a step of detecting in a biological
sample obtained from said subject nucleic acid molecules, if any,
comprising a nucleotide sequence that is at least 95% identical to
a sequence of at least 50 contiguous nucleotides in a sequence
selected from the group consisting of: a nucleotide sequence of SEQ
ID NO:X wherein X is any integer as defined in Table 1; and a
nucleotide sequence encoded by a human cDNA clone identified by a
cDNA Clone Identifier in Table 1 and contained in the deposit with
the ATCC.TM. Deposit Number shown for said cDNA clone in Table
1.
[1703] The method for diagnosing a pathological condition can
comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide
sequences, wherein at least one sequence in said panel is at least
95% identical to a sequence of at least 50 contiguous nucleotides
in a sequence selected from said group.
[1704] Also preferred is a composition of matter comprising
isolated nucleic acid molecules wherein the nucleotide sequences of
said nucleic acid molecules comprise a panel of at least two
nucleotide sequences, wherein at least one sequence in said panel
is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a sequence selected from the group consisting of: a
nucleotide sequence of SEQ ID NO:X wherein X is any integer as
defined in Table 1; and a nucleotide sequence encoded by a human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC.TM. Deposit Number shown for
said cDNA clone in Table 1. The nucleic acid molecules can comprise
DNA molecules or RNA molecules.
[1705] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 90% identical to a sequence of at
least about 10 contiguous amino acids in the amino acid sequence of
SEQ ID NO:Y wherein Y is any integer as defined in Table 1.
[1706] Also preferred is a polypeptide, wherein said sequence of
contiguous amino acids is included in the amino acid sequence of
SEQ ID NO:Y in the range of positions beginning with the residue at
about the position of the First Amino Acid of the Secreted Portion
and ending with the residue at about the Last Amino Acid of the
Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
[1707] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 30 contiguous amino acids in the amino acid sequence of
SEQ ID NO:Y.
[1708] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 100 contiguous amino acids in the amino acid sequence
of SEQ ID NO:Y.
[1709] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to the complete amino
acid sequence of SEQ ID NO:Y.
[1710] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 90% identical to a sequence of at
least about 10 contiguous amino acids in the complete amino acid
sequence of a secreted protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC.TM. Deposit Number shown for said cDNA
clone in Table 1.
[1711] Also preferred is a polypeptide wherein said sequence of
contiguous amino acids is included in the amino acid sequence of a
secreted portion of the secreted protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC.TM. Deposit Number shown for
said cDNA clone in Table 1.
[1712] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 30 contiguous amino acids in the amino acid sequence of
the secreted portion of the protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC.TM. Deposit Number shown for said cDNA
clone in Table 1.
[1713] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 100 contiguous amino acids in the amino acid sequence
of the secreted portion of the protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC.TM. Deposit Number shown for
said cDNA clone in Table 1.
[1714] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to the amino acid
sequence of the secreted portion of the protein encoded by a human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC.TM. Deposit Number shown for
said cDNA clone in Table 1.
[1715] Further preferred is an isolated antibody which binds
specifically to a polypeptide comprising an amino acid sequence
that is at least 90% identical to a sequence of at least 10
contiguous amino acids in a sequence selected from the group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is
any integer as defined in Table 1; and a complete amino acid
sequence of a protein encoded by a human cDNA clone identified by a
cDNA Clone Identifier in Table 1 and contained in the deposit with
the ATCC.TM. Deposit Number shown for said cDNA clone in Table
1.
[1716] Further preferred is a method for detecting in a biological
sample a polypeptide comprising an amino acid sequence which is at
least 90% identical to a sequence of at least 10 contiguous amino
acids in a sequence selected from the group consisting of: an amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC.TM. Deposit Number shown
for said cDNA clone in Table 1; which method comprises a step of
comparing an amino acid sequence of at least one polypeptide
molecule in said sample with a sequence selected from said group
and determining whether the sequence of said polypeptide molecule
in said sample is at least 90% identical to said sequence of at
least 10 contiguous amino acids.
[1717] Also preferred is the above method wherein said step of
comparing an amino acid sequence of at least one polypeptide
molecule in said sample with a sequence selected from said group
comprises determining the extent of specific binding of
polypeptides in said sample to an antibody which binds specifically
to a polypeptide comprising an amino acid sequence that is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC.TM. Deposit Number shown
for said cDNA clone in Table 1.
[1718] Also preferred is the above method wherein said step of
comparing sequences is performed by comparing the amino acid
sequence determined from a polypeptide molecule in said sample with
said sequence selected from said group.
[1719] Also preferred is a method for identifying the species,
tissue or cell type of a biological sample which method comprises a
step of detecting polypeptide molecules in said sample, if any,
comprising an amino acid sequence that is at least 90% identical to
a sequence of at least 10 contiguous amino acids in a sequence
selected from the group consisting of: an amino acid sequence of
SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a
complete amino acid sequence of a secreted protein encoded by a
human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC.TM. Deposit Number shown
for said cDNA clone in Table 1.
[1720] Also preferred is the above method for identifying the
species, tissue or cell type of a biological sample, which method
comprises a step of detecting polypeptide molecules comprising an
amino acid sequence in a panel of at least two amino acid
sequences, wherein at least one sequence in said panel is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the above group.
[1721] Also preferred is a method for diagnosing in a subject a
pathological condition associated with abnormal structure or
expression of a gene encoding a secreted protein identified in
Table 1, which method comprises a step of detecting in a biological
sample obtained from said subject polypeptide molecules comprising
an amino acid sequence in a panel of at least two amino acid
sequences, wherein at least one sequence in said panel is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC.TM. Deposit
Number shown for said cDNA clone in Table 1.
[1722] In any of these methods, the step of detecting said
polypeptide molecules includes using an antibody.
[1723] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a nucleotide sequence encoding a polypeptide wherein said
polypeptide comprises an amino acid sequence that is at least 90%
identical to a sequence of at least 10 contiguous amino acids in a
sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC.TM. Deposit
Number shown for said cDNA clone in Table 1.
[1724] Also preferred is an isolated nucleic acid molecule, wherein
said nucleotide sequence encoding a polypeptide has been optimized
for expression of said polypeptide in a prokaryotic host.
[1725] Also preferred is an isolated nucleic acid molecule, wherein
said polypeptide comprises an amino acid sequence selected from the
group consisting of: an amino acid sequence of SEQ ID NO:Y wherein
Y is any integer as defined in Table 1; and a complete amino acid
sequence of a secreted protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC.TM. Deposit Number shown for said cDNA
clone in Table 1.
[1726] Further preferred is a method of making a recombinant vector
comprising inserting any of the above isolated nucleic acid
molecule into a vector. Also preferred is the recombinant vector
produced by this method. Also preferred is a method of making a
recombinant host cell comprising introducing the vector into a host
cell, as well as the recombinant host cell produced by this
method.
[1727] Also preferred is a method of making an isolated polypeptide
comprising culturing this recombinant host cell under conditions
such that said polypeptide is expressed and recovering said
polypeptide. Also preferred is this method of making an isolated
polypeptide, wherein said recombinant host cell is a eukaryotic
cell and said polypeptide is a secreted portion of a human secreted
protein comprising an amino acid sequence selected from the group
consisting of: an amino acid sequence of SEQ ID NO:Y beginning with
the residue at the position of the First Amino Acid of the Secreted
Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1
and said position of the First Amino Acid of the Secreted Portion
of SEQ ID NO:Y is defined in Table 1; and an amino acid sequence of
a secreted portion of a protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC.TM. Deposit Number shown for said cDNA
clone in Table 1. The isolated polypeptide produced by this method
is also preferred.
[1728] Also preferred is a method of treatment of an individual in
need of an increased level of a secreted protein activity, which
method comprises administering to such an individual a
pharmaceutical composition comprising an amount of an isolated
polypeptide, polynucleotide, or antibody of the claimed invention
effective to increase the level of said protein activity in said
individual.
[1729] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[1730] In specific embodiments of the invention, for each "Contig
ID" listed in the fourth column of Table 2, preferably excluded are
one or more polynucleotides comprising, or alternatively consisting
of, a nucleotide sequence referenced in the fifth column of Table 2
and described by the general formula of a-b, whereas a and b are
uniquely determined for the corresponding SEQ ID NO:X referred to
in column 3 of Table 2. Further specific embodiments are directed
to polynucleotide sequences excluding one, two, three, four, or
more of the specific polynucleotide sequences referred to in the
fifth column of Table 2. In no way is this listing meant to
encompass all of the sequences which may be excluded by the general
formula, it is just a representative example. All references
available through these accessions are hereby incorporated by
reference in their entirety.
TABLE-US-00003 TABLE 2 NT Gene cDNA Clone SEQ ID Contig No. ID NO:
X ID Public Accession Numbers 1 HTTEZ21 11 706584 R37023, R46612,
R53545, R42200, R46612, H16400, H19801, H29808, N39235, N39576,
N75064 1 HTTEZ21 197 637737 R37023, R42200, R46612, R53545, R42200,
R46612, H16400, H19801, H29808, N39235, N39576, N75064 1 HTTEZ21
198 319919 R37023, R42200, R46612, R53545, R42200, R46612, H16400,
H19801, H29808, N39235, N39576, N75064 2 HBGBW52 199 330742 H95265,
H95253 7 HCUIM14 17 330779 H92793 9 HEIAX07 201 330847 T96944 10
HSAXR76 202 330900 H14823, H24074, W15573, W31649 12 HCFAW04 204
331039 H18999, H19100, H49530, H51389 13 HLMAV65 23 646698 T56702,
N32010, N32399, AA076337, AA076338, AA425636, AA424836 13 HLMAV65
206 331549 T56702, N32010, N32399, AA076337, AA076338 13 HTXEF04
207 513807 T56702, N32010, N32399, AA076337, AA076338 14 HPMFD84
208 332678 R63484, R69614, R69634, R69704, R69731, H01317, R87745,
H70638, N22190, N91941, W24315, W46759, W47175, W49748, W89186,
AA010780, AA011188, AA056460, AA149744, AA149912 15 HE6DB26 209
333590 R22155, R78386, W32024, W44434, W57584, AA054473 16 HHFFL33
26 331218 T64856, T72598, R19472, R21257, R43813, R51233, R51234,
R43813, H07887, H07978, H88789, H89008, H88789, N34394, N48632,
N67378, W47273, AA131246, AA131514, AA167333 21 HCFME41 211 338913
H56624, H80486, H85080, H87334, H90056, AA079169, AA079170,
AA172091 23 HOSEX08, 212 339067 T61276, R39946, R44197, R44197,
R61214, R61334, R79100, N22563, N33794, N75784 HOSEX91 25 HEBEB69
213 340233 AA084458 26 HE6EH18 214 340242 R38946, R45145, R45976,
R45976, R45145, N51406 29 HBMVK68 39 340290 R12213, T79175, T79259,
R39934, R52525, R52573, H90609, N34140, N52014, N54627, AA007443,
AA007495, AA075148, AA075130, AA125964 30 HMKDC66 40 340310 R72889,
R87492, N59283 31 HMKCU94 215 340313 T55569, T55648, N92368,
AA069305, AA098911 32 HRDEW41 42 621126 R16110, R16010, R59375,
R59433, R69306, R69307, R80568, R80569, H48373, N25999, N36631,
N59357, N76458, N95556, W52821, AA025244, AA026038, AA027997,
AA034306, AA034307, AA043521, AA134138, AA130293, AA132093,
AA132193 32 HRDEW41 216 340315 T69152, T69213, T80080, T80327,
R19043, R27520, R38534, R38898, R44031, R44031, R67769, H11493,
H11852, H13644, H22161, H39529, H43488, N32678, N50022, N51861,
N54126, N54677, W16972, W32896, W35293, W38598, N89624, N90277,
AA027830, AA027892, AA035739, AA055806, AA069223, AA078890,
AA078891, AA099437, AA099478, AA101431, AA112543, AA121794,
AA129629, AA136251, AA143110, AA150576, AA157125, AA158242,
AA158709, AA159975, AA159976, AA160357, AA159491, AA160534,
AA160629, AA165150, AA165151, AA164643, AA166799, AA169647,
AA169822, AA173082, AA187009 33 HTOJN06 43 340319 T65361, R63807,
R64413, H13435, H13484, N20133, W67880, AA013276, AA017015,
AA027808, AA029304, AA056160 34 HBGDA21 217 340334 T51241, T51249,
T51356, T51363, R11160, R11161, H45288, H51300, H97524, N72234,
W40241, W40244, W45480, W45486, W46818, W60803, W60934, W95170,
W95133, AA166967, AA167045, AA191454 35 HFGAK75 218 340384 R12864,
R38437, R49877, R49878, R56104, R56274, R56369, R56497, R62408,
R62409, R80227, R80333, H22843, H22844, N40663, N80562, W07685,
AA039646, AA039645, AA084546, AA143714, AA143751, AA179782,
AA180189, AA180499, AA181066, AA181067 36 HHPBD40 46 340399 T64787,
T72540, R17957, R17989, R19224, R43447, R43480, R44487, R54708,
R43447, R43480, R44487, H02892, H03792, H08062, H39801, R96240,
R96296, H55925, H61904, H79118, H79209, AA004660, AA004665,
AA004855, AA032203, AA032270, AA116120, AA116121, AA193583,
AA193627 37 HOVCL83 47 340407 R38234, N21541, N50827 39 HBICM48 49
497206 T66994, T66995, T90274, T81704, R16104, R26725, R26952,
H09075, H09424, R94094, R96182, R96183, N79107 39 HBICM48 219
340480 T66994, T90274, R16104, R26725, R26952, H09075, R94094,
R96182, R96183, N79107 40 HLTCL35 50 635471 T81400, T81399, T97001,
T97115, H80498, H81416, H91376, AA258096 40 HLTCL35 220 340536
T81400, T81399, T97001, T97115, H80498, H81416, H91376 41 HLHCK50
51 792650 T49685, T69050, T85769, R00655, R20782, R20827, R20891,
R27806, R27898, R33703, R42007, R52046, R52047, R42007, R66604,
R66605, R66641, R72373, R72374, H00427, H00428, H00955, H00956,
H15928, H25414, H27488, H28652, H28790, H28764, H45792, H46560,
R94564, R94565, H51645, H62694, H97650, N70289, N81184, N94729,
N98562, W61307, W65372, AA009816, AA009817, AA057612, AA057696,
AA078845, AA079279, AA079449, AA080946, AA129723, AA133263,
AA133975, AA134086, AA134087, AA130451, AA147199, AA149934,
AA148549, AA150474, AA158715, AA159354, AA159543, AA160770,
AA256558 41 HLHCK50 221 340581 R33703, R66605, R72374, H00956,
H15928, H27488, H45792, H51645, H97650, N70289, N81184, N98562,
AA009816, AA009817 42 HRSAN45 52 340592 T86115, R16132, N78938,
N94960, AA062910, AA128760, AA129755, AA133232, AA135673 43 HSNBB14
222 340597 R33866, H03918, H03919, H04645, H04665, H20263, H20335,
H40675, AA015653, AA045174, AA045293 44 HMABL38 54 638246 W44379 45
HSKDK47 55 340647 R30992, N43847, N50721, N73309, W31785, W32138,
W44470 46 HOSFH03 56 586413 R13900, R20375, R21746, R21791, R24287,
R24396, R26219, R26218, R43762, R43762, H13483, H18658, H18745,
H23200, H25669, H26256, H27373, H28341, N20036, N21618, N28857,
N34176, N34653, N35351, N44596, N63461, N63485, N64276, N72203,
N74518, N94754, N94784, W00446, W02907, W70310, W78207, AA056747,
AA058427, AA069438, AA069465, AA082046, AA151448, AA151449,
AA227737, AA228122, AA232358 46 HOSFH03 224 340661 R13900, R20375,
R21746, R21791, R24287, R24396, R26219, R26218, R43762, R43762,
H13483, H18658, H18745, H23200, H25669, H26256, H27373, H28341,
N20036, N21618, N28857, N34176, N34653, N35351, N44596, N63461,
N63485, N64276, N72203, N94754, N94784, W00446, W02907, W70310,
W78207, AA056747, AA058427, AA069438, AA069465, AA082046, AA151448,
AA151449 47 HOGAV75 57 589232 R17443, R56086, R56198, H24103,
H28916, R97505, R97504, H48742, H67668, H98111, N23177, N30825,
N31695, N32642, N42454, W16865, W19323, W19290, W24066, W25486,
W25532, W76176, AA021599, AA069784, AA084777, AA101750, AA101662,
AA148552, AA148551, AA150175, AA161441, AA164926, AA227354,
AA227355, AA232813, AA463377 47 HOGAV75 225 340663 R17443, R56086,
R56198, H24103, H28916, R97505, R97504, H48742, H67668, H98111,
N23177, N30825, N31695, N32642, N41350, W16865, W19323, W19290,
W24066, W25486, W25532, W76176, AA021599, AA069784, AA069834,
AA081911, AA084777, AA101750, AA101662, AA148552, AA148551,
AA150175, AA161441, AA164926 48 HFCAI74 58 340677 H49529, H49767,
W90083, W90209, AA031975, AA031974, AA084096 49 HAGBI17 59 652566
T40566, R10098, T82863, T27093, H06498, H06555, H22066, H60162,
H60205, H88668, H88715, N52473, N63318, N77435, N95489, W16826,
W49660, W49744, W84518, AA025137, AA025153, AA037221, AA037220,
AA088241, AA137103, AA150492, AA172393, AA173824, AA196791,
AA235338, AA235769, AA427925 49 HAGBI17 226 340679 T40566, R10098,
T82863, T27093, T27149, H06498, H06555, H22066, H60162, H60205,
H88668, H88715, N25857, N52473, N63318, N77435, N95489, W16826,
W49660, W49744, W84518, AA025137, AA025153, AA037221, AA037220,
AA088241, AA137103, AA150492, AA156695, AA172393, AA173824 51
HPRCA31 61 581445 T66819, T66820, T85679, R13750, R13931, R25695,
R38596, R53117, R53215, R83626, R83773, R89758, R89781, H55813,
H60033, W69796, W69884, W73236, W73291, W94781, W95297, AA071091,
AA156167, AA156502, AA223256, AA223356, AA458973, AA459189 51
HPRCA31 227 340708 T66819, T66820, T85679, R13750, R13931, R25695,
R38596, R45667, R53117, R53215, R45667, R83626, R83773, R89758,
R89781, H60033, W69796, W69884, W73236, W73291, W94781, W95297,
AA071091, AA156167, AA156502 52 HPRCE95 62 646288 T75226, R05772,
R05858, R35378, R44456, R51239, R51240, R44456, R67589, R99125,
R99667, H80457, H81377, H90424, N22077, N75320, W04771, W40159,
W45375, W57547, W78826, W80725, AA001824, AA026071, AA026072,
AA099306, AA099816, AA160137, AA169306, AA171668, AA173427,
AA193330, AA194113, AA430050, AA430259 52 HPRCE95 228 340712
T75226, R05772, R05858, R19669, R35378, R44456, R51239, R51240,
R44456, R67589, R99125, R99667, H62128, H80457, H81377, H90424,
H90480, N22077, N75320, W04771, W40159, W45375, W57547, W78826,
W80725, AA002028, AA001824, AA026071, AA026072, AA099306, AA099816,
AA160137, AA169306, AA171668, AA173517, AA173427, AA193330 53
HHTLC66 63 340716 T93117, T93217, R18513, R35994, R36368, R54402,
R54501, R78179, H21223, H30750, H43172, R90745, R90785, H94364,
N51284, W02098, W32304, AA017239, AA017330, AA025447, AA025448,
AA083964, AA112163, AA127498, AA127520, AA147192, AA158669,
AA158670, AA158668, AA158671, AA160391, AA181678, AA190725,
AA190909, AA191485 54 HMADJ02 64 665037 T55474, T85674, R32236,
R35180, R81909, H18878, H20360, H22508, H41039, H46678, R89209,
R99132, H58424, H69768, H72482, N42965, N44128, N46497, N57338,
W25680, W25676, W52136, W67531, W92351, AA018287, AA018347,
AA127971, AA151257 54 HMADJ02 229 340733 T55474, T55401, T60074,
T91252, T85674, R32185, R32236, R34238, R35180, R35565, R50840,
R81805, R81909, R82743, H18878, H18879, H20171, H20360, H22469,
H22508, H29277, H40286, H41039, H41469, H42928, H46679, H46678,
R89101, R89209, R99132, H48432, H58424, H58425, H69768, H72482,
H72883, N30666, N32244, N36122, N36966, N38925, N42965, N44128,
N46497, N47999, N51206, N57338, N69681, N94289, N98733, W02508,
W02840, W25680, W25676, W30822, W31172, W37751, W37750, W52136,
W67532, W67531, W92351, AA001534, AA018287, AA018347, AA018348,
AA025474, AA025393, AA054937, AA055147, AA084018, AA112174,
AA127920, AA127971, AA127062, AA125939, AA142846, AA151257,
AA151258 55 HPRCU93 230 340745 AA044652 56 HSAXS65 231 340750
R39323, H05070, H41666, N31947, N42783, N63271, N90340, AA044807,
AA044784, AA099107, AA131748, AA131836, AA143351 57 HKTAG35 67
750765 T63010, T63155, N80942, W78114, W79821, AA027951, AA027916
57 HKTAG35 232 422830 T63010, T63155, N80942, W78114, W79821,
AA027951, AA027916 57 HMEFX42 233 340769 T63010, T63155, N80942,
W78114, W79821, AA027951, AA027916 58 HHFHN61 68 340778 R39487,
R39488, R70790, R70844, H65872, H65871, N35912, W16998, AA007279,
AA165622, AA165658, AA193643 62 HATDT67 72 637512 W95483, W95374,
AA464846 62 HATDT67 236 340872 W95483, W95374 64 HMWEX24 74 636164
T63776, T63811, R23451, R26050, R54566, R71431, H14303, H21785,
H21786, H25266, H30712, H41912, H44413, R88668, R88667, H83339,
H99419, N20174, N29075, N33561, N33856, N34443, N37060, N42851,
N46372, N51305, N62831, N67782, N72280, W01367, W03024, W03584,
W20085, W72602, W76567, N91480, AA036951, AA036992, AA069144,
AA069232, AA074084, AA075977, AA127754, AA179521, AA179344,
AA179568, AA179576, AA236305, AA252367, AA252744, AA256845,
AA256211, AA256433, AA262880, AA418903, AA421392 64 HMWEX24 238
340878 T63776, T63811, R23451, R26050, R54565, R54566, R71431,
H83339, H99419, N20174, N29075, N34443, N37060, N42851, N46372,
N51305, N62831, N72280, W01367, W03024, W20085, W72602, W76567,
N91480, AA074084, AA075977, AA179521, AA179344, AA179568, AA179576
65 HSGBA84 75 340917 T70586, T70850, H45252, H45316, W94688, W95194
66 HTOCD52 239 340926 R96763 68 HKIXR69 78 498091 T27138, T27139,
R77447, R77540, H13397, H24086, H24199, N93267, W25026, AA033849,
AA034353, AA041406, AA236384, AA262937 68 HKIXR69 240 340959
T27138, H24086 69 HETGJ09 79 340981 H15437, H98603, N36770, N45930,
N49865, N49875, N51072, N51762, N70334, W94942, AA056608, AA137182
70 HOBNC61 80 340986 R41269, R41269, R69456, R70443, H38923,
N59126,
N59131, N75166, N75171, W15406, W39519 71 HFFAH94 81 341007 T40317,
T51739, T53472, T46971, T47506, T48920, T48921, T49015, T49016,
T49175, T49298, T49299, T49695, T49701, T49997, T50027, T50057,
T52080, T52658, T52659, T53330, T53331, T53627, T53663, T53758,
T54085, T54152, T54175, T63372, T75017, R06020, R23398, R33890,
R63243, R63756, R75655, R75765, H00189, H00241, H09883, H17683,
H42345, R86098, R87290, R89690, R91869, H52013, H64772, H73693,
H75892, H84586, H89190, N29678, N32054, N42026, N47931, N56634,
N57153, W07751, W07769, W20151, W31959, W38588, W44842, AA001770,
AA058334, AA081895, AA083423, AA083636, AA083919, AA084837,
AA085235, AA115438, AA122406, AA133479, AA132347, AA136452,
AA146919, AA147421, AA151894, AA152160, AA156975, AA157489,
AA157487, AA157934, AA159425, AA159732, AA164481, AA165483,
AA172314, AA179193, AA181685, AA187840, AA188093, AA186492,
AA186783, AA186793, AA186918, AA186935, AA187007, AA187053 73
HSQEL25 83 639101 R85294, AA150488 73 HSQEL25 241 781516 R85294,
AA150488, AA186761 73 HSQEL25 242 341015 R48156, R72687, R85294,
AA150488, AA187629 74 HEBEG68 84 341023 T67556, T69552, T84413,
T85269, T96893, R46761, R46856, H01797, H69563, H70019, H84661,
H85196, H92136, H92137, H93474, H93473, H96838, N51712, N51801,
N62956, N63622, N75603, N80289, N95238, W00738, W05621, W07127,
W21067, W94925, W92196, W95269, W95270, AA179187 75 HBIAB39 85
667525 T64303, T64688, R66180, R67060, H19829, H19859, H29834,
H87148, H87649, H89959, N35923, N36775, N42201, N45936, N72214,
W00490, AA036653, AA037218, AA064615, AA064673, AA082867, AA085339,
AA121400, AA242888, AA252242, AA262055 75 HBIAB39 244 341025
T64303, T64688, R66180, R67060, H19829, H19859, H29834, H87649,
H89959, N35923, N36775, N45936, W00490, AA036653, AA037218,
AA064615, AA064673, AA082867, AA085339, AA121400 76 HTXDU73 86
341029 T66720, R16261, H84276, H85593, AA148543, AA156634 77
HOEAS24 245 634500 T67464, T69497, T86573, T86750, T87354, T78865,
T95251, T95252, R51805, R51806, R60720, R60721, R61717, H25779,
H61825, H61824, N48718, W44894, AA010204, AA010241, AA043867,
AA044312, AA460349, AA460778 77 HOEAS24 246 341034 T67464, T69497,
T86573, T86750, T87354, T78865, T95251, T95252, R51806, R60720,
R60721, R61716, R61717, H25779, H61825, H61824, N48718, W44894,
AA010204, AA010241, AA043867, AA044312 78 HTEIY30 88 341110 R12814,
R20726, R40236, R20726, R40236, H09494, H09495, N21561, N31709,
N31708, N42363 79 HSKNE46 247 341121 R74441, R86080 80 HPMFL27 90
341124 T48869, T48870, T49904, T53681, R25109, R25110, R25671,
R27528, R31935, R31981, R33395, R36105, R36203, R68383, R68588,
R73906, R73999, R75632, R75732, R75957, R78948, R79050, H02580,
H02668, H03502, H03868, H03869, H12451, H12498, H12839, H51263,
H51833, N50695, N58781, N77468, AA035486, AA035018, AA147247,
AA147933 81 HMWDN32 91 341126 R02692 82 HPRAX55 248 341130 R46337,
R54837, R72518, H41918, H41962, H58330, H58719, H82709, N32438,
N72856, AA004403, AA053671, AA164257, AA164368 83 HHFFW36 93 341162
T65334, R06680, R32977, R32978, R33001, R33000, R64154, R64264,
R66122, R67730, H01946, H09092, H09441, H12088, H12089, H39822,
H88622, H88622, H99675, N28430, N33410, N44873, N72029, N99481,
AA128356, AA128519, AA192635, AA192784, AA192783 84 HE2PL77 94
341180 T65492, T69710, T70367, R14102, H13508, H24901, H30495,
R84506, R85296, R92833, H53504, H53874, H58116, H64947, H64948,
H83182, H88075, H88075, N20214, N29281, N33314, N44846, N67850,
N74617, W00890, W03453, W73139, W73119, AA039436, AA039435,
AA143645 85 HSDFV29 95 726747 T93478, T96318, H65329, H65527,
N20594, N24306, N26872, N28990, AA040017, AA056928, AA057074 85
HSDFV29 249 426853 T93478, T96318, R02524, H65329, H65527, N20594,
N24306, N26872, N28990, N53061, N73248, W93593, AA010653, AA037624,
AA040017, AA056928, AA057074, AA134768 85 HCQAV53 250 341197
T93478, T96318, R02524, N20594, N26872, N53061, N73248, W93593,
AA010653, AA037624, AA040017, AA057074, AA134768 86 HTPEG42 96
655475 R68003, R68002, H92758, W20199, W38589, W74674, W74780,
N90213, N90553, AA043957, AA043823, AA126598, AA157016 86 HTPEG42
251 341206 W38589, W74674, W74780, N90213, AA043957, AA043823,
AA126598, AA157016 87 HLHDR57 97 341213 R60239, R60474, N70344,
W04155, AA037509, AA039803, AA054769, AA112956, AA172011, AA191112
88 HAUAV32 252 341214 N95062, W19506, W38899, W52820, N89912,
AA035640 89 HNEBI60 253 341219 R08642, R08738, T78434, T96975,
T97087, R27984, R46404, R46404, R63138, H21791, H21792, H26134,
H26399, H41136, H41403, H43201, H43240, R98484, R98531, H56649,
H56726, H78891, N54287, AA150592 90 HSHCJ16 100 341227 T63745,
T74291, T77596, T91992, T92076, R01666, R02606, R30785, R50817,
H14952, H17656, H17773, R88235, R88234, R96151, W37867, W37858,
W52057, W60602, AA126008, AA126114, AA134190, AA134208, AA173351 91
HTSEL31 101 341228 T77424, T86561, T86739, T86066, R17360, R38366,
R42711, R44414, R42711, R44414, R69488, R70382, R75611, R75701,
H26361, H27471, H40753, AA074108, AA074906, AA074981, AA133487,
AA148391, AA148392, AA187336, AA188462 92 HAUBL57 254 630116
T87331, T87432, R16493, R16793, R20740, R22276, R22746, R34517,
R39968, R52851, R20740, R39968, H30186, H42692, H39131, H43926,
H44586, H46389, R99312, R99404, H84964, N41538, N42109, N98255,
W01018, W19986, W81009, W99356, W99386, N90413, AA009837, AA010386,
AA010322, AA019377, AA033587, AA056148, AA079689, AA081447,
AA100074, AA100159, AA114264, AA134510, AA134519, AA157284,
AA157365, AA161199, AA161200, AA181973, AA187561, AA227076,
AA227062, AA232373, AA233577, AA233594, AA256297, AA256403,
AA417901, AA418057, AA430035, AA430244 92 HAUBL57 255 341229
T50378, T51069, T87331, T87432, T80282, R16493, R16793, R16889,
R16943, R20740, R22221, R22276, R22746, R27597, R27696, R34517,
R36581, R36582, R39968, R43467, R43494, R46644, R49072, R49692,
R52816, R52851, R49072, R43467, R43494, R20740, R46644, R39968,
R49692, R62955, R63008, R66425, R66819, R67917, H21718, H27063,
H30186, H42692, H39131, H43926, H44586, H46389, R99312, R99404,
H64752, H64751, H84964, H97190, H64752, H97608, N26236, N28697,
N33047, N33556, N41538, N42109, N70229, N79806, N98255, W01018,
W19986, W44335, W44449, W81010, W81009, W90684, W90779, W99356,
W99386, N90413, AA009837, AA010386, AA010322, AA019377, AA019378,
AA026243, AA029623, AA033587, AA033588, AA056089, AA056148,
AA079689, AA081447, AA081752, AA082528, AA084728, AA100074,
AA100159, AA113183, AA114264, AA127588, AA134519, AA156575,
AA157284, AA157365, AA161199, AA161200, AA181973, AA187561 93
HODAS59 103 341231 T50466, T54712, T66739, T66740, T82266, T83553,
T85787, R01232, R01346, R15302, T26641, R22176, R31989, R69835,
R70071, H48712, H53486, H69014, H69699, H70119, H70952, H71292,
H71306, H75396, H94030, N58909 94 HE6CT48 256 341236 H93834,
N25183, N25233, N63979, W21048, AA045563, AA143229, AA143285 95
HMDAA61 257 341237 R46285, H01667, H01668, H03202, H03998, H91660,
H98944, H98943, N32616, N34435, N34436, N35623, N47837, N48668,
N66778, N72838, N76146, N93528, W05651, W21334, AA023016, AA022490,
AA043501, AA044658 96 HAQBK61 106 670590 R48595, R48697, R51237,
R51238, R52061, R52060, R73248, R81473, R81668, H44607, R88507,
R95696, R95697, R97751, R97750, H52217, H56556, H63572, H63620,
H65821, H65907, H66997, H66998, H69525, H69980, H70482, H87168,
H87670, H89849, H90696, H94311, H96238, N40986, N44995, N45109,
N47641, N49294, N53837, N57496, N67811, N69411, W81597, W81592,
N91442, AA031470, AA031612, AA126236, AA126401, AA130831, AA130946
96 HAQBK61 258 341240 R48595, R48697, R51237, R51238, R52061,
R52060, R73248, R81473, R81668, H44607, R88507, R95696, R95697,
R97751, R97750, H52217, H56556, H63572, H63620, H65821, H65907,
H66997, H66998, H69525, H69980, H70482, H87168, H87670, H89849,
H90696, H94311, H96238, N40986, N44995, N45109, N47641, N49294,
N53837, N57496, N67811, N69411, W81597, W81592, N91442, AA031470,
AA031612, AA126236, AA126401, AA130831, AA130946 97 HAQBF73 260
341243 T62952, T92192, T92225, T77665, R11188, T81487, T81700,
R00485, R00584, R02320, R02422, R20298, R22227, R23678, R40975,
R42968, R43639, R42968, R40975, R43639, R67269, R68907, H02952,
H04250, R93173, R93265, R94343, H56904, H56992, H83900, H97837,
N29328, N30433, N42347, N47549, N47550, N47895, N49858, N51150,
N56938, N57020, N59159, N63879, N66463, N71798, N75895, N76091,
N77882, N92023, W04435, W05139, W05635, W19637, W57588, W73239,
W73294, W92204, W92205, N89882, AA074753, AA074836, AA171536,
AA192742, AA192815 98 HAQBT94 108 341244 R00206, R01689, R02630,
N30938, N34891, N99874, W25308 99 HETHE07 261 634935 R24661,
R25164, R31031, R31521, R36666, R39748, R41825, R41825, R76400,
R76739, H03481, H03576, H29806, H73483, H75574, H96448, H96862,
W67151, W67394, W81230, W81266, AA040399, AA054370, AA054507,
AA074719, AA075225, AA076083, AA076164, AA115461, AA115066,
AA165265, AA165266, AA164408, AA164409, AA164486, AA164492,
AA164493, AA164487, AA169668, AA169281, AA169420, AA173414,
AA173443, AA173439, AA173493, AA173499, AA173504 99 HETHE07 262
341247 R24661, R25164, R31031, R31521, R36666, R39748, R41825,
R41825, R76400, R76739, H03481, H03576, H29806, H73483, H75574,
H96448, H96862, W67151, W67394, W81230, W81266, AA040399, AA054370,
AA054507, AA074719, AA075225, AA076083, AA076164, AA115461,
AA115066, AA165265, AA165266, AA164408, AA164409, AA164486,
AA164492, AA164493, AA164487, AA169668, AA169281, AA169420,
AA173414, AA173443, AA173439, AA173493, AA173499, AA173504 100
HLQAB52 263 341250 H43350, N27368 101 HCRAM28 111 341252 T65107,
T97334, R53476, H09583, H30548, H71011, H95075, N29391, N30834,
N41362, N74154, N75079, N92831, N95057, W19524, W72254, AA025307,
AA024537, AA025223, AA160270, AA160271, AA167140, AA167276,
AA188817 101 HIBEK16 265 638538 T65107, T97334, R53475, R53476,
H09583, H30548, H71011, H95075, N29391, N30834, N41362, N57317,
N74154, N75079, N92831, N95057, W04321, W19524, W25059, W72254,
AA025307, AA024537, AA025223, AA160270, AA160271, AA167276,
AA188817 102 HE2BG03 266 341253 N49408, N49498, N55047, W03070,
W67229, W67339, W77952 103 HEBDJ82 113 341263 R11788, T96282,
T96281, R18963, R33113, R33261, R37707, R43974, R52405, R52406,
R52462, R52463, R43974, R55831, R55917, R56079, R56190, R63506,
R63551, R66072, R67672, H04652, H04674, H14855, H88794, H89018,
H88794, N29740, N57160, W45643, W45596, W69792, W69880, AA043192,
AA043594 104 HCUBC79 114 639176 R26344, R66398, R67132, R67854,
R68609, R69202, R74181, R74266, H13077, R93558, H87000, H95448,
H96357, W39616, N91087, AA134291, AA176656, AA176854, AA188764,
AA256577 104 HCUBC79 267 587008 R26344, R66398, R67132, R67854,
R68609, R69202, R74181, R74266, H13077, R93558, H87000, H95448,
H96357, W39616, N91087, AA134291, AA176656, AA176854, AA188764,
AA256577 104 HCUBC79 268 341271 R26344, R66398, R67132, R67854,
R68609, R69202, R74181, R74266, H13077, R93558, H65931, H87000,
H95448, H96357, W39616, N91087, AA134291, AA176656, AA176854,
AA188764 105 HSVAF07 115 647593 T49542, T49543, N79115, N93812,
W44963, W65318, W65317, AA005085, AA099521, AA223969, AA426123,
AA425831, AA428347 105 HSVAF07 269 637678 T49542, T49543, N42222,
N62353, N79115, W44963, W44467, W65318, W65317, AA005085, AA099521,
AA099520, AA223969 105 HSVAF07 270 341274 T49543, N42222, N62353,
N79115, N93812, W44428, W44467, W65318, AA005085, AA099521,
AA099520 106 HT3AM65 271 550213 R50060, H22175, R84872, R85806,
R86903, R86915, H54447, H54537, H71458, H71587, N52360, N64314,
N75131, W52304, W56703, AA025637, AA025638, AA033583, AA033584,
AA035172, AA043426, AA043427, AA148635, AA148636, AA164764,
AA164765, AA215808 106 HT3AM65 272 341277 R50060, H22175, R84872,
R85806, R86903, R86915, H54447, H54537, H71458, H71587, N52360,
N64314, N75131, W52304, W56703, AA025637, AA025638, AA033583,
AA033584, AA035172, AA043426, AA043427, AA148635, AA148636,
AA164765 107 HE6DK18 117 341297 R01532, N41434, N70473, N70471,
N72861, W03083, W03834, W45060, W45070, W48640, W51956, N91018,
AA039299, AA039300, AA046006, AA046007 108 HEBEK93 118 656435
T63347, T63427, T63669, R39259, R68780, R70392, R73655, R73737,
R80228, N26735, N27000, N30045,
N40122, N62562, N63462, N79017, N95323, N99129, W19257, W24778,
W42778, W42893, W74164, W76535, AA025427, AA037007, AA083212,
AA083311, AA099834, AA115588, AA115078, AA135669, AA135684,
AA143000, AA159331, AA159430, AA171693, AA171808, AA173590,
AA173737, AA173920 108 HEBEK93 273 341298 T63347, T63427, T63669,
R39259, R68780, R70392, R73655, R73737, R80228, R80334, N26735,
N27000, N30045, N40122, N62562, N63462, N79017, N95323, N99129,
W19257, W24778, W42778, W42893, W74164, W76535, AA025427, AA037007,
AA083212, AA083311, AA099304, AA099834, AA115588, AA115078,
AA135669, AA135684, AA143000, AA159331, AA159430, AA171693,
AA171808, AA173590, AA173737, AA173920 109 HJPCM10 119 671656
T52165, T52241, T90189, T84079, R32492, R32602, H57835, H86933,
H86932, N36854, N58903, N66193, N66878, N93552, N93556, N95615,
N99190, AA043182, AA043183, AA042984, AA243295 109 HJPCM10 275
341315 T52165, T52241, T90189, T84079, R32492, R32602, H57835,
H86933, H86932, N36854, N58903, N66193, N66878, N93552, N93556,
N95615, N99190, AA043182, AA043183, AA042984, AA043039 110 HSXBL78
120 341338 T74710, T75297, R11916, R13124, R14338, R25727, R39185,
R40603, R40664, R43583, R40664, R43583, R40603, R62471, R75755,
H05460, H07011, H10315, H10314, H27091, H29792, H29791, R95668,
N29515, N57459, W86201, W86202, AA082028 111 HOEAW81 121 638133
T83147, T84144, H87571, H91807, N30742, N41841, N94464, N94563,
W15644, W30743, W30854, W31005, AA081589, AA115690, AA133509,
AA134307, AA134308, AA134315, AA134316 111 HOEAW81 276 341360
T89384, T90621, T83147, T84144, H87571, H91708, H91807, H92083,
N30742, N41841, N80826, N94563, N98724, W15644, W30743, W30854,
W31005, AA081589, AA084667, AA115690, AA133509, AA134307, AA134308,
AA134315, AA134316 112 HOEAP41 122 341363 R98994, N51380, N78067,
W19481, W42782, W42897, W52206, W69436, W69519, W73362, W73439,
W94387, N90296, AA037138, AA046403, AA069722, AA082067, AA082024,
AA157654, AA157755 113 HEAAR60 123 667582 T62691, T62842, T81833,
T99606, N24671, N35462, W90766, W90699, AA188889, AA189105,
AA460709, AA460883 113 HEAAR60 277 341368 T62842, T81833, T99606,
N24671, N35462, W90766, W90699, AA188889, AA189105 114 HTXGS75 124
341369 T47597, T47598, T90304, T82977, T99735, T99736, R17706,
R38999, R42722, R42786, R42722, R42786, H85229, H95203, N24689,
N29773, N30154, N35767, N36667, N57192, N70880, N76099, N98288,
W05394, W06883, W19087, AA056547, AA056668, AA121234, AA121235,
AA149160, AA149159, AA182535, AA188679, AA191046 115 HOVBA03 125
638141 H83533, AA143176, AA159616, AA165673, AA159698 115 HOVBA03
278 341382 H83533, N47656, N47744, AA143176, AA159616, AA165673,
AA159698 117 HBMUW78 127 656003 T53926, T92254, T92553, T92625,
T93804, T93855, R32495, R32605, R40235, R40235, R63831, R64443,
H13418, H13461, H22555, H24408, R94238, R94335, H57449, H59016,
H59017, H64002, H64051, H70912, H70995, H78449, H70912, N20822,
N25219, N30547, N39398, N44197, N62369, N62884, N62878, N77785,
N77791, N79139, W70226, AA059074, AA133688, AA169568, AA169873,
AA195055, AA195678, AA195679, AA213529, AA214545 117 HBMUW78 280
341396 T92254, T92553, T92625, T93804, T93855, R32495, R32605,
R40235, R40235, R63831, R64443, H13418, H13461, H22555, H24408,
R94238, R94335, H57449, H59016, H59017, H64002, H64051, H70912,
H70995, H78449, H70912, N20822, N25219, N30547, N39398, N44197,
N62369, N62884, N62878, N77785, N77791, N79139, W70226, AA059074,
AA133688, AA169568, AA169873 118 HASAS24 128 341406 T52688, T52687,
R50972, R59542, H00394, H11046, H71487, H71487, N36814, N46032,
N69321, AA099572, AA101976, AA150971, AA151080 119 HSIDN55 129
341411 T47196, T90531, T81327, T83066, R02389, R49397, R49397,
R73170, H69590, N46989, N49400, N49489, N51462, N57532, N66084,
N79047, N92406, N98473, W24197, W24631, W55871, W56299, W65365,
W72845, W76092, AA029710, AA029642, AA039563, AA039564 120 HGBGZ64
130 341422 T77791, R10242, T84472, R89115, R94929, R94990, R95013,
N51474, N53615, N80601, W42706, AA040741, AA040742, AA043440,
AA053029, AA084287, AA084288, AA151396, AA151397 121 H6EBJ64 131
637481 R51043, R51157, R79165, R79266, H05444, R91173, R91222,
R91701, R92139, R99529, H63402, H63455, H65251, H65864, H65874,
H69798, H70976, W42837, W84415, AA029221, AA029222, AA031485,
AA031464, AA037310, AA045771, AA045962, AA193397, AA253178,
AA253078 121 H6EBJ64 281 341423 R51043, R51157, R79165, R79266,
H05444, R91173, R91222, R91701, R92139, R99528, R99529, H63402,
H63455, H65251, H65252, H65863, H65864, H65873, H65874, H69798,
H70976, W42747, W42837, W84415, AA029221, AA029222, AA031485,
AA031464, AA037310, AA045771, AA045962, AA193397 122 HOECP43 132
341430 H20033, H20113, H50094, H50095, N44916, N66466, W81365,
W81624, AA131047, AA130966 123 H2CBV31 133 341436 T94047, T94797,
T75357, R10575, T80348, T80455, T81292, R01189, R01300, R17953,
R20854, R21132, R34459, R38683, R39040, R39061, R42034, R43442,
R46727, R42034, R43442, R46727, R67985, H19293, H19292, H78408,
H78506, N39442, N48310, N48554, N49035, N94078, W02832, W30814,
W49543, W49774, AA039485, AA039484, AA062802, AA082406, AA088216,
AA189147 124 HPCAD23 134 341438 T39517, T40647, R08890, T79580,
T79658, R39186, R44549, R44549, H29275, H29360, H68472, N29403,
N36337, W92928, W92929, AA012938, AA012885, AA017220, AA017313,
AA037431, AA037247, AA037535 125 HSPAG15 135 341444 N24480, N35123,
N62648, N68160, N80371, N80618, AA036900 126 HELGH31 136 341446
R66409, R67143, R77266, R77313, H01414, H01413, H98804, H99892,
N24566, N25024, N30234, N34009, N57126, N66135, N66161, N98658,
N98686 127 HUSHH48 282 341470 R13839, R36985, H03004, H05634,
H12993, H50184, H99245, N26368, N27512, N29876, N42211, N42520,
N58934, N64797, N73798, N75644, N95515, N99424, W95429, W95556,
AA031322, AA031321, AA035435, AA035436, AA101558, AA101559,
AA179484, AA179505, AA179536, AA179525, AA189168 128 HLYAU95 138
341473 T70334, T70418, R32765, R62187, H01280, H05481, H27058,
H61678, H61677, H73249, H98909, N22192, N24242, N32166, N32167,
N32933, N71233, W01111, AA028126, AA044276, AA044252, AA047430,
AA047469 129 HHSCV65 139 341476 R55035, R55065, R59065, R59066,
H11375, H19623, H19622, H22362, H22393, H40210, H40211, H46219,
H46469, H47010, N41006, N45484, N47600, W06944, W86913, N90740,
AA133763, AA133764 130 HTTAD57 140 341482 R62307, H98858, N36520,
N50449, N52248, N62504, N62512, N62869, N63975, N66790, N67455,
N75852, N77775, N79652, W07436, W60913, W68604, W68801, W69914,
W70179, W73347, W73467, W79509, W79608 132 HEBFU93 283 341488
R59976, H39102, R85866 133 HSGSC60 143 341490 T64219, T64405,
R36668, R39750, R71095, R71442, H56180, H56396, H99262, N40260,
N42314, N80584, W07706, W37443, W37568, W37623, W37624, W73084,
W73188, AA082510, AA099421, AA100989, AA134925, AA135041 134
HPMGD24 144 341495 T55461, T75312, T75315, R07965, R35914, R38680,
R49434, R49434, H24097, H29201, H29202, H51145, N40044, N49410,
N49500, N50862, W74365, W79169, AA031988, AA037809, AA037810,
AA133953, AA134020, AA148740, AA148739, AA181720, AA190790,
AA190382 135 HPTVC60 145 592550 R17247, R35296, H60973, W44347,
AA010018 135 HPTVC60 284 341499 R17247, R35296, H60973, W44347,
AA010018 136 HSKNE18 146 615251 H92812, N39336, N45183, W05346,
W24987, W63719, W67309, W67213, AA423922 136 HSKNE18 285 341502
H92812, N39336, N45183, N95495, W05346, W24987, W63719, W67309,
W67213 137 HMWIF35 147 611997 T78146, R61278, R61279, R73549,
H43446, H43445, H78315, H98875, N23168, N25102, N33160, N42291,
N57672, N57687, N59658, N68645, N69875, N77530, N91661, N98635,
W52629, W52630, W56233, W88468, W89033, AA420965, AA421063 137
HMWIF35 286 341506 T78146, R61278, R61279, R73549, H43445, H78315,
H98875, N23168, N25102, N33160, N42291, N57672, N57687, N59658,
N68645, N69875, N77530, N91661, N98635, W52629, W52630, W56233,
W88468, W89033 138 HMWGI25 148 341507 T99927, R00277, R62966,
R63020, H02510, H02511, H61375, H62397, H83984, N53025, N59209,
N75235, W67237 139 HSKGF03 149 668029 H20149, H20243, R94133,
H93190, N28258, N30555, N32545, N32551, N41895, N52973, N57208,
W02873, W03036, AA025259, AA025337, AA127243, AA126526, AA148192,
AA243272, AA418257 139 HSKGF03 287 645277 H20149, H20243, R94133,
H93190, N30555, N32545, N32551, N41895, N57208, W02873, W03036,
AA025337, AA127243, AA126526, AA148192, AA243272, AA418257 139
HSKGF03 288 341510 H20149, H20243, N28258, N30555, N32545, N32551,
N41895, N52973, AA025259, AA025337, AA148192 140 HMSKE75 150 341517
T39685, T58334, T58375, T95040, T95136, R01008, R01009, R01079,
R01806, R32520, R32634, R62417, R62608, R62657, H21569, H50551,
H50552, H97933, N45317, N54767, N62531, N71780, N79429, W60291,
W60382, W99324, AA039317, AA039237, AA126906, AA127110, AA133949,
AA133950, AA150269, AA150371, AA159341, AA159411, AA166691,
AA166802 141 HCMSH30 151 645267 T52564, T55946, T73366, W05488,
W21489, W23461 141 HCMSH30 289 341524 T48350, T48349, T52564,
T55946, T57088, T57193, T73366, T85101, T97261, T97372, R00232,
R00339, R34368, R34486, R36253, R36254, R82314, R82315, H13839,
H13885, H22323, H22324, R91853, R92692, N22767, N30057, N30064,
N30060, N40765, N40768, N70641, N71006, N71091, N76132, N76459,
N91781, W01941, W05460, W05488, W20019, W21489, W23461, W31478,
W31481, W32000, W32023, W32273, W44853, W72460, W76518, AA001714,
AA001805, AA005320, AA004243, AA010720, AA011167, AA024893,
AA024894, AA025007, AA024806, AA025085, AA027095, AA044819,
AA044844, AA056733, AA058387, AA099737, AA099736, AA134033 142
HTWCB92 152 341529 R12145, R15078, R59983, R67165, N50425, N50481,
N58860, N66880, N72725, W19898, N91244, AA023009, AA022475,
AA098971, AA099874, AA131860, AA131754, AA182936, AA181586 143
HBMDM46 153 669036 T89546, T79378, T83501, T96827, R48892, H47139,
H67305, H83557, H92560, N33828, N34173, N35285, N39763, N44626,
N56596, W03073, W07402, AA156699, AA236327, AA253287, AA417777 143
HBMDM46 290 341532 T89457, T79808, T83354, T96828, R48785, R78498,
R78548, H00257, H00258, H47139, H66023, H68516, H83449, H92561,
N24951, N25572, N26320, N29642, N32983, N32991, N39763, N56596,
N57800, N66437, N80274, W85944, AA041383 144 HFAMG13 154 341533
T64876, T90951, T85837, R17002, N59579, N73645, N75336, W21293,
W95777, AA025785, AA147507 145 HFXHL79 155 638175 R13316, H08897,
R98996, W07340, AA131594, AA157620, AA180960 145 HFXHL79 291 341534
R13316, H08817, H08897, R98996, W07340, AA131594, AA157620,
AA180960 146 HSNAK17 156 582244 T53909, R10445, T85536, R72952,
H15770, H62143, W42808, AA031603, AA031637, AA182758, AA232173 146
HSNAK17 292 341541 T53909, R10445, T85536, R72952, H15770, H62143,
W42808, AA031603, AA031637, AA182758 147 HCFBC03 157 635552 R32283,
R32295, R32329, R32338, R53752, R53753, R66100, R67703, H42383,
H43148, W90075, AA001927, AA424984 147 HCFBC03 293 341543 R32283,
R32295, R32329, R32338, R53752, R53753, R66100, R67703, H42383,
H43148, W90075, W90193, AA001927 147 HSJAP03 294 464384 R32283,
R32295, R32329, R32338, R53752, R53753, R66100, R67703, H42383,
H43148, W90075, AA001927 148 HSKGO26 158 341548 T54320, T90772,
T83224, T83638, T83802, T84784, R01903, R01952, R61603, H03357,
H04040, N44934, N71086, N71095, W05465, W38751, AA037109, AA081457,
AA081456, AA160292, AA160293 149 HCQAV96 159 341554 T51727, T51937,
R01243, R01357, R25494, R45561, R45561, R71014, H13671, H13718,
H17162, H17947, H38744, H89126, H89233, H89126, H98057, N45220,
N50863, N64600, N79925, W39199, W47233, N90258, AA028057, AA028058,
AA084220, AA084221 150 HSHCC16 160 341568 T52645, T56803, T77815,
R19061, R44103, R48608, R48711, R44103, R76418, R76417, H21046,
H50569, H62124, H88678, H96335, N28505, N31055, N35202, N36658,
N39983, N44201, N75851, N95279, W24554, W38410, W39503, W46946,
W47243, W47289, W56401, W60889, W94931, W92202, AA082353, AA100058,
AA102513, AA115079, AA115191, AA115928, AA115927, AA121322,
AA132576, AA132889, AA135237, AA143499, AA156981, AA160994,
AA167060, AA167165, AA167318, AA181572, AA186382 151 HTLEF62 295
341595 R15404, AA047517 152 HTLAD94 296 341646 R06128, R16166,
R15426, R17678, R18984, R26888, R27120, R35870, R35871, R44869,
R51276, R44869, R66882, R67967, H13315, H22113, H27381, H28345,
H37951, H38579, R93605, R97908, R97907, H53653,
H61431, H61432, H62657, H63776, H63826, H65287, H65810, H89508,
H89654, N29760, N57179, N74909, W23437, AA015926, AA026270,
AA026558, AA045484, AA046516, AA055479, AA070895, AA070985,
AA086047, AA086129, AA102052, AA102053, AA100978, AA128868,
AA133807, AA133806, AA134215, AA134849, AA152266, AA150204,
AA171825, AA173393, AA173394, AA182536, AA182582 153 HTSFQ12 163
341653 T78257, T81592, T83408, T98857, T99453, R24503, R24602,
R34836, R34932, R52437, R52501, R52500, H10205, H12773, H12827,
H20540, H20632, H40525, R92740, H51257, H51827, H78114, H78113,
H93565, N30243, N30795, N41570, N57137, N67626, N71081, N75985,
N76340, W01320, W04402, W04410, W52430, W52471, W61358, W60048,
AA040527, AA040528, AA044739, AA044794, AA133224, AA130908,
AA130822, AA142867, AA151675, AA151755 154 HE6FL83 164 616853
T73457, T73524, T65316, T65344, R21668, R24170, R46558, R52243,
R52244, R46558, H10183, H19602, H20914, H20913, H23732, H23751,
H29842, H29931, H30765, H41436, H41563, H38484, R84972, R87333,
R88049, R90853, N59099, N66303, N73535, N92979, W23841, W25041,
W40499, W45502, W56621, W56651, W67369, W67629, W72130, W77854,
W94427, W92638, AA043732, AA055978, AA056006 154 HE6FL83 297 341654
T73524, T65316, T65344, R21668, R24170, R46558, R52243, R46558,
H10183, H20914, H20913, H23751, H29931, H30765, H41436, H41563,
H38484, R84972, R87333, R88049, R90853, N59099, N92979, W23841,
W25041, W40499, W45502, W56621, W67369, W67629, W72130, W77854,
W94427, W92638, AA043732, AA055978, AA056006 155 HTXFJ55 165 670981
T49430, T92075, R00570, R00674, R12889, R16317, R17166, R17919,
R18012, R21041, R34226, R35552, R41283, R43263, R41283, R43263,
H10590, H15663, H17419, H17418, H60450, H60497, H83595, N31466,
N44274, N51210, N72512, W38423, W40215, W44751, W52648, W80752,
W80840, W85690, W85743, AA009995, AA009996, AA052977, AA053566,
AA053470, AA065021, AA069177, AA069242, AA076347, AA113353,
AA113434, AA134898, AA223817, AA243151 155 HTXFJ55 298 341661
T49430, T39608, T39610, T40697, T49431, T91991, T92075, R00570,
R00674, R12889, R16317, R17166, R17919, R18012, R21041, R34226,
R35552, R41283, R41394, R43263, R41283, R41394, R43263, R69121,
H10384, H10590, H15663, H17419, H17418, H60450, H60497, H83595,
H95242, N31466, N44274, N51210, N72512, W38423, W40215, W44751,
W45437, W52648, W58732, W67761, W67741, W72214, W74143, W77958,
W80752, W80840, W85690, W85743, AA009995, AA009996, AA052977,
AA053566, AA053470, AA065021, AA069177, AA069242, AA076347,
AA113353, AA113434, AA134930, AA134898, AA172161, AA172312 156
HJPCJ76 166 341664 T39919, T60946, T72529, T73752, R09799, R11461,
R12389, R12763, R20500, R45320, R45320, R60969, R61636, R70489,
R70579, R77628, R77629, R99728, H59663, H85076, N21970, N21980,
N44811, N50899, N51455, N52600, N53595, N59331, N71174, N71184,
N77139, N78503, W05163, W57664, W72044, W76396, AA007568, AA007569,
AA021623, AA021217, AA035589, AA046604, AA046719, AA059114,
AA059172, AA062948, AA127212, AA127195, AA135430, AA158353,
AA176606, AA176879 157 HLTED27 299 341672 R14927, R19442, R20366,
R38545, R39202, R43752, R44690, R51296, R51408, R43752, R81517,
R81767, H10945, H15061, H15997, H16577, H16621, H44519, H53352,
H53396, H68444, H99530, H99531, N29833, N47446, N47447, N49036,
N57251, N64206, N64268, N66379, N92787, N94418, N95423, N98459,
W24035, W30978, W32201, W32256, W56159, W58059, W58387, W60462,
W94562, W92646, N90558, AA026570, AA031679, AA031680, AA031834,
AA031835, AA043396, AA045186, AA046907, AA055036, AA055153,
AA056450, AA074783, AA074784, AA074607, AA075380, AA075429,
AA084527, AA099356, AA101888, AA121182, AA121222, AA127716,
AA127771, AA127762, AA149004, AA149005, AA165300, AA165299,
AA165450, AA169451, AA171961, AA171746, AA188146, AA188223 158
HMKBA64 168 341674 T96889, T96890, T97468, T97469, R97096, H64964,
H93009, H94235, N47960, N49171, N59386, N77074, W01926, AA035208,
AA035209, AA040430, AA040431, AA040644, AA045294, AA045521,
AA058688, AA088175, AA099553, AA101990, AA160278, AA160279 159
HNFIP24 169 351534 H04128, AA099288, AA101983 160 HCELB21 170
670664 R11975, R25294, R39817, H08811, H08888, H12161, H62686,
H73328, H78750, H79523, H90226, H90880, N72184, W00465, W38433,
W44822, AA016301, AA033738, AA033739 160 HCELB21 300 351542 R11975,
R25294, H08811, H08888, H73328, H79523, H90880, N72184, W00465,
W38433, W44822, AA016301, AA033738, AA033739 161 HAWBA28 171 351550
T86822, R18174, R42341, R54362, R54363, R42341, R72609, R72678,
H19127, H19126, N30662, W52710, W61287, W65383, AA027835, AA027878,
AA029269, AA053333, AA058647, AA114991, AA148794, AA148525,
AA156780, AA156849 162 HSAAS44 172 670221 T79117, H09893, H10480,
H86006, N40237, AA017440, AA132358, AA187785 162 HSAAS44 301 351553
T78632, T79117, R40722, R73748, H09723, H09804, H09893, H10480,
H11320, H85873, H86006, N40237, N50800, N55157, AA017440, AA017441,
AA029770, AA029769, AA031922, AA031993, AA088220, AA132238,
AA132358, AA169653, AA169833, AA171744, AA171956, AA187785 163
HAFAL73 173 660102 R73802, H24981, H44193, H44192, H51123, AA234319
163 HAFAL73 302 351554 R73802, H24981, H44193, H44192, H51123 164
HSAWF26 174 626839 T92615, H65155 164 HSAWF26 303 361212 H65155 165
HEAAL31 175 639007 AA151656, AA151652, AA151733, AA151736 165
HEAAL31 304 361221 T56046, T56062, T56080, T56096, T90475, T90482,
T90571, T90577, T74331, T89037, R10054, R11653, R54570, H22902,
H24285, H24287, H50611, H50610, H67882, H67927, N27199, N36263,
N39942, N40544, N44078, N46591, N58937, N73790, N94307, N95519,
N99415, W02128, W19438, W32393, W57988, W58072, W73132, W90660,
N90316, AA009898, AA022614, AA022615, AA025590, AA081613, AA084503,
AA085189 166 HFKFX55 176 361222 R50327, N53166, N80292, N92405,
N93437, W24630, W69468, AA013428, AA016267, AA132452, AA132636 167
H2LAO11 177 361224 T77193, R24954, R34527, R35684, R42813, R45217,
R49037, R45217, R69485, R70378, H10333, H10332, R83327, R83424,
H52395, H52396, H86524, H86605, H90386, H90437, H99168, N27057,
N34842, N43917, N55352, N62688, N63631, N68510, N69539, N77359,
N79085, N80462, N80477, W00825, W01972, W19934, W21605, W94195,
AA018111, AA018112, AA040014, AA058863, AA063281, AA063266,
AA086194, AA112364, AA112365, AA128903, AA134835 169 HPTTU11 179
653251 T47595, T47596, T74295, T90111, T82935, R34627, H40736,
N39397, W45441, W45652, W44582, W44631, W47414, W47609, AA034253,
AA035455, AA037139, AA059320, AA063639, AA226954, AA459767 169
HPTTU11 306 361247 T47596, T74295, T90111, T82935, H40736, N39397,
W45441, W45652, W44582, W44631, W47414, W47609, AA034253, AA035455,
AA037139, AA059320, AA059373, AA063639 170 HCFAE79 180 361318
T46931, T50673, T39801, T40801, T53401, T53402, R21169, R22220,
R22275, R22787, R24273, R24382, R24908, R32461, R33226, R33227,
R55432, R72796, R76252, R76535, R79988, R80091, R81411, R81412,
R82779, R82786, H00624, H00630, H12733, H12734, H12738, H12739,
H15831, H16139, H17120, H25890, H25891, H26787, H27500, H27572,
H27734, H29299, H58288, H69891, H69892, H83814, H83952, H95016,
H69892, H97790, N20245, N26081, N29310, N32053, N32279, N36118,
N36720, N38746, N44124, N45024, N75811, N78841, N80850, W07334,
W07515, W17199, W24810, W24844, W45102, W45152, W46243, W57629,
W58578, W60475, W69835, W69945, W79132, W94310, W95456, W95564,
N89582, AA032187, AA033661, AA036714, AA039761, AA053224, AA055646,
AA057354, AA062720, AA075568, AA075604, AA079402, AA081343,
AA081342, AA084535, AA086350, AA086062, AA086063, AA088496,
AA088680, AA102649, AA101660, AA101659, AA114157, AA127925,
AA131829, AA131995, AA143316, AA143333, AA143365, AA149758,
AA156474, AA157072, AA157160, AA157305, AA157842, AA161119,
AA161300, AA171710, AA179721, AA180474, AA181468, AA181640,
AA181881, AA186914 172 HODCW06 182 361346 T57053, R94753, N57860,
N64100 173 HFTAR26 183 361363 T89342, R32023, R32076, H00913,
H73617, N30331, N30338, N56674, N99888, AA007590, AA055091,
AA121507, AA127732 174 H2MBF44 184 663171 T51838, T51884, T92411,
T65619, T75302, T80328, T81433, T81434, T96979, T97091, R00998,
R07112, R13827, R13922, R35406, R39034, R49203, R53033, R49203,
R61594, H00151, H01276, H05079, H07041, H14693, H16360, H38255,
N21123, N31138, W47578, W47599, W56427, W90765, W90698, AA001816,
AA004544, AA021298, AA081577, AA102004, AA112823, AA113108,
AA137149, AA150268, AA150370, AA165473, AA181003, AA187989,
AA259097 174 H2MBF44 308 361365 T51838, T51884, T92411, T65552,
T65619, T75302, T80328, T81433, T81434, T96979, T97091, R00998,
R07112, R13827, R13922, R27577, R27676, R35406, R37686, R38011,
R38672, R39034, R49203, R51542, R53033, R49203, R61540, R61594,
H00151, H01276, H05079, H07042, H07041, H14588, H14693, H16360,
H37896, H38255, H99405, N21123, N31138, N34542, N75155, N80252,
W07378, W42462, W45201, W47578, W47599, W56427, W74265, W79879,
W90765, W90698, AA001746, AA001816, AA004544, AA021240, AA021298,
AA069253, AA069252, AA081577, AA084448, AA086275, AA099494,
AA102004, AA112823, AA113108, AA128022, AA135400, AA137149,
AA150268, AA150370, AA165393, AA165473, AA181003, AA181189,
AA182857, AA187989, AA187990, AA188268 175 HE8BI92 185 662220
R14311, R24143, R62968, R63250, R64668, R72480, R73256, R80927,
H26458, R93403, R93450, H57481, H58002, H58409, H99384, N24149,
N40700, N54068, N92774, W19427, W52093, W52957, W59973, W60435,
AA099083, AA131685, AA131990, AA136214, AA136404, AA173574,
AA173533, AA190478, AA191014 175 HE8BI92 309 361376 R14311, R24143,
R62968, R63250, R64668, R72480, R73256, R80927, H26458, R93403,
R93450, H57481, H58002, H58409, H99384, N24149, N40700, N54068,
N92774, W19427, W52093, W52957, W59973, W60435, AA099083, AA131685,
AA131990, AA136214, AA136404, AA158610, AA173574, AA173533,
AA190478, AA191014 176 HFTBR48 186 661994 R10708, H98169, H99191,
N21971, N24474, N28580, N29775, N32622, N47841, N49576, N57194,
N71175, W19349, AA027991, AA191432, AA224194, AA224296, AA261907,
AA262544, AA463932, AA464068 176 HFTBR48 310 361380 R10708, H98169,
H99191, N21971, N24474, N28580, N29775, N32622, N47841, N49576,
N57194, N71175, W19349, AA027991, AA191432 177 HE9CM64 187 605123
T53630, T53631, T75521, T78612, T79093, T85687, R19218, R44482,
R44482, H03595, H28113, H41562, H46641, N33914, N80225, N93335,
W38515, AA039786, AA039785, AA047032, AA047250, AA075305, AA463938,
AA464013, AA464074, AA464722 177 HE9CM64 311 361382 T53630, T53631,
T75521, T78612, T79093, T85687, R19218, R44482, R44482, H03595,
H28113, H41562, H46641, N33914, N80225, N93335, W38515, AA039786,
AA039785, AA047032, AA047250, AA075305 178 HATAV51 188 668035
T60208, T65846, T66000, R07954, R19068, R20084, R27055, R27054,
R27789, R27878, R44110, R44110, R70833, R82660, R82711, H00472,
H00526, H02416, H04214, H09792, H09878, H12753, H12795, H27575,
R94131, H81849, H81850, H95618, N33235, N34207, N36442, N41951,
N66133, N79969, N93411, W21452, W37638, W37856, W38928, W48827,
W49808, W56224, W58537, W58503, W94176, AA025349, AA025357,
AA039951, AA040002, AA055533, AA063282, AA158356, AA158429,
AA158430, AA164832, AA166790, AA193584, AA193628, AA234543,
AA236154, AA236123, AA429773 178 HATAV51 312 361385 T60208, T65846,
T66000, R07954, R20084, R27055, R27054, R27789, R27878, R44110,
R44110, R70781, R70833, R82660, R82711, H00472, H00526, H02416,
H04214, H09792, H12753, H12795, R94131, H81849, H81850, H95618,
N33235, N34207, N36442, N41951, N66133, N79969, N93411, W37375,
W37638, W49808, W58503, AA025349, AA039951, AA055533, AA063282,
AA158430, AA166675, AA166790, AA193584, AA193628 179 HAQAF27 189
361386 H27958, H27959, W86977, AA040831, AA043447, AA043448 180
HCEEK08 190 622186 N23815, N28919, W79250, AA025462, AA034130,
AA262825 180 HCEEK08 313 361389 R38720, N22616, N63156, N63214,
N79453, W74316, W76541, AA009980, AA022500, AA025463, AA039676,
AA056428, AA173931 181 HAFAU18 191 666320 H20101, H40615, H49128,
H50364, H50404, H51392, H99578, N23395, N24224, N24475, N26729,
N28029, N29128, N34617, N35179, W38665, W44731, W92818, W92895,
AA236416, AA236417, AA459710 181 HBGBT42 314 637497 H20101, H40615,
H49128, H50364, H50404, H51392, N23395, N24224, N26729, N28029,
N29128, N34617, N35179, W38665, W44731, W92895, AA236416, AA236417,
AA459710 181 HAFAU18 315 361390 H99192, H99578, N21284, N35179,
W92818, W92895, AA121981, AA121980 182 HETBY74 192 361396 R23770,
R26913, R32172, R32216, R38317, R39395, R63720, R63767, R70906,
R70940, R70993, R77169, R82584, R82585, H01942, H04495, H21696,
H21906, H97740, H99748, N62092, N95055, W38893, W93362, W93394,
W93606, AA016203, AA016243, AA076610, AA079633, AA079807, AA086218,
AA158761, AA159815, AA159816, AA159710, AA159711, AA159565,
AA160381, AA186897, AA188520 183 HTOAF35 193 661824 T39317, T48625,
T48626, T51203, T51306, T51521, T51668, T51998, T52109, T53727,
T54321, T57602, T91590, T91631, T92608, T92874, R44474, R44474,
R72793, R75886, R76062, H01272, H28132, H41188, R86158, H51133,
H51210, H85775, H92916, H95863, H95864, H98904, N23098, N24931,
N25285, N29927, N29934, N40471, N48509, N53513, N66520, N70329,
N75555, N92546, W02222, W04277, W05583, W39032, W84492, AA019752,
AA043261, AA074472, AA076072, AA076029, AA079109, AA079148,
AA088186, AA088656, AA099308, AA099818, AA126104, AA130889,
AA130784, AA149583, AA165424, AA165588, AA169434, AA173473,
AA173474, AA187661, AA186720 183 HTOAF35 316 361392 T39317, T48626,
T51203, T51306, T51521, T51668, T51998, T52109, T53727, T54321,
T57602, T91590, T91631, T92608, T92874, R44474, R44474, R72793,
R75886, R76062, H01272, H28132, H41188, R86158, H51133, H51210,
H85775, H92916, H95863, H95864, N23098, N24931, N25285, N29927,
N29934, N40471, N48509, N53513, N66520, N70329, N75555, N92546,
W02222, W04277, W05583, W39032, W84492, AA019752, AA043261,
AA074472, AA076072, AA076029, AA079109, AA088186, AA088656,
AA099308, AA099818, AA126104, AA130889, AA130784, AA149583,
AA165424, AA165588, AA169434, AA173473, AA173474, AA187661,
AA186720 184 HCRBX32 194 495055 T61851, T72629, T77182, T88844,
R10094, R10194, T82253, T83530, R01229, R01343, R20727, R24715,
R31457, R31458, R32713, R32714, R39272, R39439, R45449, R51552,
R51553, R54400, R54499, R41197, R20727, R45449, R59399, R59459,
H14539, H15683, H19939, H20326, H22915, H24093, H43536, R98988,
R99766, H48909, H48910, H72736, H72735, N20635, N29007, N31914,
N41658, N42699, N57556, N64823, N67014, N67536, N67783, N75675,
N95768, W20500, W19698, W24881, W78750, W80488, W86081, N89954,
AA015900, AA015899, AA046100, AA045995, AA099206, AA253083,
AA253253, AA256913, AA255489 184 HCRBX32 317 361397 T61851, T72629,
T72699, T77182, T88844, R10094, R10194, T82253, T83530, R01229,
R01343, R20727, R24715, R31457, R31458, R32713, R32714, R39272,
R39439, R41197, R45449, R51552, R51553, R54400, R54499, R41197,
R20727, R45449, R59399, R59459, H14539, H15683, H19939, H20326,
H22915, H24093, H43536, R98988, R99766, H48909, H48910, H72736,
H72735, N20635, N29007, N31914, N41658, N42699, N57556, N64823,
N67014, N67536, N67783, N75675, N95768, W20500, W19698, W24881,
W78750, W80488, W86081, N89954, AA015900, AA015899, AA046100,
AA045995, AA099206 185 HEBGB80 195 636011 R53950, W68693, W68786
185 HEBGB80 318 361398 R53950, W68693, W68786 186 HFAMH74 196
638302 R68577, R77269, H22571, H45827, H48519, H48687, N73815,
N78323, W69528, AA122266, AA126601 186 HFAMH74 319 361402 R68372,
R68577, R77269, R77319, H12718, H12719, H22571, H44679, H45826,
H45827, H45875, H45877, R87058, H48519, H48687, H51661, H65843,
H65930, H66425, H66424, H69570, H70028, N39190, N47250, N62091,
N73815, N78323, W69445, W69528, AA122266, AA126601, AA131324,
AA131790, AA131840, AA136024
[1731] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Isolation of a Selected cDNA Clone From the Deposited Sample
[1732] Each cDNA clone in a cited ATCC.TM. deposit is contained in
a plasmid vector. Table 1 identifies the vectors used to construct
the cDNA library from which each clone was isolated. In many cases,
the vector used to construct the library is a phage vector from
which a plasmid has been excised. The table immediately below
correlates the related plasmid for each phage vector used in
constructing the cDNA library. For example, where a particular
clone is identified in Table 1 as being isolated in the vector
"Lambda Zap," the corresponding deposited clone is in
"pBluescript.TM.."
TABLE-US-00004 Corresponding Vector Used to Deposited Construct
Library Plasmid Lambda Zap pBluescript .TM. (pBS) Uni-Zap XR
pBluescript .TM. (pBS) Zap Express pBK lafmid BA plafmid BA pSport1
pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR
.RTM. 2.1 pCR .RTM. 2.1
[1733] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),
Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express
(U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript.TM. (pBS)
(Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988);
Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494
(1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61
(1992)) are commercially available from STRATAGENE.TM. Cloning
Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037.
pBS contains an ampicillin resistance gene and pBK contains a
neomycin resistance gene. Both can be transformed into E. coli
strain XL-1 Blue, also available from STRATAGENE.TM.. pBS comes in
4 forms SK+, SK-, KS+ and KS. The S and K refers to the orientation
of the polylinker to the T7 and T3 primer sequences which flank the
polylinker region ("S" is for Sad and "K" is for KpnI which are the
first sites on each respective end of the linker). "+" or "-" refer
to the orientation of the f1 origin of replication ("on"), such
that in one orientation, single stranded rescue initiated from the
f1 on generates sense strand DNA and in the other, antisense.
[1734] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from LIFE TECHNOLOGIES.TM., Inc., P.O. Box 6009,
Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin
resistance gene and may be transformed into E. coli strain DH10B,
also available from LIFE TECHNOLOGIES.TM.. (See, for instance,
Gruber, C. E., et al., Focus 15:59 (1993).) Vector lafmid BA (Bento
Soares, Columbia University, NY) contains an ampicillin resistance
gene and can be transformed into E. coli strain XL-1 Blue. Vector
pCR.RTM.2.1, which is available from Invitrogen, 1600 Faraday
Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance
gene and may be transformed into E. coli strain DH10B, available
from LIFE TECHNOLOGIES.TM.. (See, for instance, Clark, J. M., Nuc.
Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology
9: (1991).) Preferably, a polynucleotide of the present invention
does not comprise the phage vector sequences identified for the
particular clone in Table 1, as well as the corresponding plasmid
vector sequences designated above.
[1735] The deposited material in the sample assigned the ATCC.TM.
Deposit Number cited in Table 1 for any given cDNA clone also may
contain one or more additional plasmids, each comprising a cDNA
clone different from that given clone. Thus, deposits sharing the
same ATCC.TM. Deposit Number contain at least a plasmid for each
cDNA clone identified in Table 1. Typically, each ATCC.TM. deposit
sample cited in Table 1 comprises a mixture of approximately equal
amounts (by weight) of about 50 plasmid DNAs, each containing a
different cDNA clone; but such a deposit sample may include
plasmids for more or less than 50 cDNA clones, up to about 500 cDNA
clones.
[1736] Two approaches can be used to isolate a particular clone
from the deposited sample of plasmid DNAs cited for that clone in
Table 1. First, a plasmid is directly isolated by screening the
clones using a polynucleotide probe corresponding to SEQ ID
NO:X.
[1737] Particularly, a specific polynucleotide with 30-40
nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
is labeled, for instance, with .sup.32P-.gamma.-ATP using T4
polynucleotide kinase and purified according to routine methods.
(E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid
mixture is transformed into a suitable host, as indicated above
(such as XL-1 Blue (STRATAGENE.TM.)) using techniques known to
those of skill in the art, such as those provided by the vector
supplier or in related publications or patents cited above. The
transformants are plated on 1.5% agar plates (containing the
appropriate selection agent, e.g., ampicillin) to a density of
about 150 transformants (colonies) per plate. These plates are
screened using Nylon membranes according to routine methods for
bacterial colony screening (e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor
Laboratory Press, pages 1.93 to 1.104), or other techniques known
to those of skill in the art.
[1738] Alternatively, two primers of 17-20 nucleotides derived from
both ends of the SEQ ID NO:X (i.e., within the region of SEQ ID
NO:X bounded by the 5' NT and the 3' NT of the clone defined in
Table 1) are synthesized and used to amplify the desired cDNA using
the deposited cDNA plasmid as a template. The polymerase chain
reaction is carried out under routine conditions, for instance, in
25 ul of reaction mixture with 0.5 ug of the above cDNA template. A
convenient reaction mixture is 1.5-5 mM MgCl.sub.2, 0.01% (w/v)
gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each
primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation at 94 degree C. for 1 min; annealing at 55 degree C.
for 1 min; elongation at 72 degree C. for 1 min) are performed with
a Perkin-Elmer Cetus automated thermal cycler. The amplified
product is analyzed by agarose gel electrophoresis and the DNA band
with expected molecular weight is excised and purified. The PCR
product is verified to be the selected sequence by subcloning and
sequencing the DNA product.
[1739] Several methods are available for the identification of the
5' or 3' non-coding portions of a gene which may not be present in
the deposited clone. These methods include but are not limited to,
filter probing, clone enrichment using specific probes, and
protocols similar or identical to 5' and 3' "RACE" protocols which
are well known in the art. For instance, a method similar to 5'
RACE is available for generating the missing 5' end of a desired
full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.
21(7):1683-1684 (1993).)
[1740] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the gene of interest is used to PCR amplify the 5'
portion of the desired full-length gene. This amplified product may
then be sequenced and used to generate the full length gene.
[1741] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[1742] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the desired gene.
Example 2
Isolation of Genomic Clones Corresponding to a Polynucleotide
[1743] A human genomic P1 library (Genomic Systems, Inc.) is
screened by PCR using primers selected for the cDNA sequence
corresponding to SEQ ID NO:X., according to the method described in
Example 1. (See also, Sambrook.)
Example 3
Tissue Distribution of Polypeptide
[1744] Tissue distribution of mRNA expression of polynucleotides of
the present invention is determined using protocols for Northern
blot analysis, described by, among others, Sambrook et al. For
example, a cDNA probe produced by the method described in Example 1
is labeled with P.sup.32 using the REDIPRIME.TM. DNA labeling
system (Amersham Life Science), according to manufacturer's
instructions. After labeling, the probe is purified using CHROMA
SPIN-100.TM. column (CLONTECH.TM. Laboratories, Inc.), according to
manufacturer's protocol number PT1200-1. The purified labeled probe
is then used to examine various human tissues for mRNA
expression.
[1745] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM)
(CLONTECH.TM.) are examined with the labeled probe using
EXPRESSHYB.TM. hybridization solution (CLONTECH.TM.) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at -70
degree C. overnight, and the films developed according to standard
procedures.
Example 4
Chromosomal Mapping of the Polynucleotides
[1746] An oligonucleotide primer set is designed according to the
sequence at the 5' end of SEQ ID NO:X. This primer preferably spans
about 100 nucleotides. This primer set is then used in a polymerase
chain reaction under the following set of conditions: 30 seconds,
95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This
cycle is repeated 32 times followed by one 5 minute cycle at 70
degree C. Human, mouse, and hamster DNA is used as template in
addition to a somatic cell hybrid panel containing individual
chromosomes or chromosome fragments (Bios, Inc). The reactions is
analyzed on either 8% polyacrylamide gels or 3.5% agarose gels.
Chromosome mapping is determined by the presence of an
approximately 100 bp PCR fragment in the particular somatic cell
hybrid.
Example 5
Bacterial Expression of a Polypeptide
[1747] A polynucleotide encoding a polypeptide of the present
invention is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' ends of the DNA sequence, as
outlined in Example 1, to synthesize insertion fragments. The
primers used to amplify the cDNA insert should preferably contain
restriction sites, such as BamHI and XbaI, at the 5' end of the
primers in order to clone the amplified product into the expression
vector. For example, BamHI and XbaI correspond to the restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic
resistance (Amp.sup.r), a bacterial origin of replication (on), an
IPTG-regulatable promoter/operator (P/O), a ribosome binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning
sites.
[1748] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, which
expresses the lad repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies are
selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
[1749] Clones containing the desired constructs are grown overnight
(0/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The 0/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lad
repressor, clearing the P/O leading to increased gene
expression.
[1750] Cells are grown for an extra 3 to 4 hours. Cells are then
harvested by centrifugation (20 mins at 6000 Xg). The cell pellet
is solubilized in the chaotropic agent 6 Molar Guanidine HCl by
stirring for 3-4 hours at 4 degree C. The cell debris is removed by
centrifugation, and the supernatant containing the polypeptide is
loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin column (available from QIAGEN, Inc., supra). Proteins with a
6.times.His tag bind to the Ni-NTA resin with high affinity and can
be purified in a simple one-step procedure (for details see: The
QIAexpressionist (1995) QIAGEN, Inc., supra).
[1751] Briefly, the supernatant is loaded onto the column in 6 M
guanidine-HCl, pH 8, the column is first washed with 10 volumes of
6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M
guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M
guanidine-HCl, pH 5.
[1752] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins are eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4 degree C. or frozen at -80 degree C.
[1753] In addition to the above expression vector, the present
invention further includes an expression vector comprising phage
operator and promoter elements operatively linked to a
polynucleotide of the present invention, called pHE4a. (ATCC.TM.
Accession Number 209645, deposited on Feb. 25, 1998.) This vector
contains: 1) a neomycinphosphotransferase gene as a selection
marker, 2) an E. coli origin of replication, 3) a T5 phage promoter
sequence, 4) two lac operator sequences, 5) a Shine-Delgarno
sequence, and 6) the lactose operon repressor gene (lacIq). The
origin of replication (oriC) is derived from pUC19 (LTI,
Gaithersburg, Md.). The promoter sequence and operator sequences
are made synthetically.
[1754] DNA can be inserted into the pHEa by restricting the vector
with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted
product on a gel, and isolating the larger fragment (the stuffer
fragment should be about 310 base pairs). The DNA insert is
generated according to the PCR protocol described in Example 1,
using PCR primers having restriction sites for NdeI (5' primer) and
XbaI, BamHI, XhoI, or Asp718 (3' primer). The PCR insert is gel
purified and restricted with compatible enzymes. The insert and
vector are ligated according to standard protocols.
[1755] The engineered vector could easily be substituted in the
above protocol to express protein in a bacterial system.
Example 6
Purification of a Polypeptide from an Inclusion Body
[1756] The following alternative method can be used to purify a
polypeptide expressed in E coli when it is present in the form of
inclusion bodies. Unless otherwise specified, all of the following
steps are conducted at 4-10 degree C.
[1757] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10 degree C. and the
cells harvested by continuous centrifugation at 15,000 rpm (Heraeus
Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste and the amount of purified protein required,
an appropriate amount of cell paste, by weight, is suspended in a
buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are dispersed to a homogeneous suspension using a high shear
mixer.
[1758] The cells are then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[1759] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the polypeptide containing supernatant is incubated at 4 degree
C. overnight to allow further GuHCl extraction.
[1760] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4 degree C. without mixing for 12 hours prior to further
purification steps.
[1761] To clarify the refolded polypeptide solution, a previously
prepared tangential filtration unit equipped with 0.16 um membrane
filter with appropriate surface area (e.g., Filtron), equilibrated
with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample
is loaded onto a cation exchange resin (e.g., Poros HS-50,
Perseptive Biosystems). The column is washed with 40 mM sodium
acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500
mM NaCl in the same buffer, in a stepwise manner. The absorbance at
280 nm of the effluent is continuously monitored. Fractions are
collected and further analyzed by SDS-PAGE.
[1762] Fractions containing the polypeptide are then pooled and
mixed with 4 volumes of water. The diluted sample is then loaded
onto a previously prepared set of tandem columns of strong anion
(Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20,
Perseptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are
washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20
column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M
NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under
constant A.sub.280 monitoring of the effluent. Fractions containing
the polypeptide (determined, for instance, by 16% SDS-PAGE) are
then pooled.
[1763] The resultant polypeptide should exhibit greater than 95%
purity after the above refolding and purification steps. No major
contaminant bands should be observed from Commassie blue stained
16% SDS-PAGE gel when 5 ug of purified protein is loaded. The
purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml
according to LAL assays.
Example 7
Cloning and Expression of a Polypeptide in a Baculovirus Expression
System
[1764] In this example, the plasmid shuttle vector pA2 is used to
insert a polynucleotide into a baculovirus to express a
polypeptide. This expression vector contains the strong polyhedrin
promoter of the Autographa californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites such as BamHI,
Xba I and Asp718. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For easy selection
of recombinant virus, the plasmid contains the beta-galactosidase
gene from E. coli under control of a weak Drosophila promoter in
the same orientation, followed by the polyadenylation signal of the
polyhedrin gene. The inserted genes are flanked on both sides by
viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate a viable virus that express the
cloned polynucleotide.
[1765] Many other baculovirus vectors can be used in place of the
vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[1766] Specifically, the cDNA sequence contained in the deposited
clone, including the AUG initiation codon and the naturally
associated leader sequence identified in Table 1, is amplified
using the PCR protocol described in Example 1. If the naturally
occurring signal sequence is used to produce the secreted protein,
the pA2 vector does not need a second signal peptide.
Alternatively, the vector can be modified (pA2 GP) to include a
baculovirus leader sequence, using the standard methods described
in Summers et al., "A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures," Texas Agricultural Experimental
Station Bulletin No. 1555 (1987).
[1767] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[1768] The plasmid is digested with the corresponding restriction
enzymes and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("GENECLEAN.TM." BIO 101 Inc., La Jolla, Calif.).
[1769] The fragment and the dephosphorylated plasmid are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (STRATAGENE.TM. Cloning Systems, La
Jolla, Calif.) cells are transformed with the ligation mixture and
spread on culture plates. Bacteria containing the plasmid are
identified by digesting DNA from individual colonies and analyzing
the digestion product by gel electrophoresis. The sequence of the
cloned fragment is confirmed by DNA sequencing.
[1770] Five ug of a plasmid containing the polynucleotide is
co-transfected with 1.0 ug of a commercially available linearized
baculovirus DNA ("BACULOGOLD.TM. baculovirus DNA", Pharmingen, San
Diego, Calif.), using the lipofection method described by Felgner
et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of
BACULOGOLD.TM. virus DNA and 5 ug of the plasmid are mixed in a
sterile well of a microtiter plate containing 50 ul of serum-free
Grace's medium (LIFE TECHNOLOGIES.TM. Inc., Gaithersburg, Md.).
Afterwards, 10 ul LIPOFECTIN.TM. plus 90 ul Grace's medium are
added, mixed and incubated for 15 minutes at room temperature. Then
the transfection mixture is added drop-wise to Sf9 insect cells
(ATCC.TM. CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate is then incubated for 5
hours at 27 degrees C. The transfection solution is then removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. Cultivation is then continued at 27
degrees C. for four days.
[1771] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (LIFE TECHNOLOGIES.TM. Inc.,
Gaithersburg) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by LIFE TECHNOLOGIES.TM. Inc.,
Gaithersburg, page 9-10.) After appropriate incubation, blue
stained plaques are picked with the tip of a micropipettor (e.g.,
Eppendorf). The agar containing the recombinant viruses is then
resuspended in a microcentrifuge tube containing 200 ul of Grace's
medium and the suspension containing the recombinant baculovirus is
used to infect Sf9 cells seeded in 35 mm dishes. Four days later
the supernatants of these culture dishes are harvested and then
they are stored at 4 degree C.
[1772] To verify the expression of the polypeptide, Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus containing
the polynucleotide at a multiplicity of infection ("MOI") of about
2. If radiolabeled proteins are desired, 6 hours later the medium
is removed and is replaced with SF900 II medium minus methionine
and cysteine (available from LIFE TECHNOLOGIES.TM. Inc., Rockville,
Md.). After 42 hours, 5 uCi of .sup.35S-methionine and 5 uCi
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[1773] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the produced protein.
Example 8
Expression of a Polypeptide in Mammalian Cells
[1774] The polypeptide of the present invention can be expressed in
a mammalian cell. A typical mammalian expression vector contains a
promoter element, which mediates the initiation of transcription of
mRNA, a protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription is achieved with the early
and late promoters from SV40, the long terminal repeats (LTRs) from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus (CMV). However, cellular elements can also be used
(e.g., the human actin promoter).
[1775] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as pSVL and
pMSG (PHARMACIA.TM., Uppsala, Sweden), pRSVcat (ATCC.TM. 37152),
pSV2dhfr (ATCC.TM. 37146), pBC12MI (ATCC.TM. 67109), pCMVSport 2.0,
and pCMVSport 3.0. Mammalian host cells that could be used include,
human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese
hamster ovary (CHO) cells.
[1776] Alternatively, the polypeptide can be expressed in stable
cell lines containing the polynucleotide integrated into a
chromosome. The co-transfection with a selectable marker such as
dhfr, gpt, neomycin, hygromycin allows the identification and
isolation of the transfected cells.
[1777] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful in developing cell lines that carry several
hundred or even several thousand copies of the gene of interest.
(See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370
(1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta,
1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology
9:64-68 (1991).) Another useful selection marker is the enzyme
glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using
these markers, the mammalian cells are grown in selective medium
and the cells with the highest resistance are selected. These cell
lines contain the amplified gene(s) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
[1778] Derivatives of the plasmid pSV2-dhfr (ATCC.TM. Accession No.
37146), the expression vectors pC4 (ATCC.TM. Accession No. 209646)
and pC6 (ATCC.TM. Accession No. 209647) contain the strong promoter
(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and
Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors also contain the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene, and the mouse DHFR gene under control of the SV40 early
promoter.
[1779] Specifically, the plasmid pC6, for example, is digested with
appropriate restriction enzymes and then dephosphorylated using
calf intestinal phosphates by procedures known in the art. The
vector is then isolated from a 1% agarose gel.
[1780] A polynucleotide of the present invention is amplified
according to the protocol outlined in Example 1. If the naturally
occurring signal sequence is used to produce the secreted protein,
the vector does not need a second signal peptide. Alternatively, if
the naturally occurring signal sequence is not used, the vector can
be modified to include a heterologous signal sequence. (See, e.g.,
WO 96/34891.)
[1781] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[1782] The amplified fragment is then digested with the same
restriction enzyme and purified on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed
and bacteria are identified that contain the fragment inserted into
plasmid pC6 using, for instance, restriction enzyme analysis.
[1783] Chinese hamster ovary cells lacking an active DHFR gene is
used for transfection. Five .mu.g of the expression plasmid pC6a
pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using
LIPOFECTIN.TM. (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the neo gene from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of metothrexate plus 1 mg/ml G418. After about 10-14 days single
clones are trypsinized and then seeded in 6-well petri dishes or 10
ml flasks using different concentrations of methotrexate (50 nM,
100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well
plates containing even higher concentrations of methotrexate (1 uM,
2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until
clones are obtained which grow at a concentration of 100-200 uM.
Expression of the desired gene product is analyzed, for instance,
by SDS-PAGE and Western blot or by reversed phase HPLC
analysis.
Example 9
Protein Fusions
[1784] The polypeptides of the present invention are preferably
fused to other proteins. These fusion proteins can be used for a
variety of applications. For example, fusion of the present
polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose binding protein facilitates purification. (See Example 5;
see also EP A 394,827; Traunecker, et al., Nature 331:84-86
(1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases
the halflife time in vivo. Nuclear localization signals fused to
the polypeptides of the present invention can target the protein to
a specific subcellular localization, while covalent heterodimer or
homodimers can increase or decrease the activity of a fusion
protein. Fusion proteins can also create chimeric molecules having
more than one function. Finally, fusion proteins can increase
solubility and/or stability of the fused protein compared to the
non-fused protein. All of the types of fusion proteins described
above can be made by modifying the following protocol, which
outlines the fusion of a polypeptide to an IgG molecule, or the
protocol described in Example 5.
[1785] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian expression vector.
[1786] For example, if pC4 (Accession No. 209646) is used, the
human Fc portion can be ligated into the BamHI cloning site. Note
that the 3' BamHI site should be destroyed. Next, the vector
containing the human Fc portion is re-restricted with BamHI,
linearizing the vector, and a polynucleotide of the present
invention, isolated by the PCR protocol described in Example 1, is
ligated into this BamHI site. Note that the polynucleotide is
cloned without a stop codon, otherwise a fusion protein will not be
produced.
[1787] If the naturally occurring signal sequence is used to
produce the secreted protein, pC4 does not need a second signal
peptide. Alternatively, if the naturally occurring signal sequence
is not used, the vector can be modified to include a heterologous
signal sequence. (See, e.g., WO 96/34891.)
[1788] Human IgG Fc region:
TABLE-US-00005 (SEQ ID NO: 1)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
TTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
TCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 10
Production of an Antibody from a Polypeptide
[1789] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing a polypeptide of the
present invention is administered to an animal to induce the
production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the secreted protein is prepared and
purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to
produce polyclonal antisera of greater specific activity.
[1790] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or protein binding fragments
thereof). Such monoclonal antibodies can be prepared using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In
general, such procedures involve immunizing an animal (preferably a
mouse) with polypeptide or, more preferably, with a secreted
polypeptide-expressing cell. Such cells may be cultured in any
suitable tissue culture medium; however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented with
10% fetal bovine serum (inactivated at about 56 degrees C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
[1791] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP2O), available
from the ATCC.TM.. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981).) The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the polypeptide.
[1792] Alternatively, additional antibodies capable of binding to
the polypeptide can be produced in a two-step procedure using
anti-idiotypic antibodies. Such a method makes use of the fact that
antibodies are themselves antigens, and therefore, it is possible
to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the protein-specific antibody can
be blocked by the polypeptide. Such antibodies comprise
anti-idiotypic antibodies to the protein-specific antibody and can
be used to immunize an animal to induce formation of further
protein-specific antibodies.
[1793] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, secreted protein-binding fragments can
be produced through the application of recombinant DNA technology
or through synthetic chemistry.
[1794] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. (See, for review, Morrison, Science 229:1202 (1985); Oi et
al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).)
Example 11
Production of Secreted Protein for High-Throughput Screening
Assays
[1795] The following protocol produces a supernatant containing a
polypeptide to be tested. This supernatant can then be used in the
Screening Assays described herein.
[1796] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim)
stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or
magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml.
Add 200 ul of this solution to each well (24 well plates) and
incubate at RT for 20 minutes. Be sure to distribute the solution
over each well (note: a 12-channel pipetter may be used with tips
on every other channel). Aspirate off the Poly-D-Lysine solution
and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should
remain in the well until just prior to plating the cells and plates
may be poly-lysine coated in advance for up to two weeks.
[1797] Plate 293T cells (do not carry cells past P+20) at
2.times.10.sup.5 cells/well in 0.5 ml DMEM (Dulbecco's Modified
Eagle Medium) (with 4.5 G/L glucose and L-glutamine (12-604F
Biowhittaker))/10% heat inactivated FBS (14-503F
Biowhittaker)/1.times. Penstrep (17-602E Biowhittaker). Let the
cells grow overnight.
[1798] The next day, mix together in a sterile solution basin: 300
ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070
Gibco/BRL)/96-well plate. With a small volume multi-channel
pipetter, aliquot approximately 2 ug of an expression vector
containing a polynucleotide insert, produced by the methods
described in Examples 8 or 9, into an appropriately labeled 96-well
round bottom plate. With a multi-channel pipetter, add 50 ul of the
Lipofectamine/Optimem I mixture to each well. Pipette up and down
gently to mix. Incubate at RT 15-45 minutes. After about 20
minutes, use a multi-channel pipetter to add 150 ul Optimem Ito
each well. As a control, one plate of vector DNA lacking an insert
should be transfected with each set of transfections.
[1799] Preferably, the transfection should be performed by
tag-teaming the following tasks. By tag-teaming, hands on time is
cut in half, and the cells do not spend too much time on PBS.
First, person A aspirates off the media from four 24-well plates of
cells, and then person B rinses each well with 0.5-1 ml PBS. Person
A then aspirates off PBS rinse, and person B, using a12-channel
pipetter with tips on every other channel, adds the 200 ul of
DNA/Lipofectamine/Optimem I complex to the odd wells first, then to
the even wells, to each row on the 24-well plates. Incubate at 37
degrees C. for 6 hours.
[1800] While cells are incubating, prepare appropriate media,
either 1% BSA in DMEM with 1.times. penstrep, or CHO-5 media (116.6
mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO.sub.4-5H.sub.2O; 0.050
mg/L of Fe(NO.sub.3).sub.3-9H.sub.2O; 0.417 mg/L of
FeSO.sub.4-7H.sub.2O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl.sub.2;
48.84 mg/L of MgSO.sub.4; 6995.50 mg/L of NaCl; 2400.0 mg/L of
NaHCO.sub.3; 62.50 mg/L of NaH.sub.2PO.sub.4--H.sub.2O; 71.02 mg/L
of Na.sub.2HPO4; 0.4320 mg/L of ZnSO.sub.4-7H.sub.2O; 0.002 mg/L of
Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of
DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010
mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic
Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20
mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of
L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of
L-Asparagine-H.sub.2O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml
of L-Cystine-2HCL-H.sub.2O; 31.29 mg/ml of L-Cystine-2HCL; 7.35
mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml
of Glycine; 52.48 mg/ml of L-Histidine-HCL-H.sub.2O; 106.97 mg/ml
of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of
L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine;
101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79
mg/ml of L-Tryrosine-2Na-2H.sub.2O; 99.65 mg/ml of L-Valine; 0.0035
mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of
i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL;
0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L
of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin
B.sub.12; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine;
0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM
of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of
Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of
Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of
Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine
and 1.times. penstrep. (BSA (81-068-3 BAYER.TM.) 100 gm dissolved
in 1 L DMEM for a 10% BSA stock solution). Filter the media and
collect 50 ul for endotoxin assay in 15 ml polystyrene conical.
[1801] The transfection reaction is terminated, preferably by
tag-teaming, at the end of the incubation period. Person A
aspirates off the transfection media, while person B adds 1.5 ml
appropriate media to each well. Incubate at 37 degrees C. for 45 or
72 hours depending on the media used: 1% BSA for 45 hours or CHO-5
for 72 hours.
[1802] On day four, using a 300 ul multichannel pipetter, aliquot
600 ul in one 1 ml deep well plate and the remaining supernatant
into a 2 ml deep well. The supernatants from each well can then be
used in the assays described in Examples 13-20.
[1803] It is specifically understood that when activity is obtained
in any of the assays described below using a supernatant, the
activity originates from either the polypeptide directly (e.g., as
a secreted protein) or by the polypeptide inducing expression of
other proteins, which are then secreted into the supernatant. Thus,
the invention further provides a method of identifying the protein
in the supernatant characterized by an activity in a particular
assay.
Example 12
Construction of GAS Reporter Construct
[1804] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[1805] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATs
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[1806] The STATs are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[1807] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Schidler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor
family, capable of activating Jaks, is divided into two groups: (a)
Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-.alpha., IFN-g, and
IL-10. The Class 1 receptors share a conserved cysteine motif (a
set of four conserved cysteines and one tryptophan) and a WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ
ID NO:2)).
[1808] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway.
[1809] Therefore, activation of the Jaks-STATs pathway, reflected
by the binding of the GAS or the ISRE element, can be used to
indicate proteins involved in the proliferation and differentiation
of cells. For example, growth factors and cytokines are known to
activate the Jaks-STATs pathway. (See Table below.) Thus, by using
GAS elements linked to reporter molecules, activators of the
Jaks-STATs pathway can be identified.
TABLE-US-00006 JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements)
or ISRE IFN family IFN-a/B + + - - 1, 2, 3 ISRE IFN-g + + - 1 GAS
(IRF1 > Lys6 > IFP) Il-10 + ? ? - 1, 3 gp130 family IL-6
(Pleiotropic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP)
Il-11(Pleiotropic) ? + ? ? 1, 3 OnM(Pleiotropic) ? + + ? 1, 3
LIF(Pleiotropic) ? + + ? 1, 3 CNTF(Pleiotropic) -/+ + + ? 1, 3
G-CSF(Pleiotropic) ? + ? ? 1, 3 IL-12(Pleiotropic) + - + + 1, 3 g-C
family IL-2 (lymphocytes) - + - + 1, 3, 5 GAS IL-4 (lymph/myeloid)
- + - + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) - +
- + 5 GAS IL-9 (lymphocytes) - + - + 5 GAS IL-13 (lymphocyte) - + ?
? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) - - + - 5
GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid) - - + - 5 GAS Growth hormone family GH ? - + - 5
PRL ? +/- + - 1, 3, 5 EPO ? - + - 5 GAS (B-CAS > IRF1 = IFP
>> Ly6) Receptor Tyrosine Kinases EGF ? + + - 1, 3 GAS (IRF1)
PDGF ? + + - 1, 3 CSF-1 ? + + - 1, 3 GAS (not IRF1)
[1810] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 13-14,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATs upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00007 (SEQ ID NO: 3)
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCC
CGAAATATCTGCCATCTCAATTAG:3'
[1811] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
TABLE-US-00008 (SEQ ID NO: 4) 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
[1812] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
CLONTECH.TM.. The resulting PCR fragment is digested with XhoI/Hind
III and subcloned into BLSK2-. (STRATAGENE.TM..) Sequencing with
forward and reverse primers confirms that the insert contains the
following sequence:
TABLE-US-00009 (SEQ ID NO: 5)
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAA
ATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC
CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCG
CCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAG
CTT:3'
[1813] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can be instead of SEAP, in
this or in any of the other Examples. Well known reporter molecules
that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by an antibody.
[1814] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
CLONTECH.TM. using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[1815] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (CLONTECH.TM.), using these restriction sites in the
multiple cloning site, to create the GAS-SEAP/Neo vector. Once this
vector is transfected into mammalian cells, this vector can then be
used as a reporter molecule for GAS binding as described in
Examples 13-14.
[1816] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing NFK-B and EGR
promoter sequences are described in Examples 15 and 16. However,
many other promoters can be substituted using the protocols
described in these Examples. For instance, SRE, IL-2, NFAT, or
Osteocalcin promoters can be substituted, alone or in combination
(e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct
activity, such as HELA (epithelial), HUVEC (endothelial), Reh
(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or
Cardiomyocyte.
Example 13
High-Throughput Screening Assay for T-Cell Activity
[1817] The following protocol is used to assess T-cell activity by
identifying factors, and determining whether supernate containing a
polypeptide of the invention proliferates and/or differentiates
T-cells. T-cell activity is assessed using the GAS/SEAP/Neo
construct produced in Example 12. Thus, factors that increase SEAP
activity indicate the ability to activate the Jaks-STATS signal
transduction pathway. The T-cell used in this assay is Jurkat
T-cells (ATCC.TM. Accession No. TIB-152), although Molt-3 cells
(ATCC.TM. Accession No. CRL-1552) and Molt-4 cells (ATCC.TM.
Accession No. CRL-1582) cells can also be used.
[1818] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In
order to generate stable cell lines, approximately 2 million Jurkat
cells are transfected with the GAS-SEAP/neo vector using DMRIE-C
(LIFE TECHNOLOGIES.TM.) (transfection procedure described below).
The transfected cells are seeded to a density of approximately
20,000 cells per well and transfectants resistant to 1 mg/ml
genticin selected. Resistant colonies are expanded and then tested
for their response to increasing concentrations of interferon
gamma. The dose response of a selected clone is demonstrated.
[1819] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM.TM.
(LIFE TECHNOLOGIES.TM.) with 10 ug of plasmid DNA in a T25 flask.
Add 2.5 ml OPTI-MEM.TM. containing 50 ul of DMRIE-C and incubate at
room temperature for 15-45 mins.
[1820] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM.TM. to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM.TM.
to T25 flask and incubate at 37 degrees C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.
[1821] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are
treated with supernatants containing polypeptides of the invention
and/or induced polypeptides of the invention as produced by the
protocol described in Example 11.
[1822] On the day of treatment with the supernatant, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of supernatants being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[1823] Transfer the cells to a triangular reservoir boat, in order
to dispense the cells into a 96 well dish, using a 12 channel
pipette. Using a 12 channel pipette, transfer 200 ul of cells into
each well (therefore adding 100,000 cells per well).
[1824] After all the plates have been seeded, 50 ul of the
supernatants are transferred directly from the 96 well plate
containing the supernatants into each well using a 12 channel
pipette. In addition, a dose of exogenous interferon gamma (0.1,
1.0, 10 ng) is added to wells H9, H10, and H11 to serve as
additional positive controls for the assay.
[1825] The 96 well dishes containing Jurkat cells treated with
supernatants are placed in an incubator for 48 hrs (note: this time
is variable between 48-72 hrs). 35 ul samples from each well are
then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using cellophane
covers) and stored at -20 degrees C. until SEAP assays are
performed according to Example 17. The plates containing the
remaining treated cells are placed at 4 degrees C. and serve as a
source of material for repeating the assay on a specific well if
desired.
[1826] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
[1827] The above protocol may be used in the generation of both
transient, as well as, stable transfected cells, which would be
apparent to those of skill in the art.
Example 14
High-Throughput Screening Assay Identifying Myeloid Activity
[1828] The following protocol is used to assess myeloid activity by
determining whether polypeptides of the invention proliferates
and/or differentiates myeloid cells. Myeloid cell activity is
assessed using the GAS/SEAP/Neo construct produced in Example 12.
Thus, factors that increase SEAP activity indicate the ability to
activate the Jaks-STATS signal transduction pathway. The myeloid
cell used in this assay is U937, a pre-monocyte cell line, although
TF-1, HL60, or KG1 can be used.
[1829] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 12, a DEAE-Dextran method (Kharbanda
et. al., 1994, Cell Growth & Differentiation, 5:259-265) is
used. First, harvest 2.times.10e.sup.7 U937 cells and wash with
PBS. The U937 cells are usually grown in RPMI 1640 medium
containing 10% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 mg/ml
streptomycin.
[1830] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na.sub.2HPO.sub.4.7H.sub.2O, 1
mM MgCl.sub.2, and 675 uM CaCl.sub.2. Incubate at 37 degrees C. for
45 min.
[1831] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37 degrees
C. for 36 hr.
[1832] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[1833] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[1834] Add 50 ul of the supernatant prepared by the protocol
described in Example 11. Incubate at 37 degrees C. for 48 to 72 hr.
As a positive control, 100 Unit/ml interferon gamma can be used
which is known to activate U937 cells. Over 30 fold induction is
typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 17.
Example 15
High-Throughput Screening Assay Identifying Neuronal Activity
[1835] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR1 is responsible for such induction.
Using the EGR1 promoter linked to reporter molecules, activation of
cells can be assessed.
[1836] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells can be assessed.
[1837] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to
+1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR
amplified from human genomic DNA using the following primers:
TABLE-US-00010 (SEQ ID NO: 6) 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG
-3' (SEQ ID NO: 7) 5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'
[1838] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS: SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR1
promoter.
[1839] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[1840] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat.
#12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a pre coated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed from the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[1841] Transfect the EGR/SEAP/Neo construct into PC12 using the
Lipofectamine protocol described in Example 11. EGR-SEAP/PC12
stable cells are obtained by growing the cells in 300 ug/ml G418.
The G418-free medium is used for routine growth but every one to
two months, the cells should be re-grown in 300 ug/ml G418 for
couple of passages.
[1842] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[1843] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.10.sup.5
cells/ml.
[1844] Add 200 ul of the cell suspension to each well of 96-well
plate (equivalent to 1.times.10.sup.5 cells/well). Add 50 ul
supernatant produced by Example 11, 37.degree. C. for 48 to 72 hr.
As a positive control, a growth factor known to activate PC12 cells
through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor
(NGF). Over fifty-fold induction of SEAP is typically seen in the
positive control wells. SEAP assay the supernatant according to
Example 17.
Example 16
High-Throughput Screening Assay for T-Cell Activity
[1845] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[1846] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1 MHC.
[1847] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the supernatants produced in Example 11.
Activators or inhibitors of NF-KB would be useful in treating
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[1848] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:8), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00011 (SEQ ID NO: 9)
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGC
CATCTCAATTAG:3'
[1849] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site:
TABLE-US-00012 (SEQ ID NO: 4) 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
[1850] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
CLONTECH.TM.. The resulting PCR fragment is digested with XhoI and
Hind III and subcloned into BLSK2-. (STRATAGENE.TM.) Sequencing
with the T7 and T3 primers confirms the insert contains the
following sequence:
TABLE-US-00013 (SEQ ID NO: 10)
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCA
ATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC
ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGC
TATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3'
[1851] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (CLONTECH.TM.) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[1852] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (CLONTECH.TM.),
replacing the GFP gene, after restricting pGFP-1 with SalI and
NotI.
[1853] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 13. Similarly, the method for assaying
supernatants with these stable Jurkat T-cells is also described in
Example 13. As a positive control, exogenous TNF alpha (0.1, 1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 17
Assay for SEAP Activity
[1854] As a reporter molecule for the assays described in Examples
13-16, SEAP activity is assayed using the Tropix Phospho-light Kit
(Cat. BP-400) according to the following general procedure. The
Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction
Buffers used below.
[1855] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 ul of 2.5.times. dilution buffer into Optiplates
containing 35 ul of a supernatant. Seal the plates with a plastic
sealer and incubate at 65 degree C. for 30 min. Separate the
Optiplates to avoid uneven heating.
[1856] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the table below). Add 50 ul
Reaction Buffer and incubate at room temperature for 20 minutes.
Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on
luminometer, one should treat 5 plates at each time and start the
second set 10 minutes later.
[1857] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity.
TABLE-US-00014 Reaction Buffer Formulation: Rxn buffer # of plates
diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14
80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110
5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140
7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170
8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200
10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44
230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255
12.75 50 260 13
Example 18
High-Throughput Screening Assay Identifying Changes in Small
Molecule Concentration and Membrane Permeability
[1858] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify
supernatants which bind to receptors of a particular cell. Although
the following protocol describes an assay for calcium, this
protocol can easily be modified to detect changes in potassium,
sodium, pH, membrane potential, or any other small molecule which
is detectable by a fluorescent probe.
[1859] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog no. F-14202), used here.
[1860] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with clear bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 ul of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after
the final wash.
[1861] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic
acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4
is added to each well. The plate is incubated at 37 degrees C. in a
CO.sub.2 incubator for 60 min. The plate is washed four times in
the Biotek washer with HBSS leaving 100 ul of buffer.
[1862] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37 degrees C. water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate
is then washed once in Denley Cell Wash with 200 ul, followed by an
aspiration step to 100 ul final volume.
[1863] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-4. The supernatant is added to the well, and
a change in fluorescence is detected.
[1864] To measure the fluorescence of intracellular calcium, the
FLIPR is set for the following parameters: (1) System gain is
300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event which has resulted in an increase in
the intracellular Ca.sup.++ concentration.
Example 19
High-Throughput Screening Assay Identifying Tyrosine Kinase
Activity
[1865] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[1866] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and non-receptor linked and cytosolic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[1867] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, the identification of novel
human secreted proteins capable of activating tyrosine kinase
signal transduction pathways are of interest. Therefore, the
following protocol is designed to identify those novel human
secreted proteins capable of activating the tyrosine kinase signal
transduction pathways.
[1868] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well LOPRODYNE.TM.
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from SIGMA.TM. Chemicals (St. Louis, Mo.) or 10%
MATRIGEL.TM. purchased from Becton Dickinson (Bedford, Mass.), or
calf serum, rinsed with PBS and stored at 4 degree C. Cell growth
on these plates is assayed by seeding 5,000 cells/well in growth
medium and indirect quantitation of cell number through use of
ALAMAR BLUE.TM. as described by the manufacturer Alamar
Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate
covers #3071 from Becton Dickinson (Bedford, Mass.) are used to
cover the LOPRODYNE.TM. Silent Screen Plates. Falcon Microtest III
cell culture plates can also be used in some proliferation
experiments.
[1869] To prepare extracts, A431 cells are seeded onto the nylon
membranes of LOPRODYNE.TM. plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example
11, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4,
2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170)
obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to
each well and the plate is shaken on a rotating shaker for 5
minutes at 4 degrees C. The plate is then placed in a vacuum
transfer manifold and the extract filtered through the 0.45 mm
membrane bottoms of each well using house vacuum. Extracts are
collected in a 96-well catch/assay plate in the bottom of the
vacuum manifold and immediately placed on ice. To obtain extracts
clarified by centrifugation, the content of each well, after
detergent solubilization for 5 minutes, is removed and centrifuged
for 15 minutes at 4 degrees C. at 16,000.times.g.
[1870] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[1871] Generally, the tyrosine kinase activity of a supernatant is
evaluated by determining its ability to phosphorylate a tyrosine
residue on a specific substrate (a biotinylated peptide).
Biotinylated peptides that can be used for this purpose include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase
cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin).
Both peptides are substrates for a range of tyrosine kinases and
are available from Boehringer Mannheim.
[1872] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 ul of 5 uM
Biotinylated Peptide, then 10 ul ATP/Mg.sub.2+ (5 mM ATP/50 mM
MgCl.sub.2), then 10 ul of 5.times. Assay Buffer (40 mM imidazole
hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100
mM MgCl.sub.2, 5 mM MnCl.sub.2, 0.5 mg/ml BSA), then 5 ul of Sodium
Vanadate (1 mM), and then 5 ul of water. Mix the components gently
and preincubate the reaction mix at 30 degrees C. for 2 min.
Initial the reaction by adding 10 ul of the control enzyme or the
filtered supernatant.
[1873] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
[1874] Tyrosine kinase activity is determined by transferring 50 ul
aliquot of reaction mixture to a microtiter plate (MTP) module and
incubating at 37 degrees C. for 20 min. This allows the
streptavadin coated 96 well plate to associate with the
biotinylated peptide. Wash the MTP module with 300 ul/well of PBS
four times. Next add 75 ul of anti-phosphotyrosine antibody
conjugated to horse radish peroxidase (anti-P-Tyr-POD (0.5 u/ml))
to each well and incubate at 37 degrees C. for one hour. Wash the
well as above.
[1875] Next add 100 ul of peroxidase substrate solution (Boehringer
Mannheim) and incubate at room temperature for at least 5 mins (up
to 30 min) Measure the absorbance of the sample at 405 nm by using
ELISA reader. The level of bound peroxidase activity is quantitated
using an ELISA reader and reflects the level of tyrosine kinase
activity.
Example 20
High-Throughput Screening Assay Identifying Phosphorylation
Activity
[1876] As a potential alternative and/or compliment to the assay of
protein tyrosine kinase activity described in Example 19, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase
(MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[1877] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr
at room temp, (RT). The plates are then rinsed with PBS and blocked
with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4 degrees C. until use.
[1878] A431 cells are seeded at 20,000/well in a 96-well
LOPRODYNE.TM. filterplate and
[1879] cultured overnight in growth medium. The cells are then
starved for 48 hr in basal medium (DMEM) and then treated with EGF
(6 ng/well) or 50 ul of the supernatants obtained in Example 11 for
5-20 minutes. The cells are then solubilized and extracts filtered
directly into the assay plate.
[1880] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
ug/ml) which specifically recognizes the phosphorylated epitope of
the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation.
Example 21
Method of Determining Alterations in a Gene Corresponding to a
Polynucleotide
[1881] RNA isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease) is be
isolated. cDNA is then generated from these RNA samples using
protocols known in the art. (See, Sambrook.) The cDNA is then used
as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO:X. Suggested PCR conditions consist of 35
cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58
degrees C.; and 60-120 seconds at 70 degrees C., using buffer
solutions described in Sidransky et al., Science 252:706
(1991).
[1882] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase. (Epicentre Technologies). The intron-exon borders of
selected exons is also determined and genomic PCR products analyzed
to confirm the results. PCR products harboring suspected mutations
is then cloned and sequenced to validate the results of the direct
sequencing.
[1883] PCR products is cloned into T-tailed vectors as described in
Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced
with T7 polymerase (United States Biochemical). Affected
individuals are identified by mutations not present in unaffected
individuals.
[1884] Genomic rearrangements are also observed as a method of
determining alterations in a gene corresponding to a
polynucleotide. Genomic clones isolated according to Example 2 are
nick-translated with digoxigenindeoxy-uridine 5'-triphosphate
(Boehringer Manheim), and FISH performed as described in Johnson et
al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the
labeled probe is carried out using a vast excess of human cot-1 DNA
for specific hybridization to the corresponding genomic locus.
[1885] Chromosomes are counterstained with
4,6-diamino-2-phenylidole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75
(1991).) Image collection, analysis and chromosomal fractional
length measurements are performed using the ISee Graphical Program
System. (Inovision Corporation, Durham, N.C.) Chromosome
alterations of the genomic region hybridized by the probe are
identified as insertions, deletions, and translocations. These
alterations are used as a diagnostic marker for an associated
disease.
Example 22
Method of Detecting Abnormal Levels of a Polypeptide in a
Biological Sample
[1886] A polypeptide of the present invention can be detected in a
biological sample, and if an increased or decreased level of the
polypeptide is detected, this polypeptide is a marker for a
particular phenotype. Methods of detection are numerous, and thus,
it is understood that one skilled in the art can modify the
following assay to fit their particular needs.
[1887] For example, antibody-sandwich ELISAs are used to detect
polypeptides in a sample, preferably a biological sample. Wells of
a microtiter plate are coated with specific antibodies, at a final
concentration of 0.2 to 10 ug/ml. The antibodies are either
monoclonal or polyclonal and are produced by the method described
in Example 10. The wells are blocked so that non-specific binding
of the polypeptide to the well is reduced.
[1888] The coated wells are then incubated for >2 hours at RT
with a sample containing the polypeptide. Preferably, serial
dilutions of the sample should be used to validate results. The
plates are then washed three times with deionized or distilled
water to remove unbounded polypeptide.
[1889] Next, 50 ul of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[1890] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution to each well and
incubate 1 hour at room temperature. Measure the reaction by a
microtiter plate reader. Prepare a standard curve, using serial
dilutions of a control sample, and plot polypeptide concentration
on the X-axis (log scale) and fluorescence or absorbance of the
Y-axis (linear scale). Interpolate the concentration of the
polypeptide in the sample using the standard curve.
Example 23
Formulation
[1891] The invention also provides methods of treatment and/or
prevention of diseases or disorders (such as, for example, any one
or more of the diseases or disorders disclosed herein) by
administration to a subject of an effective amount of a
Therapeutic. By therapeutic is meant polynucleotides or
polypeptides of the invention (including fragments and variants),
agonists or antagonists thereof, and/or antibodies thereto, in
combination with a pharmaceutically acceptable carrier type (e.g.,
a sterile carrier).
[1892] The Therapeutic will be formulated and dosed in a fashion
consistent with good medical practice, taking into account the
clinical condition of the individual patient (especially the side
effects of treatment with the Therapeutic alone), the site of
delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[1893] As a general proposition, the total pharmaceutically
effective amount of the Therapeutic administered parenterally per
dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of
patient body weight, although, as noted above, this will be subject
to therapeutic discretion. More preferably, this dose is at least
0.01 mg/kg/day, and most preferably for humans between about 0.01
and 1 mg/kg/day for the hormone. If given continuously, the
Therapeutic is typically administered at a dose rate of about 1
ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day
or by continuous subcutaneous infusions, for example, using a
mini-pump. An intravenous bag solution may also be employed. The
length of treatment needed to observe changes and the interval
following treatment for responses to occur appears to vary
depending on the desired effect.
[1894] Therapeutics can be are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any. The term "parenteral" as used herein refers to
modes of administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
[1895] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The term "parenteral" as used herein refers
to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
[1896] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics include suitable polymeric materials
(such as, for example, semi-permeable polymer matrices in the form
of shaped articles, e.g., films, or mirocapsules), suitable
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, and sparingly soluble derivatives
(such as, for example, a sparingly soluble salt).
[1897] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[1898] Sustained-release Therapeutics also include liposomally
entrapped Therapeutics of the invention (see generally, Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing the Therapeutic are prepared by methods known
per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA)
82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal Therapeutic.
[1899] In yet an additional embodiment, the Therapeutics of the
invention are delivered by way of a pump (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574
(1989)).
[1900] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[1901] For parenteral administration, in one embodiment, the
Therapeutic is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to the Therapeutic.
[1902] Generally, the formulations are prepared by contacting the
Therapeutic uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[1903] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[1904] The Therapeutic is typically formulated in such vehicles at
a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of polypeptide salts.
[1905] Any pharmaceutical used for therapeutic administration can
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutics generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[1906] Therapeutics ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampoules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10-ml vials are filled with 5
ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized Therapeutic using
bacteriostatic Water-for-Injection.
[1907] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the Therapeutics of the invention. Associated with
such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. In addition, the Therapeutics may be employed in
conjunction with other therapeutic compounds.
[1908] The Therapeutics of the invention may be administered alone
or in combination with adjuvants. Adjuvants that may be
administered with the Therapeutics of the invention include, but
are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE
(Biocine Corp.), QS21 (GENENTECH.TM., Inc.), BCG (e.g.,
THERACYS.RTM.), MPL and nonviable preparations of Corynebacterium
parvum. In a specific embodiment, Therapeutics of the invention are
administered in combination with alum. In another specific
embodiment, Therapeutics of the invention are administered in
combination with QS-21. Further adjuvants that may be administered
with the Therapeutics of the invention include, but are not limited
to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21,
QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant
technology. Vaccines that may be administered with the Therapeutics
of the invention include, but are not limited to, vaccines directed
toward protection against MMR (measles, mumps, rubella), polio,
varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus
influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease,
rotavirus, cholera, yellow fever, Japanese encephalitis,
poliomyelitis, rabies, typhoid fever, and pertussis. Combinations
may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[1909] The Therapeutics of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the Therapeutics of
the invention, include but not limited to, chemotherapeutic agents,
antibiotics, steroidal and non-steroidal anti-inflammatories,
conventional immunotherapeutic agents, and/or therapeutic
treatments described below. Combinations may be administered either
concomitantly, e.g., as an admixture, separately but simultaneously
or concurrently; or sequentially. This includes presentations in
which the combined agents are administered together as a
therapeutic mixture, and also procedures in which the combined
agents are administered separately but simultaneously, e.g., as
through separate intravenous lines into the same individual.
Administration "in combination" further includes the separate
administration of one of the compounds or agents given first,
followed by the second.
[1910] In certain embodiments, Therapeutics of the invention are
administered in combination with antiretroviral agents,
nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or
protease inhibitors (PIs). NRTIs that may be administered in
combination with the Therapeutics of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). NNRTIs that may be administered in
combination with the Therapeutics of the invention, include, but
are not limited to, VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM.
(delavirdine), and SUSTIVA.TM. (efavirenz). Protease inhibitors
that may be administered in combination with the Therapeutics of
the invention, include, but are not limited to, CRIXIVAN.TM.
(indinavir), NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and
VIRACEPT.TM. (nelfinavir). In a specific embodiment, antiretroviral
agents, nucleoside reverse transcriptase inhibitors, non-nucleoside
reverse transcriptase inhibitors, and/or protease inhibitors may be
used in any combination with Therapeutics of the invention to treat
AIDS and/or to prevent or treat HIV infection.
[1911] Additional NRTIs include LODENOSINE.TM. (F-ddA; an
acid-stable adenosine NRTI; Triangle/ABBOTT.TM.; COVIRACIL.TM.
(emtricitabine/FTC; structurally related to lamivudine (3TC) but
with 3- to 10-fold greater activity in vitro; Triangle/ABBOTT.TM.);
dOTC (BCH-10652, also structurally related to lamivudine but
retains activity against a substantial proportion of
lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused
approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON.RTM. (Adefovir Dipivoxil, the active prodrug of adefovir;
its active form is PMEA-pp); TENOFOVIR.TM. (bis-POC PMPA, a PMPA
prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/ABBOTT.TM.); D-D4FC (related to 3TC, with activity against
AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN.TM.
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'
azido-2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl
(SATE)-bearing prodrug forms of .beta.-L-FD4C and .beta.-L-FddC (WO
98/17281).
[1912] Additional NNRTIs include COACTINON.TM. (Emivirine/MKC-442,
potent NNRTI of the HEPT class; Triangle/ABBOTT.TM.);
CAPRAVIRINE.TM. (AG-1549/S-1153, a next generation NNRTI with
activity against viruses containing the K103N mutation;
AGOURON.TM.); PNU-142721 (has 20- to 50-fold greater activity than
its predecessor delavirdine and is active against K103N mutants;
PHARMACIA.TM. & Upjohn); DPC-961 and DPC-963 (second-generation
derivatives of efavirenz, designed to be active against viruses
with the K103N mutation; DUPONT.TM.); GW-420867X (has 25-fold
greater activity than HBY097 and is active against K103N mutants;
Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the
latex tree; active against viruses containing either or both the
Y181C and K103N mutations); and Propolis (WO 99/49830).
[1913] Additional protease inhibitors include LOPINAVIR.TM.
(ABT378/r; ABBOTT.TM. Laboratories); BMS-232632 (an azapeptide;
Bristol-Myres Squibb); TIPRANAVIR.TM. (PNU-140690, a non-peptic
dihydropyrone; PHARMACIA.TM. & Upjohn); PD-178390 (a
nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide;
Bristol-Myers Squibb); L-756,423 (an indinavir analog; MERCK.TM.);
DMP-450 (a cyclic urea compound; Avid & DUPONT.TM.); AG-1776 (a
peptidomimetic with in vitro activity against protease
inhibitor-resistant viruses; AGOURON.TM.); VX-175/GW-433908
(phosphate prodrug of amprenavir; Vertex & Glaxo Welcome);
CGP61755 (Ciba); and AGENERASE.TM. (amprenavir; Glaxo Wellcome
Inc.).
[1914] Additional antiretroviral agents include fusion
inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include
T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane
protein ectodomain which binds to gp41 in its resting state and
prevents transformation to the fusogenic state; Trimeris) and
T-1249 (a second-generation fusion inhibitor; Trimeris).
[1915] Additional antiretroviral agents include fusion
inhibitors/chemokine receptor antagonists. Fusion
inhibitors/chemokine receptor antagonists include CXCR4 antagonists
such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and
the T22 analogs T134 and T140; CCR5 antagonists such as RANTES
(9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4
antagonists such as NSC 651016 (a distamycin analog). Also included
are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists
such as RANTES, SDF-1, MIP-1.alpha., MIP-1.beta., etc., may also
inhibit fusion.
[1916] Additional antiretroviral agents include integrase
inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA)
acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIR.TM. (AR
177, an oligonucleotide that probably acts at cell surface rather
than being a true integrase inhibitor; Arondex); and naphthols such
as those disclosed in WO 98/50347.
[1917] Additional antiretroviral agents include hydroxyurea-like
compounds such as BCX-34 (a purine nucleoside phosphorylase
inhibitor; Biocryst); ribonucleotide reductase inhibitors such as
DIDOX.TM. (Molecules for Health); inosine monophosphate
dehydrogenase (IMPDH) inhibitors such as VX-497 (Vertex); and
mycopholic acids such as CellCept (mycophenolate mofetil;
ROCHE.TM.).
[1918] Additional antiretroviral agents include inhibitors of viral
integrase, inhibitors of viral genome nuclear translocation such as
arylene bis(methylketone) compounds; inhibitors of HIV entry such
as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble
complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100;
nucleocapsid zinc finger inhibitors such as dithiane compounds;
targets of HIV Tat and Rev; and pharmacoenhancers such as
ABT-378.
[1919] Other antiretroviral therapies and adjunct therapies include
cytokines and lymphokines such as MIP-1.alpha., MIP-1.beta.,
SDF-1.alpha., IL-2, PROLEUKIN.TM. (aldesleukin/L2-7001;
CHIRON.TM.), IL-4, IL-10, IL-12, and IL-13; interferons such as
IFN-.alpha.2a; antagonists of TNFs, NF.kappa.B, GM-CSF, M-CSF, and
IL-10; agents that modulate immune activation such as cyclosporin
and prednisone; vaccines such as Remune.TM. (HIV Immunogen), APL
400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E)
recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2
rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched
synthetic peptide derived from discontinuous gp120 C3/C4 domain,
fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines;
gene-based therapies such as genetic suppressor elements (GSEs; WO
98/54366), and intrakines (genetically modified CC chemokines
targeted to the ER to block surface expression of newly synthesized
CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med.
3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5,
the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and
PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3
antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D,
268-D and 50.1, anti-Tat antibodies, anti-TNF-.alpha. antibodies,
and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor
agonists and antagonists such as TCDD,
3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and
.alpha.-naphthoflavone (WO 98/30213); and antioxidants such as
.gamma.-L-glutamyl-L-cysteine ethyl ester (.gamma.-GCE; WO
99/56764).
[1920] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the Therapeutics of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[1921] In other embodiments, Therapeutics of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the Therapeutics of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLET.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
Therapeutics of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat or prevent an
opportunistic Pneumocystis carinii pneumonia infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with ISONIAZID.TM., RIFAMPIN.TM., PYRAZINAMIDE.TM.,
and/or ETHAMBUTOL.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium avium complex infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or
AZITHROMYCIN.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium tuberculosis infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with
FLUCONAZOLE.TM. ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat or prevent an opportunistic fungal
infection. In another specific embodiment, Therapeutics of the
invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection.
In another specific embodiment, Therapeutics of the invention are
used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplasma gondii infection. In another specific
embodiment, Therapeutics of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat or prevent an opportunistic bacterial
infection.
[1922] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the Therapeutics of
the invention include, but are not limited to, amoxicillin,
beta-lactamases, aminoglycosides, beta-lactam (glycopeptide),
beta-lactamases, Clindamycin, chloramphenicol, cephalosporins,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole, penicillins, quinolones, rapamycin, rifampin,
streptomycin, sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamethoxazole, and vancomycin.
[1923] In other embodiments, Therapeutics of the invention are
administered in combination with immunosuppressive agents
Immunosuppressive agents that may be administered in combination
with the Therapeutics of the invention include, but are not limited
to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone, prednisone, azathioprine, FK-506,
15-deoxyspergualin, and other immunosuppressive agents that act by
suppressing the function of responding T cells. Other
immunosuppressive agents that may be administered in combination
with the Therapeutics of the invention include, but are not limited
to, prednisolone, methotrexate, thalidomide, methoxsalen,
rapamycin, leflunomide, mizoribine (BREDININ.TM.), brequinar,
deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT.RTM. 3
(muromonab-CD3), SANDIMMUNE.TM., NEORAL.TM., SANGDYA.TM.
(cyclosporine), PROGRAF.RTM. (FK506, tacrolimus), CELLCEPT.RTM.
(mycophenolate motefil, of which the active metabolite is
mycophenolic acid), IMURAN.TM. (azathioprine),
glucocorticosteroids, adrenocortical steroids such as DELTASONE.TM.
(prednisone) and HYDELTRASOL.TM. (prednisolone), FOLEX.TM. and
MEXATE.TM. (methotrxate), OXSORALEN-ULTRA.TM. (methoxsalen) and
RAPAMUNE.TM. (sirolimus). In a specific embodiment,
immunosuppressants may be used to prevent rejection of organ or
bone marrow transplantation.
[1924] In an additional embodiment, Therapeutics of the invention
are administered alone or in combination with one or more
intravenous immune globulin preparations. Intravenous immune
globulin preparations that may be administered with the
Therapeutics of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM.,
ATGAM.TM. (antithymocyte glubulin), and GAMIMUNE.TM.. In a specific
embodiment, Therapeutics of the invention are administered in
combination with intravenous immune globulin preparations in
transplantation therapy (e.g., bone marrow transplant).
[1925] In certain embodiments, the Therapeutics of the invention
are administered alone or in combination with an anti-inflammatory
agent. Anti-inflammatory agents that may be administered with the
Therapeutics of the invention include, but are not limited to,
corticosteroids (e.g. betamethasone, budesonide, cortisone,
dexamethasone, hydrocortisone, methylprednisolone, prednisolone,
prednisone, and triamcinolone), nonsteroidal anti-inflammatory
drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen,
floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen,
oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam,
tiaprofenic acid, and tolmetin.), as well as antihistamines,
aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,
arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic
acid derivatives, pyrazoles, pyrazolones, salicylic acid
derivatives, thiazinecarboxamides, e-acetamidocaproic acid,
S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,
bendazac, benzydamine, bucolome, difenpiramide, ditazol,
emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,
oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole,
and tenidap.
[1926] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-angiogenic agent. Anti-angiogenic agents that may be
administered with the compositions of the invention include, but
are not limited to, Angiostatin (ENTREMED.TM., Rockville, Md.),
Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel
(TAXOL.TM.), Suramin, Tissue Inhibitor of Metalloproteinase-1,
Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen
Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and
various forms of the lighter "d group" transition metals.
[1927] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[1928] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[1929] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[1930] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include, but are not limited to, platelet
factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from queen crab shells), (Murata et al., Cancer Res.
51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine; modulators of matrix metabolism, including for
example, proline analogs, cishydroxyproline,
d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,
aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992));
Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992));
Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin
(Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate
("GST"; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, (1987)); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, (1992)); and
metalloproteinase inhibitors such as BB94.
[1931] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(CELGENE.TM., Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem
and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin
alpha v beta 3 antagonist (C. Storgard et al., J. Clin. Invest.
103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole
(CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin
A-4 (CA4P) (OXiGENE.TM., Boston, Mass.); Squalamine (Magainin
Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (TAP
PHARMACEUTICALS.TM., Deerfield, Ill.); ZD-0101 ASTRAZENECA.TM.
(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555);
CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA;
Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839);
Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint;
PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644);
Tamoxifen (NOLVADEX.TM.); Tazarotene; Tetrathiomolybdate;
XELODA.TM. (Capecitabine); and 5-Fluorouracil.
[1932] Anti-angiogenic agents that may be administered in
combination with the compounds of the invention may work through a
variety of mechanisms including, but not limited to, inhibiting
proteolysis of the extracellular matrix, blocking the function of
endothelial cell-extracellular matrix adhesion molecules, by
antagonizing the function of angiogenesis inducers such as growth
factors, and inhibiting integrin receptors expressed on
proliferating endothelial cells. Examples of anti-angiogenic
inhibitors that interfere with extracellular matrix proteolysis and
which may be administered in combination with the compositions of
the invention include, but are not limited to, AG-3340
(AGOURON.TM., La Jolla, Calif.), BAY-12-9566 (BAYER.TM., West
Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.),
CGS-27032A (NOVARTIS.TM., East Hanover, N.J.), Marimastat (British
Biotech, Oxford, UK), and METASTAT.TM. (AETERNA.TM., St-Foy,
Quebec). Examples of anti-angiogenic inhibitors that act by
blocking the function of endothelial cell-extracellular matrix
adhesion molecules and which may be administered in combination
with the compositions of the invention include, but are not limited
to, EMD-121974 (MERCK.TM. KcgaA Darmstadt, Germany) and VITAXIN.TM.
(IXSYS.TM., La Jolla, Calif./MEDIMMUNE.TM., Gaithersburg, Md.).
Examples of anti-angiogenic agents that act by directly
antagonizing or inhibiting angiogenesis inducers and which may be
administered in combination with the compositions of the invention
include, but are not limited to, Angiozyme (Ribozyme, Boulder,
Colo.), Anti-VEGF antibody (GENENTECH.TM., S. San Francisco,
Calif.), PTK-787/ZK-225846 (NOVARTIS.TM., Basel, Switzerland),
SU-101 (SUGEN.TM., S. San Francisco, Calif.), SU-5416
(SUGEN.TM./PHARMACIA.TM. Upjohn, Bridgewater, N.J.), and SU-6668
(SUGEN.TM.). Other anti-angiogenic agents act to indirectly inhibit
angiogenesis. Examples of indirect inhibitors of angiogenesis which
may be administered in combination with the compositions of the
invention include, but are not limited to, IM-862 (CYTRAN.TM.,
Kirkland, Wash.), Interferon-alpha, IL-12 (ROCHE.TM., Nutley,
N.J.), and Pentosan polysulfate (Georgetown University, Washington,
D.C.).
[1933] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
an autoimmune disease, such as for example, an autoimmune disease
described herein.
[1934] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use of compositions
of the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
rheumatoid arthritis.
[1935] In another embodiment, the polynucleotides encoding a
polypeptide of the present invention are administered in
combination with an angiogenic protein, or polynucleotides encoding
an angiogenic protein. Examples of angiogenic proteins that may be
administered with the compositions of the invention include, but
are not limited to, acidic and basic fibroblast growth factors,
VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta,
platelet-derived endothelial cell growth factor, platelet-derived
growth factor, tumor necrosis factor alpha, hepatocyte growth
factor, insulin-like growth factor, colony stimulating factor,
macrophage colony stimulating factor, granulocyte/macrophage colony
stimulating factor, and nitric oxide synthase.
[1936] In additional embodiments, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
Therapeutics of the invention include, but are not limited to
alkylating agents such as nitrogen mustards (for example,
Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide,
Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and
methylmelamines (for example, Hexamethylmelamine and Thiotepa),
alkyl sulfonates (for example, Busulfan), nitrosoureas (for
example, Carmustine (BCNU), Lomustine (CCNU), Semustine
(methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for
example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)),
folic acid analogs (for example, Methotrexate (amethopterin)),
pyrimidine analogs (for example, Fluorouacil (5-fluorouracil;
5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine
(cytosine arabinoside)), purine analogs and related inhibitors (for
example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine
(6-thioguanine; TG), and Pentostatin (2'-deoxycoformycin)), vinca
alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and
Vincristine (vincristine sulfate)), epipodophyllotoxins (for
example, Etoposide and Teniposide), antibiotics (for example,
Dactinomycin (actinomycin D), Daunorubicin (daunomycin;
rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and
Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase),
biological response modifiers (for example, Interferon-alpha and
interferon-alpha-2b), platinum coordination compounds (for example,
Cisplatin (cis-DDP) and Carboplatin), anthracenedione
(Mitoxantrone), substituted ureas (for example, Hydroxyurea),
methylhydrazine derivatives (for example, Procarbazine
(N-methylhydrazine; MIH), adrenocorticosteroids (for example,
Prednisone), progestins (for example, Hydroxyprogesterone caproate,
Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol
acetate), estrogens (for example, Diethylstilbestrol (DES),
Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol),
antiestrogens (for example, Tamoxifen), androgens (Testosterone
proprionate, and Fluoxymesterone), antiandrogens (for example,
Flutamide), gonadotropin-releasing hormone analogs (for example,
Leuprolide), other hormones and hormone analogs (for example,
methyltestosterone, estramustine, estramustine phosphate sodium,
chlorotrianisene, and testolactone), and others (for example,
dicarbazine, glutamic acid, and mitotane).
[1937] In one embodiment, the compositions of the invention are
administered in combination with one or more of the following
drugs: infliximab (also known as Remicade.TM. Centocor, Inc.),
Trocade (ROCHE.TM., RO-32-3555), Leflunomide (also known as
Arava.TM. from HOECHST MARION ROUSSEL.TM.), Kineret.TM. (an IL-1
Receptor antagonist also known as Anakinra from AMGEN.TM.,
Inc.)
[1938] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or combination of one or
more of the components of CHOP. In one embodiment, the compositions
of the invention are administered in combination with anti-CD20
antibodies, human monoclonal anti-CD20 antibodies. In another
embodiment, the compositions of the invention are administered in
combination with anti-CD.sub.20 antibodies and CHOP, or
anti-CD.sub.20 antibodies and any combination of one or more of the
components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, compositions of the invention
are administered in combination with Rituximab. In a further
embodiment, compositions of the invention are administered with
Rituximab and CHOP, or Rituximab and any combination of one or more
of the components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, compositions of the invention
are administered in combination with tositumomab. In a further
embodiment, compositions of the invention are administered with
tositumomab and CHOP, or tositumomab and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. The anti-CD.sub.20 antibodies may optionally be
associated with radioisotopes, toxins or cytotoxic prodrugs.
[1939] In another specific embodiment, the compositions of the
invention are administered in combination Zevalin.TM.. In a further
embodiment, compositions of the invention are administered with
Zevalin.TM. and CHOP, or Zevalin.TM. and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. Zevalin.TM. may be associated with one or more
radisotopes. Particularly preferred isotopes are .sup.90Y and
.sup.111In.
[1940] In an additional embodiment, the Therapeutics of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the Therapeutics of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
In another embodiment, Therapeutics of the invention may be
administered with any interleukin, including, but not limited to,
IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, and IL-21.
[1941] In one embodiment, the Therapeutics of the invention are
administered in combination with members of the TNF family. TNF,
TNF-related or TNF-like molecules that may be administered with the
Therapeutics of the invention include, but are not limited to,
soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known
as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), OPG, and
neutrokine-alpha (International Publication No. WO 98/18921, OX40,
and nerve growth factor (NGF), and soluble forms of Fas, CD30,
CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[1942] In an additional embodiment, the Therapeutics of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the Therapeutics
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (P1GF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (P1GF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular
Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in
International Publication Number WO 96/26736; Vascular Endothelial
Growth Factor-D (VEGF-D), as disclosed in International Publication
Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D),
as disclosed in International Publication Number WO 98/07832; and
Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in
German Patent Number DE19639601. The above mentioned references are
herein incorporated by reference in their entireties.
[1943] In an additional embodiment, the Therapeutics of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the Therapeutics of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[1944] In an additional embodiment, the Therapeutics of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the Therapeutics of the invention include, but are not limited to,
granulocyte macrophage colony stimulating factor (GM-CSF)
(sargramostim, LEUKINE.TM., PROKINE.TM.), granulocyte colony
stimulating factor (G-CSF) (filgrastim, NEUPOGEN.TM.), macrophage
colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin
alfa, EPOGEN.TM., PROCRIT.TM.), stem cell factor (SCF, c-kit
ligand, steel factor), megakaryocyte colony stimulating factor,
PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any
one or more of IL-1 through IL-12, interferon-gamma, or
thrombopoietin.
[1945] In certain embodiments, Therapeutics of the present
invention are administered in combination with adrenergic blockers,
such as, for example, acebutolol, atenolol, betaxolol, bisoprolol,
carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol,
pindolol, propranolol, sotalol, and timolol.
[1946] In another embodiment, the Therapeutics of the invention are
administered in combination with an antiarrhythmic drug (e.g.,
adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin,
diliazem, disopyramide, esmolol, flecamide, lidocaine, mexiletine,
moricizine, phenyloin, procainamide, N-acetyl procainamide,
propafenone, propranolol, quinidine, sotalol, tocamide, and
verapamil).
[1947] In another embodiment, the Therapeutics of the invention are
administered in combination with diuretic agents, such as carbonic
anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide,
and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide,
mannitol, and urea), diuretics that inhibit
Na.sup.+-K.sup.+-2Cl.sup.- symport (e.g., furosemide, bumetanide,
azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and
torsemide), thiazide and thiazide-like diuretics (e.g.,
bendroflumethiazide, benzthiazide, chlorothiazide,
hydrochlorothiazide, hydroflumethiazide, methyclothiazide,
polythiazide, trichormethiazide, chlorthalidone, indapamide,
metolazone, and quinethazone), potassium sparing diuretics (e.g.,
amiloride and triamterene), and mineralcorticoid receptor
antagonists (e.g., spironolactone, canrenone, and potassium
canrenoate).
[1948] In one embodiment, the Therapeutics of the invention are
administered in combination with treatments for endocrine and/or
hormone imbalance disorders. Treatments for endocrine and/or
hormone imbalance disorders include, but are not limited to,
.sup.127I, radioactive isotopes of iodine such as .sup.131I and
.sup.123I; recombinant growth hormone, such as HUMATROPE.TM.
(recombinant somatropin); growth hormone analogs such as
PROTROPIN.TM. (somatrem); dopamine agonists such as PARLODEL.TM.
(bromocriptine); somatostatin analogs such as SANDOSTATIN.TM.
(octreotide); gonadotropin preparations such as PREGNYL.TM.,
A.P.L..TM. and PROFASI.TM. (chorionic gonadotropin (CG)),
PERGONAL.TM. (menotropins), and METRODIN.TM. (urofollitropin
(uFSH)); synthetic human gonadotropin releasing hormone
preparations such as FACTREL.TM. and LUTREPULSE.TM. (gonadorelin
hydrochloride); synthetic gonadotropin agonists such as LUPRON.TM.
(leuprolide acetate), SUPPRELIN.TM. (histrelin acetate),
SYNAREL.TM. (nafarelin acetate), and ZOLADEX.TM. (goserelin
acetate); synthetic preparations of thyrotropin-releasing hormone
such as RELEFACT TRH.TM. and THYPINONE.TM. (protirelin);
recombinant human TSH such as THYROGEN.TM.; synthetic preparations
of the sodium salts of the natural isomers of thyroid hormones such
as L-T.sub.4.TM., SYNTHROID.TM. and LEVOTHROID.TM. (levothyroxine
sodium), L-T.sub.3.TM., CYTOMEL.TM. and TRIOSTAT.TM. (liothyroine
sodium), and THYROLAR.TM. (liotrix); antithyroid compounds such as
6-n-propylthiouracil (propylthiouracil),
1-methyl-2-mercaptoimidazole and TAPAZOLE.TM. (methimazole),
NEO-MERCAZOLE.TM. (carbimazole); beta-adrenergic receptor
antagonists such as propranolol and esmolol; Ca.sup.2+ channel
blockers; dexamethasone and iodinated radiological contrast agents
such as TELEPAQUE.TM. (iopanoic acid) and ORAGRAFIN.TM. (sodium
ipodate).
[1949] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, estrogens or conjugated
estrogens such as ESTRACE.TM. (estradiol), ESTINYL.TM. (ethinyl
estradiol), PREMARIN.TM., ESTRATAB.TM., ORTHO-EST.TM., OGEN.TM. and
estropipate (estrone), ESTROVIS.TM. (quinestrol), ESTRADERM.TM.
(estradiol), DELESTROGEN.TM. and VALERGEN.TM. (estradiol valerate),
DEPO-ESTRADIOL CYPIONATE.TM. and ESTROJECT LA.TM. (estradiol
cypionate); antiestrogens such as NOLVADEX.TM. (tamoxifen),
SEROPHENE.TM. and CLOMID.TM. (clomiphene); progestins such as
DURALUTIN.TM. (hydroxyprogesterone caproate), MPA.TM. and
DEPO--PROVERA.TM. (medroxyprogesterone acetate), PROVERA.TM. and
CYCRIN.TM. (MPA), MEGACE.TM. (megestrol acetate), NORLUTIN.TM.
(norethindrone), and NORLUTATE.TM. and AYGESTIN.TM. (norethindrone
acetate); progesterone implants such as NORPLANT SYSTEM.TM.
(subdermal implants of norgestrel); antiprogestins such as RU
486.TM. (mifepristone); hormonal contraceptives such as ENOVID.TM.
(norethynodrel plus mestranol), PROGESTASERT.TM. (intrauterine
device that releases progesterone), LOESTRIN.TM., BREVICON.TM.,
MODICON.TM. GENORA.TM., NELONA.TM., NORINYL.TM., OVACON-35.TM. and
OVACON-50.TM. (ethinyl estradiol/norethindrone), LEVLEN.TM.,
NORDETTE.TM., TRI-LEVLEN.TM. and TRIPHASIL-21.TM. (ethinyl
estradiol/levonorgestrel) LO/OVRAL.TM. and OVRAL.TM. (ethinyl
estradiol/norgestrel), DEMULEN.TM. (ethinyl estradiol/ethynodiol
diacetate), NORINYL.TM., ORTHO-NOVUM.TM., NORETHIN.TM., GENORA.TM.,
and NELOVA.TM. (norethindrone/mestranol), DESOGEN.TM. and
ORTHO-CEPT.TM. (ethinyl estradiol/desogestrel), ORTHO-CYCLEN.TM.
and ORTHO-TRICYCLEN.TM. (ethinyl estradiol/norgestimate),
MICRONOR.TM. and NOR-QD.TM. (norethindrone), and OVRETTE.TM.
(norgestrel).
[1950] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, testosterone esters such
as methenolone acetate and testosterone undecanoate; parenteral and
oral androgens such as TESTOJECT-50.TM. (testosterone), TESTEX.TM.
(testosterone propionate), DELATESTRYL.TM. (testosterone
enanthate), DEPO-TESTOSTERONE.TM. (testosterone cypionate),
DANOCRINE.TM. (danazol), HALOTESTIN.TM. (fluoxymesterone), ORETON
METHYL.TM., TESTRED.TM. and VIRILON.TM. (methyltestosterone), and
OXANDRIN.TM. (oxandrolone); testosterone transdermal systems such
as TESTODERM.TM.; androgen receptor antagonist and
5-alpha-reductase inhibitors such as ANDROCUR.TM. (cyproterone
acetate), EULEXIN.TM. (flutamide), and PROSCAR.TM. (finasteride);
adrenocorticotropic hormone preparations such as CORTROSYN.TM.
(cosyntropin); adrenocortical steroids and their synthetic analogs
such as ACLOVATE.TM. (alclometasone dipropionate), CYCLOCORT.TM.
(amcinonide), BECLOVENT.TM. and VANCERIL.TM. (beclomethasone
dipropionate), CELESTONE.TM. (betamethasone), BENISONE.TM. and
UTICORT.TM. (betamethasone benzoate), DIPROSONE.TM. (betamethasone
dipropionate), CELESTONE PHOSPHATE.TM. (betamethasone sodium
phosphate), CELESTONE SOLUSPAN.TM. (betamethasone sodium phosphate
and acetate), BETA-VAL.TM. and VALISONE.TM. (betamethasone
valerate), TEMOVATE.TM. (clobetasol propionate), CLODERM.TM.
(clocortolone pivalate), CORTEF.TM. and HYDROCORTONE.TM. (cortisol
(hydrocortisone)), HYDROCORTONE ACETATE.TM. (cortisol
(hydrocortisone) acetate), LOCOID.TM. (cortisol (hydrocortisone)
butyrate), HYDROCORTONE PHOSPHATE.TM. (cortisol (hydrocortisone)
sodium phosphate), A-HYDROCORT.TM. and SOLU CORTEF.TM. (cortisol
(hydrocortisone) sodium succinate), WESTCORT.TM. (cortisol
(hydrocortisone) valerate), CORTISONE ACETATE.TM. (cortisone
acetate), DESOWEN.TM. and TRIDESILON.TM. (desonide), TOPICORT.TM.
(desoximetasone), DECADRON.TM. (dexamethasone), DECADRON LA.TM.
(dexamethasone acetate), DECADRON PHOSPHATE.TM. and HEXADROL
PHOSPHATE.TM. (dexamethasone sodium phosphate), FLORONE.TM. and
MAXIFLOR.TM. (diflorasone diacetate), FLORINEF ACETATE.TM.
(fludrocortisone acetate), AEROBID.TM. and NASALIDE.TM.
(flunisolide), FLUONID.TM. and SYNALAR.TM. (fluocinolone
acetonide), LIDEX.TM. (fluocinonide), FLUOR-OP.TM. and FML.TM.
(fluorometholone), CORDRAN.TM. (flurandrenolide), HALOG.TM.
(halcinonide), HMS LIZUIFILM.TM. (medrysone), MEDROL.TM.
(methylprednisolone), DEPO-MEDROL.TM. and MEDROL ACETATE.TM.
(methylprednisone acetate), A-METHAPRED.TM. and SOLUMEDROL.TM.
(methylprednisolone sodium succinate), ELOCON.TM. (mometasone
furoate), HALDRONE.TM. (paramethasone acetate), DELTA-CORTEF.TM.
(prednisolone), ECONOPRED.TM. (prednisolone acetate),
HYDELTRASOL.TM. (prednisolone sodium phosphate), HYDELTRA-T.B.A.TM.
(prednisolone tebutate), DELTASONE.TM. (prednisone), ARISTOCORT.TM.
and KENACORT.TM. (triamcinolone), KENALOG.TM. (triamcinolone
acetonide), ARISTOCORT.TM. and KENACORT DIACETATE.TM.
(triamcinolone diacetate), and ARISTOSPAN.TM. (triamcinolone
hexacetonide); inhibitors of biosynthesis and action of
adrenocortical steroids such as CYTADREN.TM. (aminoglutethimide),
NIZORAL.TM. (ketoconazole), MODRASTANE.TM. (trilostane), and
METOPIRONE.TM. (metyrapone).
[1951] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to bovine, porcine or human
insulin or mixtures thereof; insulin analogs; recombinant human
insulin such as HUMULIN.TM. and NOVOLIN.TM.; oral hypoglycemic
agents such as ORAMIDE.TM. and ORINASE.TM. (tolbutamide),
DIABINESE.TM. (chlorpropamide), TOLAMIDE.TM. and TOLINASE.TM.
(tolazamide), DYMELOR.TM. (acetohexamide), glibenclamide,
MICRONASE.TM., DIBETA.TM. and GLYNASE.TM. (glyburide),
GLUCOTROL.TM. (glipizide), and DIAMICRON.TM. (gliclazide),
GLUCOPHAGE.TM. (metformin), PRECOSE.TM. (acarbose), AMARYL.TM.
(glimepiride), and ciglitazone; thiazolidinediones (TZDs) such as
rosiglitazone, AVANDIA.TM. (rosiglitazone maleate) ACTOS.TM.
(piogliatazone), and troglitazone; alpha-glucosidase inhibitors;
bovine or porcine glucagon; somatostatins such as SANDOSTATIN.TM.
(octreotide); and diazoxides such as PROGLYCEM.TM. (diazoxide). In
still other embodiments, Therapeutics of the invention are
administered in combination with one or more of the following: a
biguanide antidiabetic agent, a glitazone antidiabetic agent, and a
sulfonylurea antidiabetic agent.
[1952] In one embodiment, the Therapeutics of the invention are
administered in combination with treatments for uterine motility
disorders. Treatments for uterine motility disorders include, but
are not limited to, estrogen drugs such as conjugated estrogens
(e.g., PREMARIN.RTM. and ESTRATAB.RTM.), estradiols (e.g.,
CLIMARA.RTM. and ALORA.RTM.), estropipate, and chlorotrianisene;
progestin drugs (e.g., AMEN.RTM. (medroxyprogesterone),
MICRONOR.RTM. (norethidrone acetate), PROMETRIUM.RTM. progesterone,
and megestrol acetate); and estrogen/progesterone combination
therapies such as, for example, conjugated
estrogens/medroxyprogesterone (e.g., PREMPRO.TM. and
PREMPHASE.RTM.) and norethindrone acetate/ethinyl estsradiol (e.g.,
FEMHRT.TM.).
[1953] In an additional embodiment, the Therapeutics of the
invention are administered in combination with drugs effective in
treating iron deficiency and hypochromic anemias, including but not
limited to, ferrous sulfate (iron sulfate, FEOSOL.TM.), ferrous
fumarate (e.g., FEOSTAT.TM.), ferrous gluconate (e.g., FERGON.TM.),
polysaccharide-iron complex (e.g., NIFEREX.TM.), iron dextran
injection (e.g., INFED.TM.), cupric sulfate, pyroxidine,
riboflavin, Vitamin B.sub.12, cyancobalamin injection (e.g.,
REDISOL.TM., RUBRAMIN PC.TM.), hydroxocobalamin, folic acid (e.g.,
FOLVITE.TM.), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum
factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or
ferritin.
[1954] In certain embodiments, the Therapeutics of the invention
are administered in combination with agents used to treat
psychiatric disorders. Psychiatric drugs that may be administered
with the Therapeutics of the invention include, but are not limited
to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene,
clozapine, fluphenazine, haloperidol, loxapine, mesoridazine,
molindone, olanzapine, perphenazine, pimozide, quetiapine,
risperidone, thioridazine, thiothixene, trifluoperazine, and
triflupromazine), antimanic agents (e.g., carbamazepine, divalproex
sodium, lithium carbonate, and lithium citrate), antidepressants
(e.g., amitriptyline, amoxapine, bupropion, citalopram,
clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine,
imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone,
nortriptyline, paroxetine, phenelzine, protriptyline, sertraline,
tranylcypromine, trazodone, trimipramine, and venlafaxine),
antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide,
clorazepate, diazepam, halazepam, lorazepam, oxazepam, and
prazepam), and stimulants (e.g., d-amphetamine, methylphenidate,
and pemoline).
[1955] In other embodiments, the Therapeutics of the invention are
administered in combination with agents used to treat neurological
disorders. Neurological agents that may be administered with the
Therapeutics of the invention include, but are not limited to,
antiepileptic agents (e.g., carbamazepine, clonazepam,
ethosuximide, phenobarbital, phenyloin, primidone, valproic acid,
divalproex sodium, felbamate, gabapentin, lamotrigine,
levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide,
diazepam, lorazepam, and clonazepam), antiparkinsonian agents
(e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine,
pergolide, ropinirole, pramipexole, benztropine; biperiden;
ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS
therapeutics (e.g. riluzole).
[1956] In another embodiment, Therapeutics of the invention are
administered in combination with vasodilating agents and/or calcium
channel blocking agents. Vasodilating agents that may be
administered with the Therapeutics of the invention include, but
are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors
(e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril,
enalapril, enalaprilat, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, spirapril, trandolapril, and
nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide
mononitrate, and nitroglycerin). Examples of calcium channel
blocking agents that may be administered in combination with the
Therapeutics of the invention include, but are not limited to
amlodipine, bepridil, diltiazem, felodipine, flunarizine,
isradipine, nicardipine, nifedipine, nimodipine, and verapamil. In
additional embodiments, the Therapeutics of the invention are
administered in combination with other therapeutic or prophylactic
regimens, such as, for example, radiation therapy.
Example 24
Method of Treating Decreased Levels of the Polypeptide
[1957] The present invention relates to a method for treating an
individual in need of an increased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an agonist of the invention (including polypeptides of
the invention). Moreover, it will be appreciated that conditions
caused by a decrease in the standard or normal expression level of
a secreted protein in an individual can be treated by administering
the polypeptide of the present invention, preferably in the
secreted form. Thus, the invention also provides a method of
treatment of an individual in need of an increased level of the
polypeptide comprising administering to such an individual a
Therapeutic comprising an amount of the polypeptide to increase the
activity level of the polypeptide in such an individual.
[1958] For example, a patient with decreased levels of a
polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide
for six consecutive days. Preferably, the polypeptide is in the
secreted form. The exact details of the dosing scheme, based on
administration and formulation, are provided in Example 23.
Example 25
Method of Treating Increased Levels of the Polypeptide
[1959] The present invention also relates to a method of treating
an individual in need of a decreased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an antagonist of the invention (including polypeptides
and antibodies of the invention).
[1960] In one example, antisense technology is used to inhibit
production of a polypeptide of the present invention. This
technology is one example of a method of decreasing levels of a
polypeptide, preferably a secreted form, due to a variety of
etiologies, such as cancer. For example, a patient diagnosed with
abnormally increased levels of a polypeptide is administered
intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and
3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day
rest period if the treatment was well tolerated. The formulation of
the antisense polynucleotide is provided in Example 23.
Example 26
Method of Treatment Using Gene Therapy-Ex Vivo
[1961] One method of gene therapy transplants fibroblasts, which
are capable of expressing a polypeptide, onto a patient. Generally,
fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin) is added. The
flasks are then incubated at 37 degree C. for approximately one
week.
[1962] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[1963] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[1964] The cDNA encoding a polypeptide of the present invention can
be amplified using PCR primers which correspond to the 5' and 3'
end sequences respectively as set forth in Example 1 using primers
and having appropriate restriction sites and initiation/stop
codons, if necessary. Preferably, the 5' primer contains an EcoRI
site and the 3' primer includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is then used to transform bacteria HB101, which are then plated
onto agar containing kanamycin for the purpose of confirming that
the vector has the gene of interest properly inserted.
[1965] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells transduced with the vector. The
packaging cells now produce infectious viral particles containing
the gene (the packaging cells are now referred to as producer
cells).
[1966] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether protein is produced.
[1967] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 27
Gene Therapy Using Endogenous Genes Corresponding to
Polynucleotides of the Invention
[1968] Another method of gene therapy according to the present
invention involves operably associating the endogenous
polynucleotide sequence of the invention with a promoter via
homologous recombination as described, for example, in U.S. Pat.
No. 5,641,670, issued Jun. 24, 1997; International Publication NO:
WO 96/29411, published Sep. 26, 1996; International Publication NO:
WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.
Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature,
342:435-438 (1989). This method involves the activation of a gene
which is present in the target cells, but which is not expressed in
the cells, or is expressed at a lower level than desired.
[1969] Polynucleotide constructs are made which contain a promoter
and targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous polynucleotide sequence, flanking the
promoter. The targeting sequence will be sufficiently near the 5'
end of the polynucleotide sequence so the promoter will be operably
linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using
PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter.
[1970] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[1971] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[1972] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous polynucleotide sequence. This results in the
expression of polynucleotide corresponding to the polynucleotide in
the cell. Expression may be detected by immunological staining, or
any other method known in the art.
[1973] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.z HPO.sub.4, 6 mM dextrose). The cells are
recentrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[1974] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the locus
corresponding to the polynucleotide of the invention, plasmid pUC18
(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV
promoter is amplified by PCR with an XbaI site on the 5' end and a
BamHI site on the 3' end. Two non-coding sequences are amplified
via PCR: one non-coding sequence (fragment 1) is amplified with a
HindIII site at the 5' end and an Xba site at the 3' end; the other
non-coding sequence (fragment 2) is amplified with a BamHI site at
the 5' end and a HindIII site at the 3' end. The CMV promoter and
the fragments (1 and 2) are digested with the appropriate enzymes
(CMV promoter--XbaI and BamHI; fragment 1--XbaI; fragment 2--BamHI)
and ligated together. The resulting ligation product is digested
with HindIII, and ligated with the HindIII-digested pUC18
plasmid.
[1975] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5..times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[1976] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37 degree C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[1977] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 28
Method of Treatment Using Gene Therapy--In Vivo
[1978] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an
animal to increase or decrease the expression of the polypeptide.
The polynucleotide of the present invention may be operatively
linked to a promoter or any other genetic elements necessary for
the expression of the polypeptide by the target tissue. Such gene
therapy and delivery techniques and methods are known in the art,
see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622,
U.S. Pat. No. 5,705,151, U.S. Pat. No. 5,580,859; Tabata et al.,
Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res.
35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318
(1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et
al., Circulation 94(12):3281-3290 (1996) (incorporated herein by
reference).
[1979] The polynucleotide constructs may be delivered by any method
that delivers injectable materials to the cells of an animal, such
as, injection into the interstitial space of tissues (heart,
muscle, skin, lung, liver, intestine and the like). The
polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
[1980] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, LIPOFECTIN.TM.
or precipitating agents and the like. However, the polynucleotides
of the present invention may also be delivered in liposome
formulations (such as those taught in Felgner P. L. et al. (1995)
Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol.
Cell 85(1):1-7) which can be prepared by methods well known to
those skilled in the art.
[1981] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Any strong promoter known to those skilled in the art
can be used for driving the expression of DNA Unlike other gene
therapies techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature
of the polynucleotide synthesis in the cells. Studies have shown
that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to
six months.
[1982] The polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[1983] For the naked polynucleotide injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 g/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues. However, other
parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
polynucleotide constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[1984] The dose response effects of injected polynucleotide in
muscle in vivo is determined as follows. Suitable template DNA for
production of mRNA coding for polypeptide of the present invention
is prepared in accordance with a standard recombinant DNA
methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes.
The quadriceps muscles of mice are then injected with various
amounts of the template DNA.
[1985] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The template DNA is
injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over one minute, approximately 0.5 cm from the distal
insertion site of the muscle into the knee and about 0.2 cm deep. A
suture is placed over the injection site for future localization,
and the skin is closed with stainless steel clips.
[1986] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for protein expression. A time course for
protein expression may be done in a similar fashion except that
quadriceps from different mice are harvested at different times.
Persistence of DNA in muscle following injection may be determined
by Southern blot analysis after preparing total cellular DNA and
HIRT supernatants from injected and control mice. The results of
the above experimentation in mice can be use to extrapolate proper
dosages and other treatment parameters in humans and other animals
using naked DNA.
Example 29
Transgenic Animals
[1987] The polypeptides of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[1988] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, 1983, Mol. Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety.
[1989] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[1990] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric. The transgene may be integrated as a single
transgene or as multiple copies such as in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.
(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred. Briefly, when such a technique is to be utilized,
vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[1991] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[1992] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[1993] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of polypeptides of the present invention,
studying diseases, disorders, and/or conditions associated with
aberrant expression, and in screening for compounds effective in
ameliorating such diseases, disorders, and/or conditions.
Example 30
Knock-Out Animals
[1994] Endogenous gene expression can also be reduced by
inactivating or "knocking out" the gene and/or its promoter using
targeted homologous recombination. (E.g., see Smithies et al.,
Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512
(1987); Thompson et al., Cell 5:313-321 (1989); each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional polynucleotide of the invention (or a
completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous polynucleotide sequence (either the coding regions or
regulatory regions of the gene) can be used, with or without a
selectable marker and/or a negative selectable marker, to transfect
cells that express polypeptides of the invention in vivo. In
another embodiment, techniques known in the art are used to
generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art.
[1995] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[1996] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[1997] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[1998] Transgenic and "knock-out" animals of the invention have
uses which include, but are not limited to, animal model systems
useful in elaborating the biological function of polypeptides of
the present invention, studying diseases, disorders, and/or
conditions associated with aberrant expression, and in screening
for compounds effective in ameliorating such diseases, disorders,
and/or conditions.
Example 31
Production of an Antibody
[1999] Hybridoma Technology
[2000] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing polypeptide(s) of the
invention are administered to an animal to induce the production of
sera containing polyclonal antibodies. In a preferred method, a
preparation of polypeptide(s) of the invention is prepared and
purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to
produce polyclonal antisera of greater specific activity.
[2001] Monoclonal antibodies specific for polypeptide(s) of the
invention are prepared using hybridoma technology. (Kohler et al.,
Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6.511
(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a
mouse) is immunized with polypeptide(s) of the invention, or, more
preferably, with a secreted polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 g/l of nonessential
amino acids, about 1,000 U/ml of penicillin, and about 100 .mu.g/ml
of streptomycin.
[2002] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP2O), available
from the ATCC.TM.. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981)). The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the polypeptide(s) of the invention.
[2003] Alternatively, additional antibodies capable of binding
polypeptide(s) of the invention can be produced in a two-step
procedure using anti-idiotypic antibodies. Such a method makes use
of the fact that antibodies are themselves antigens, and therefore,
it is possible to obtain an antibody which binds to a second
antibody. In accordance with this method, protein specific
antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma
cells, and the hybridoma cells are screened to identify clones
which produce an antibody whose ability to bind to the
polypeptide(s) of the invention protein-specific antibody can be
blocked by polypeptide(s) of the invention. Such antibodies
comprise anti-idiotypic antibodies to the polypeptide(s) of the
invention protein-specific antibody and are used to immunize an
animal to induce formation of further polypeptide(s) of the
invention protein-specific antibodies.
[2004] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed herein.
(See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).)
[2005] Isolation of Antibody Fragments Directed Polypeptide(S) of
the Invention from a Library of ScFvs
[2006] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against polypeptide(s) of the invention to which the
donor may or may not have been exposed (see e.g., U.S. Pat. No.
5,885,793 incorporated herein by reference in its entirety).
[2007] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of
0.8 with shaking. Five ml of this culture is used to innoculate 50
ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 .mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[2008] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[2009] Panning of the Library Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[2010] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing. These ELISA positive clones may
also be further characterized by techniques known in the art, such
as, for example, epitope mapping, binding affinity, receptor signal
transduction, ability to block or competitively inhibit
antibody/antigen binding, and competitive agonistic or antagonistic
activity.
Example 32
Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
[2011] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and
IL-15. Interestingly, these signals are by themselves weak
effectors but can, in combination with various co-stimulatory
proteins, induce activation, proliferation, differentiation,
homing, tolerance and death among B cell populations.
[2012] One of the best studied classes of B-cell co-stimulatory
proteins is the TNF-superfamily. Within this family CD40, CD27, and
CD30 along with their respective ligands CD154, CD70, and CD153
have been found to regulate a variety of immune responses. Assays
which allow for the detection and/or observation of the
proliferation and differentiation of these B-cell populations and
their precursors are valuable tools in determining the effects
various proteins may have on these B-cell populations in terms of
proliferation and differentiation. Listed below are two assays
designed to allow for the detection of the differentiation,
proliferation, or inhibition of B-cell populations and their
precursors.
[2013] In Vitro Assay-Purified polypeptides of the invention, or
truncated forms thereof, is assessed for its ability to induce
activation, proliferation, differentiation or inhibition and/or
death in B-cell populations and their precursors. The activity of
the polypeptides of the invention on purified human tonsillar B
cells, measured qualitatively over the dose range from 0.1 to
10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation
assay in which purified tonsillar B cells are cultured in the
presence of either formalin-fixed Staphylococcus aureus Cowan I
(SAC) or immobilized anti-human IgM antibody as the priming agent.
Second signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220).
[2014] Various dilutions of each sample are placed into individual
wells of a 96-well plate to which are added 10.sup.5 B-cells
suspended in culture medium (RPMI 1640 containing 10% FBS,
5.times.10.sup.-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin,
and 10.sup.-5 dilution of SAC) in a total volume of 150 ul.
Proliferation or inhibition is quantitated by a 20 h pulse (1
uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor
addition. The positive and negative controls are IL2 and medium
respectively.
[2015] In Vivo Assay-BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of a polypeptide of the invention, or
truncated forms thereof. Mice receive this treatment for 4
consecutive days, at which time they are sacrificed and various
tissues and serum collected for analyses. Comparison of H&E
sections from normal spleens and spleens treated with polypeptides
of the invention identify the results of the activity of the
polypeptides on spleen cells, such as the diffusion of
peri-arterial lymphatic sheaths, and/or significant increases in
the nucleated cellularity of the red pulp regions, which may
indicate the activation of the differentiation and proliferation of
B-cell populations Immunohistochemical studies using a B cell
marker, anti-CD45R(B220), are used to determine whether any
physiological changes to splenic cells, such as splenic
disorganization, are due to increased B-cell representation within
loosely defined B-cell zones that infiltrate established T-cell
regions.
[2016] Flow cytometric analyses of the spleens from mice treated
with polypeptide is used to indicate whether the polypeptide
specifically increases the proportion of ThB+, CD45R(B220) dull B
cells over that which is observed in control mice.
[2017] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and polypeptide-treated mice.
[2018] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides of the invention (e.g., gene therapy), agonists,
and/or antagonists of polynucleotides or polypeptides of the
invention.
Example 33
T Cell Proliferation Assay
[2019] Proliferation assay for Resting PBLs.
[2020] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
microliters per well of mAb to CD3 (HIT3a, Pharmingen) or
isotype-matched control mAb (B33.1) overnight at 4.degree. C. (1
microgram/ml in 0.05M bicarbonate buffer, pH 9.5), then washed
three times with PBS. PBMC are isolated by F/H gradient
centrifugation from human peripheral blood and added to
quadruplicate wells (5.times.10.sup.4/well) of mAb coated plates in
RPMI containing 10% FCS and P/S in the presence of varying
concentrations of TNF Delta and/or TNF Epsilon protein (total
volume 200 microliters). Relevant protein buffer and medium alone
are controls. After 48 hr. culture at 37.degree. C., plates are
spun for 2 min. at 1000 rpm and 100 microliters of supernatant is
removed and stored -20.degree. C. for measurement of IL-2 (or other
cytokines) if effect on proliferation is observed. Wells are
supplemented with 100 microliters of medium containing 0.5
microcuries of .sup.3H-thymidine and cultured at 37.degree. C. for
18-24 hr. Wells are harvested and incorporation of
.sup.3H-thymidine used as a measure of proliferation. Anti-CD3
alone is the positive control for proliferation. IL-2 (100 U/ml) is
also used as a control which enhances proliferation. Control
antibody which does not induce proliferation of T cells is used as
the negative controls for the effects of TNF Delta and/or TNF
Epsilon proteins.
[2021] Alternatively, a proliferation assay on resting PBL
(peripheral blood lymphocytes) is measured by the up-take of
.sup.3H-thymidine. The assay is performed as follows. PBMC are
isolated by FICOLL.TM. (LSM, ICN Biotechnologies, Aurora, Ohio)
gradient centrifugation from human peripheral blood, and are
cultured overnight in 10% (Fetal Calf Serum, Biofluids, Rockville,
Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation
period allows the adherent cells to attach to the plastic, which
results in a lower background in the assay as there are fewer cells
that can act as antigen presenting cells or that might be producing
growth factors. The following day the non-adherent cells are
collected, washed and used in the proliferation assay. The assay is
performed in a 96 well plate using 2.times.10.sup.4 cells/well in a
final volume of 200 microliters. The supernatants (e.g., CHO or
293T supernatants) expressing the protein of interest are tested at
a 30% final dilution, therefore 60 ul are added to 140 ul of 10%
FCS/RPMI containing the cells. Control supernatants are used at the
same final dilution and express the following proteins: vector
(negative control), IL-2 (*), IFN.gamma., TNF.alpha., IL-10 and
TR2. In addition to the control supernatants, recombinant human
IL-2 (R & D Systems, Minneapolois, Minn.) at a final
concentration of 100 ng/ml is also used. After 24 hours of culture,
each well is pulsed with 1 uCi of .sup.3H-thymidine (Nen, Boston,
Mass.). Cells are then harvested 20 hours following pulsing and
incorporation of .sup.3H-thymidine is used as a measure of
proliferation. Results are expressed as an average of triplicate
samples plus or minus standard error.
[2022] (*) The amount of the control cytokines IL-2, IFN.gamma.,
TNF.alpha. and IL-10 produced in each transfection varies between
300 pg to 5 ng/ml.
[2023] Costimulation Assay.
[2024] A costimulation assay on resting PBL (peripheral blood
lymphocytes) is performed in the presence of immobilized antibodies
to CD3 and CD28. The use of antibodies specific for the invariant
regions of CD3 mimic the induction of T cell activation that would
occur through stimulation of the T cell receptor by an antigen.
Cross-linking of the TCR (first signal) in the absence of a
costimulatory signal (second signal) causes very low induction of
proliferation and will eventually result in a state of "anergy",
which is characterized by the absence of growth and inability to
produce cytokines. The addition of a costimulatory signal such as
an antibody to CD28, which mimics the action of the costimulatory
molecule. B7-1 expressed on activated APCs, results in enhancement
of T cell responses including cell survival and production of IL-2.
Therefore this type of assay allows to detect both positive and
negative effects caused by addition of supernatants expressing the
proteins of interest on T cell proliferation.
[2025] The assay is performed as follows. Ninety-six well plates
are coated with 100 ng/ml anti-CD3 and 5 ug/ml anti-CD28
(Pharmingen, San Diego, Calif.) in a final volume of 100 ul and
incubated overnight at 4 C. Plates are washed twice with PBS before
use. PBMC are isolated by FICOLL.TM. (LSM, ICN Biotechnologies,
Aurora, Ohio) gradient centrifugation from human peripheral blood,
and are cultured overnight in 10% FCS (Fetal Calf Serum, Biofluids,
Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight
incubation period allows the adherent cells to attach to the
plastic, which results in a lower background in the assay as there
are fewer cells that can act as antigen presenting cells or that
might be producing growth factors. The following day the non
adherent cells are collected, washed and used in the proliferation
assay. The assay is performed in a 96 well plate using
2.times.10.sup.4 cells/well in a final volume of 200 ul. The
supernatants (e.g., CHO or 293T supernatants) expressing the
protein of interest are tested at a 30% final dilution, therefore
60 ul are added to 140 ul of 10% FCS/RPMI containing the cells.
Control supernatants are used at the same final dilution and
express the following proteins: vector only (negative control),
IL-2, IFN.gamma., TNF.alpha., IL-10 and TR2. In addition to the
control supernatants recombinant human IL-2 (R & D Systems,
Minneapolis, Minn.) at a final concentration of 10 ng/ml is also
used. After 24 hours of culture, each well is pulsed with 1 uCi of
.sup.3H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20
hours following pulsing and incorporation of .sup.3H-thymidine is
used as a measure of proliferation. Results are expressed as an
average of triplicate samples plus or minus standard error.
[2026] Costimulation Assay: IFN .gamma. and IL-2 ELISA
[2027] The assay is performed as follows. Twenty-four well plates
are coated with either 300 ng/ml or 600 ng/ml anti-CD3 and 5 ug/ml
anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 500
ul and incubated overnight at 4 C. Plates are washed twice with PBS
before use. PBMC are isolated by FICOLL.TM. (LSM, ICN
Biotechnologies, Aurora, Ohio) gradient centrifugation from human
peripheral blood, and are cultured overnight in 10% FCS (Fetal Calf
Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg,
Md.). This overnight incubation period allows the adherent cells to
attach to the plastic, which results in a lower background in the
assay as there are fewer cells that can act as antigen presenting
cells or that might be producing growth factors. The following day
the non adherent cells are collected, washed and used in the
costimulation assay. The assay is performed in the pre-coated
twenty-four well plate using 1.times.10.sup.5 cells/well in a final
volume of 900 ul. The supernatants (293T supernatants) expressing
the protein of interest are tested at a 30% final dilution,
therefore 300 ul are added to 600 ul of 10% FCS/RPMI containing the
cells. Control supernatants are used at the same final dilution and
express the following proteins: vector only (negative control),
IL-2, IFN.gamma., IL-12 and IL-18. In addition to the control
supernatants recombinant human IL-2 (all cytokines were purchased
from R & D Systems, Minneapolis, Minn.) at a final
concentration of 10 ng/ml, IL-12 at a final concentration of 1
ng/ml and IL-18 at a final concentration of 50 ng/ml are also used.
Controls and unknown samples are tested in duplicate. Supernatant
samples (250 ul) are collected 2 days and 5 days after the
beginning of the assay. ELISAs to test for IFN.gamma. and IL-2
secretion are performed using kits purchased from R & D
Systems, (Minneapolis, Minn.). Results are expressed as an average
of duplicate samples plus or minus standard error.
[2028] Proliferation assay for preactivated-resting T cells.
[2029] A proliferation assay on preactivated-resting T cells is
performed on cells that are previously activated with the lectin
phytohemagglutinin (PHA). Lectins are polymeric plant proteins that
can bind to residues on T cell surface glycoproteins including the
TCR and act as polyclonal activators. PBLs treated with PHA and
then cultured in the presence of low doses of IL-2 resemble
effector T cells. These cells are generally more sensitive to
further activation induced by growth factors such as IL-2. This is
due to the expression of high affinity IL-2 receptors that allows
this population to respond to amounts of IL-2 that are 100 fold
lower than what would have an effect on a naive T cell. Therefore
the use of this type of cells might enable to detect the effect of
very low doses of an unknown growth factor, that would not be
sufficient to induce proliferation on resting (naive) T cells.
[2030] The assay is performed as follows. PBMC are isolated by F/H
gradient centrifugation from human peripheral blood, and are
cultured in 10% FCS (Fetal Calf Serum, Biofluids, Rockville,
Md.)/RPMI (Gibco BRL, Gaithersburg, Md.) in the presence of 2 ug/ml
PHA (SIGMA.TM., Saint Louis, Mo.) for three days. The cells are
then washed in PBS and cultured in 10% FCS/RPMI in the presence of
5 ng/ml of human recombinant IL-2 (R & D Systems, Minneapolis,
Minn.) for 3 days. The cells are washed and rested in starvation
medium (1% FCS/RPMI) for 16 hours prior to the beginning of the
proliferation assay. An aliquot of the cells is analyzed by FACS to
determine the percentage of T cells (CD3 positive cells) present;
this usually ranges between 93-97% depending on the donor. The
assay is performed in a 96 well plate using 2.times.10.sup.4
cells/well in a final volume of 200 ul. The supernatants (e.g., CHO
or 293T supernatants) expressing the protein of interest are tested
at a 30% final dilution, therefore 60 ul are added to 140 ul of in
10% FCS/RPMI containing the cells. Control supernatants are used at
the same final dilution and express the following proteins: vector
(negative control), IL-2, IFN.gamma., TNF.alpha., IL-10 and TR2. In
addition to the control supernatants recombinant human IL-2 at a
final concentration of 10 ng/ml is also used. After 24 hours of
culture, each well is pulsed with 1 uCi of .sup.3H-thymidine (Nen,
Boston, Mass.). Cells are then harvested 20 hours following pulsing
and incorporation of .sup.3H-thymidine is used as a measure of
proliferation. Results are expressed as an average of triplicate
samples plus or minus standard error.
[2031] The studies described in this example test activity of
polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides of the invention (e.g., gene therapy), agonists,
and/or antagonists of polynucleotides or polypeptides of the
invention.
Example 34
Effect of Polypeptides of the Invention on the Expression of MHC
Class II, Costimulatory and Adhesion Molecules and Cell
Differentiation of Monocytes and Monocyte-Derived Human Dendritic
Cells
[2032] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[2033] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of
polypeptides of the invention or LPS (positive control), washed
with PBS containing 1% BSA and 0.02 mM sodium azide, and then
incubated with 1:20 dilution of appropriate FITC- or PE-labeled
monoclonal antibodies for 30 minutes at 4 degrees C. After an
additional wash, the labeled cells are analyzed by flow cytometry
on a FACScan (Becton Dickinson).
[2034] Effect on the production of cytokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Th1 helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (10.sup.6/ml)
are treated with increasing concentrations of polypeptides of the
invention for 24 hours. LPS (100 ng/ml) is added to the cell
culture as positive control. Supernatants from the cell cultures
are then collected and analyzed for IL-12 content using commercial
ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The
standard protocols provided with the kits are used.
[2035] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increase expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[2036] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of polypeptides of the invention or LPS (positive
control), washed with PBS containing 1% BSA and 0.02 mM sodium
azide, and then incubated with 1:20 dilution of appropriate FITC-
or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C.
After an additional wash, the labeled cells are analyzed by flow
cytometry on a FACScan (Becton Dickinson).
[2037] Monocyte activation and/or increased survival. Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. Polypeptides, agonists,
or antagonists of the invention can be screened using the three
assays described below. For each of these assays, Peripheral blood
mononuclear cells (PBMC) are purified from single donor leukopacks
(American Red Cross, Baltimore, Md.) by centrifugation through a
HISTOPAQUE.TM. gradient (SIGMA.TM.). Monocytes are isolated from
PBMC by counterflow centrifugal elutriation.
[2038] Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from internally regulated
process (apoptosis). Addition to the culture of activating factors,
such as TNF-alpha dramatically improves cell survival and prevents
DNA fragmentation. Propidium iodide (PI) staining is used to
measure apoptosis as follows. Monocytes are cultured for 48 hours
in polypropylene tubes in serum-free medium (positive control), in
the presence of 100 ng/ml TNF-alpha (negative control), and in the
presence of varying concentrations of the compound to be tested.
Cells are suspended at a concentration of 2.times.10.sup.6/ml in
PBS containing PI at a final concentration of 5 .mu.g/ml, and then
incubated at room temperature for 5 minutes before FACScan
analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
[2039] Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.10.sup.5 cells/ml with increasing concentrations of the
a polypeptide of the invention and under the same conditions, but
in the absence of the polypeptide. For IL-12 production, the cells
are primed overnight with IFN (100 U/ml) in presence of a
polypeptide of the invention. LPS (10 ng/ml) is then added.
Conditioned media are collected after 24 h and kept frozen until
use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then
performed using a commercially available ELISA kit (e.g., R & D
Systems (Minneapolis, Minn.)) and applying the standard protocols
provided with the kit.
[2040] Oxidative burst. Purified monocytes are plated in 96-w plate
at 2-1.times.10.sup.5 cell/well. Increasing concentrations of
polypeptides of the invention are added to the wells in a total
volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and
antibiotics). After 3 days incubation, the plates are centrifuged
and the medium is removed from the wells. To the macrophage
monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10
mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM
phenol red and 19 U/ml of HRPO) is added, together with the
stimulant (200 nM PMA). The plates are incubated at 37.degree. C.
for 2 hours and the reaction is stopped by adding 20 .mu.l 1N NaOH
per well. The absorbance is read at 610 nm. To calculate the amount
of H.sub.2O.sub.2 produced by the macrophages, a standard curve of
a H.sub.2O.sub.2 solution of known molarity is performed for each
experiment.
[2041] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polypeptides, polynucleotides (e.g., gene therapy), agonists,
and/or antagonists of the invention.
Example 35
Biological Effects of Polypeptides of the Invention
[2042] Astrocyte and Neuronal Assays.
[2043] Recombinant polypeptides of the invention, expressed in
Escherichia coli and purified as described above, can be tested for
activity in promoting the survival, neurite outgrowth, or
phenotypic differentiation of cortical neuronal cells and for
inducing the proliferation of glial fibrillary acidic protein
immunopositive cells, astrocytes. The selection of cortical cells
for the bioassay is based on the prevalent expression of FGF-1 and
FGF-2 in cortical structures and on the previously reported
enhancement of cortical neuronal survival resulting from FGF-2
treatment. A thymidine incorporation assay, for example, can be
used to elucidate a polypeptide of the invention's activity on
these cells.
[2044] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary cortical neuronal
culture paradigm, the ability of a polypeptide of the invention to
induce neurite outgrowth can be compared to the response achieved
with FGF-2 using, for example, a thymidine incorporation assay.
[2045] Fibroblast and Endothelial Cell Assays.
[2046] Human lung fibroblasts are obtained from Clonetics (San
Diego, Calif.) and maintained in growth media from Clonetics.
Dermal microvascular endothelial cells are obtained from Cell
Applications (San Diego, Calif.). For proliferation assays, the
human lung fibroblasts and dermal microvascular endothelial cells
can be cultured at 5,000 cells/well in a 96-well plate for one day
in growth medium. The cells are then incubated for one day in 0.1%
BSA basal medium. After replacing the medium with fresh 0.1% BSA
medium, the cells are incubated with the test proteins for 3 days.
ALAMAR BLUE.TM. (Alamar Biosciences, Sacramento, Calif.) is added
to each well to a final concentration of 10%. The cells are
incubated for 4 hr. Cell viability is measured by reading in a
CYTOFLUOR.TM. fluorescence reader. For the PGE.sub.2 assays, the
human lung fibroblasts are cultured at 5,000 cells/well in a
96-well plate for one day. After a medium change to 0.1% BSA basal
medium, the cells are incubated with FGF-2 or polypeptides of the
invention with or without IL-1.alpha. for 24 hours. The
supernatants are collected and assayed for PGE.sub.2 by EIA kit
(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung
fibroblasts are cultured at 5,000 cells/well in a 96-well plate for
one day. After a medium change to 0.1% BSA basal medium, the cells
are incubated with FGF-2 or with or without polypeptides of the
invention IL-1.alpha. for 24 hours. The supernatants are collected
and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
[2047] Human lung fibroblasts are cultured with FGF-2 or
polypeptides of the invention for 3 days in basal medium before the
addition of ALAMAR BLUE.TM. to assess effects on growth of the
fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which
can be used to compare stimulation with polypeptides of the
invention.
[2048] Parkinson Models.
[2049] The loss of motor function in Parkinson's disease is
attributed to a deficiency of striatal dopamine resulting from the
degeneration of the nigrostriatal dopaminergic projection neurons.
An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4
phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is
taken-up by astrocytes and catabolized by monoamine oxidase B to
1-methyl-4-phenyl pyridine (MPP.sup.+) and released. Subsequently,
MPP.sup.+ is actively accumulated in dopaminergic neurons by the
high-affinity reuptake transporter for dopamine. MPP.sup.+ is then
concentrated in mitochondria by the electrochemical gradient and
selectively inhibits nicotidamide adenine disphosphate: ubiquinone
oxidoreductionase (complex I), thereby interfering with electron
transport and eventually generating oxygen radicals.
[2050] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[2051] Based on the data with FGF-2, polypeptides of the invention
can be evaluated to determine whether it has an action similar to
that of FGF-2 in enhancing dopaminergic neuronal survival in vitro
and it can also be tested in vivo for protection of dopaminergic
neurons in the striatum from the damage associated with MPTP
treatment. The potential effect of a polypeptide of the invention
is first examined in vitro in a dopaminergic neuronal cell culture
paradigm. The cultures are prepared by dissecting the midbrain
floor plate from gestation day 14 Wistar rat embryos. The tissue is
dissociated with trypsin and seeded at a density of 200,000
cells/cm.sup.2 on polyorthinine-laminin coated glass coverslips.
The cells are maintained in Dulbecco's Modified Eagle's medium and
F12 medium containing hormonal supplements (N1). The cultures are
fixed with paraformaldehyde after 8 days in vitro and are processed
for tyrosine hydroxylase, a specific marker for dopminergic
neurons, immunohistochemical staining. Dissociated cell cultures
are prepared from embryonic rats. The culture medium is changed
every third day and the factors are also added at that time.
[2052] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if a polypeptide of the invention
acts to prolong the survival of dopaminergic neurons, it would
suggest that the polypeptide may be involved in Parkinson's
Disease.
[2053] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 36
The Effect of Polypeptides of the Invention on the Growth of
Vascular Endothelial Cells
[2054] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, 8 units/ml heparin. A polypeptide having the
amino acid sequence of SEQ ID NO:Y, and positive controls, such as
VEGF and basic FGF (bFGF) are added, at varying concentrations. On
days 4 and 6, the medium is replaced. On day 8, cell number is
determined with a Coulter Counter.
[2055] An increase in the number of HUVEC cells indicates that the
polypeptide of the invention may proliferate vascular endothelial
cells.
[2056] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 37
Stimulatory Effect of Polypeptides of the Invention on the
Proliferation of Vascular Endothelial Cells
[2057] For evaluation of mitogenic activity of growth factors, the
colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)2H-tetrazolium) assay with the electron coupling reagent PMS
(phenazine methosulfate) was performed (CellTiter 96 AQ,
PROMEGA.TM.). Cells are seeded in a 96-well plate (5,000
cells/well) in 0.1 mL serum-supplemented medium and are allowed to
attach overnight. After serum-starvation for 12 hours in 0.5% FBS,
conditions (bFGF, VEGF.sub.165 or a polypeptide of the invention in
0.5% FBS) with or without Heparin (8 U/ml) are added to wells for
48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and
allowed to incubate for 1 hour at 37.degree. C. before measuring
the absorbance at 490 nm in an ELISA plate reader. Background
absorbance from control wells (some media, no cells) is subtracted,
and seven wells are performed in parallel for each condition. See,
Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).
[2058] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 38
Inhibition of PDGF-induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
[2059] HAoSMC proliferation can be measured, for example, by BrdUrd
incorporation. Briefly, subconfluent, quiescent cells grown on the
4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP.
Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd.
After 24 h, immunocytochemistry is performed by using BrdUrd
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdUrd antibody at 4
degrees C. for 2 h after being exposed to denaturing solution and
then incubated with the streptavidin-peroxidase and
diaminobenzidine. After counterstaining with hematoxylin, the cells
are mounted for microscopic examination, and the BrdUrd-positive
cells are counted. The BrdUrd index is calculated as a percent of
the BrdUrd-positive cells to the total cell number. In addition,
the simultaneous detection of the BrdUrd staining (nucleus) and the
FITC uptake (cytoplasm) is performed for individual cells by the
concomitant use of bright field illumination and dark field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem.
6:271(36):21985-21992 (1996).
[2060] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 39
Stimulation of Endothelial Migration
[2061] This example will be used to explore the possibility that a
polypeptide of the invention may stimulate lymphatic endothelial
cell migration.
[2062] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD;
Falk, W., et al., J. Immunological Methods 1980; 33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1%
gelatin for at least 6 hours at room temperature and dried under
sterile air. Test substances are diluted to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin
(BSA), and 25 ul of the final dilution is placed in the lower
chamber of the modified Boyden apparatus. Subconfluent, early
passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for
the minimum time required to achieve cell detachment. After placing
the filter between lower and upper chamber, 2.5.times.10.sup.5
cells suspended in 50 ul M199 containing 1% FBS are seeded in the
upper compartment. The apparatus is then incubated for 5 hours at
37.degree. C. in a humidified chamber with 5% CO2 to allow cell
migration. After the incubation period, the filter is removed and
the upper side of the filter with the non-migrated cells is scraped
with a rubber policeman. The filters are fixed with methanol and
stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park,
Ill.). Migration is quantified by counting cells of three random
high-power fields (40.times.) in each well, and all groups are
performed in quadruplicate.
[2063] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 40
Stimulation of Nitric Oxide Production by Endothelial Cells
[2064] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation. Thus,
activity of a polypeptide of the invention can be assayed by
determining nitric oxide production by endothelial cells in
response to the polypeptide.
[2065] Nitric oxide is measured in 96-well plates of confluent
microvascular endothelial cells after 24 hours starvation and a
subsequent 4 hr exposure to various levels of a positive control
(such as VEGF-1) and the polypeptide of the invention. Nitric oxide
in the medium is determined by use of the Griess reagent to measure
total nitrite after reduction of nitric oxide-derived nitrate by
nitrate reductase. The effect of the polypeptide of the invention
on nitric oxide release is examined on HUVEC.
[2066] Briefly, NO release from cultured HUVEC monolayer is
measured with a NO-specific polarographic electrode connected to a
NO meter (Iso-NO, World Precision Instruments Inc.) (1049).
Calibration of the NO elements is performed according to the
following equation:
2KNO.sub.2+2KI+2H.sub.2SO.sub.462NO+I.sub.2+2H.sub.2O+2K.sub.2SO.sub.4
[2067] The standard calibration curve is obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
nmol/L) into the calibration solution containing KI and
H.sub.2SO.sub.4. The specificity of the Iso-NO electrode to NO is
previously determined by measurement of NO from authentic NO gas
(1050). The culture medium is removed and HUVECs are washed twice
with Dulbecco's phosphate buffered saline. The cells are then
bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well
plates, and the cell plates are kept on a slide warmer (Lab Line
Instruments Inc.) To maintain the temperature at 37.degree. C. The
NO sensor probe is inserted vertically into the wells, keeping the
tip of the electrode 2 mm under the surface of the solution, before
addition of the different conditions. S-nitroso acetyl penicillamin
(SNAP) is used as a positive control. The amount of released NO is
expressed as picomoles per 1.times.10.sup.6 endothelial cells. All
values reported are means of four to six measurements in each group
(number of cell culture wells). See, Leak et al. Biochem. and
Biophys. Res. Comm. 217:96-105 (1995).
[2068] The studies described in this example tested activity of
polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 41
Effect of Polypeptides of the Invention on Cord Formation in
Angiogenesis
[2069] Another step in angiogenesis is cord formation, marked by
differentiation of endothelial cells. This bioassay measures the
ability of microvascular endothelial cells to form capillary-like
structures (hollow structures) when cultured in vitro.
[2070] CADMEC (microvascular endothelial cells) are purchased from
Cell Applications, Inc. as proliferating (passage 2) cells and are
cultured in Cell Applications' CADMEC Growth Medium and used at
passage 5. For the in vitro angiogenesis assay, the wells of a
48-well cell culture plate are coated with Cell Applications'
Attachment Factor Medium (200 ml/well) for 30 min. at 37.degree. C.
CADMEC are seeded onto the coated wells at 7,500 cells/well and
cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium
containing control buffer or a polypeptide of the invention (0.1 to
100 ng/ml) and the cells are cultured for an additional 48 hr. The
numbers and lengths of the capillary-like chords are quantitated
through use of the Boeckeler VIA-170 video image analyzer. All
assays are done in triplicate.
[2071] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. b-estradiol (1 ng/ml) is used as a negative control. The
appropriate buffer (without protein) is also utilized as a
control.
[2072] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 42
Angiogenic Effect on Chick Chorioallantoic Membrane
[2073] Chick chorioallantoic membrane (CAM) is a well-established
system to examine angiogenesis. Blood vessel formation on CAM is
easily visible and quantifiable. The ability of polypeptides of the
invention to stimulate angiogenesis in CAM can be examined.
[2074] Fertilized eggs of the White Leghorn chick (Gallus gallus)
and the Japanese qual (Coturnix coturnix) are incubated at
37.8.degree. C. and 80% humidity. Differentiated CAM of 16-day-old
chick and 13-day-old qual embryos is studied with the following
methods.
[2075] On Day 4 of development, a window is made into the egg shell
of chick eggs. The embryos are checked for normal development and
the eggs sealed with cellotape. They are further incubated until
Day 13. THERMANOX.TM. coverslips (Nunc, Naperville, Ill.) are cut
into disks of about 5 mm in diameter. Sterile and salt-free growth
factors are dissolved in distilled water and about 3.3 mg/5 ml are
pipetted on the disks. After air-drying, the inverted disks are
applied on CAM. After 3 days, the specimens are fixed in 3%
glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium
cacodylate buffer. They are photographed with a stereo microscope
[Wild M8] and embedded for semi- and ultrathin sectioning as
described above. Controls are performed with carrier disks
alone.
[2076] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 43
Angiogenesis Assay Using a MATRIGEL.TM. Implant in Mouse
[2077] In vivo angiogenesis assay of a polypeptide of the invention
measures the ability of an existing capillary network to form new
vessels in an implanted capsule of murine extracellular matrix
material (MATRIGEL.TM.). The protein is mixed with the liquid
MATRIGEL.TM. at 4 degree C. and the mixture is then injected
subcutaneously in mice where it solidifies. After 7 days, the solid
"plug" of MATRIGEL.TM. is removed and examined for the presence of
new blood vessels. MATRIGEL.TM. is purchased from Becton Dickinson
Labware/Collaborative Biomedical Products.
[2078] When thawed at 4 degree C. the MATRIGEL.TM. material is a
liquid. The MATRIGEL.TM. is mixed with a polypeptide of the
invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml
syringes. Female C57B1/6 mice approximately 8 weeks old are
injected with the mixture of MATRIGEL.TM. and experimental protein
at 2 sites at the midventral aspect of the abdomen (0.5 ml/site).
After 7 days, the mice are sacrificed by cervical dislocation, the
MATRIGEL.TM. plugs are removed and cleaned (i.e., all clinging
membranes and fibrous tissue is removed). Replicate whole plugs are
fixed in neutral buffered 10% formaldehyde, embedded in paraffin
and used to produce sections for histological examination after
staining with Masson's Trichrome. Cross sections from 3 different
regions of each plug are processed. Selected sections are stained
for the presence of vWF. The positive control for this assay is
bovine basic FGF (150 ng/ml). MATRIGEL.TM. alone is used to
determine basal levels of angiogenesis.
[2079] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 44
Rescue of Ischemia in Rabbit Lower Limb Model
[2080] To study the in vivo effects of polynucleotides and
polypeptides of the invention on ischemia, a rabbit hindlimb
ischemia model is created by surgical removal of one femoral
arteries as described previously (Takeshita et al., Am J. Pathol
147:1649-1660 (1995)). The excision of the femoral artery results
in retrograde propagation of thrombus and occlusion of the external
iliac artery. Consequently, blood flow to the ischemic limb is
dependent upon collateral vessels originating from the internal
iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660 (1995)).
An interval of 10 days is allowed for post-operative recovery of
rabbits and development of endogenous collateral vessels. At 10 day
post-operatively (day 0), after performing a baseline angiogram,
the internal iliac artery of the ischemic limb is transfected with
500 mg naked expression plasmid containing a polynucleotide of the
invention by arterial gene transfer technology using a
hydrogel-coated balloon catheter as described (Riessen et al. Hum
Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90:
936-944 (1992)). When a polypeptide of the invention is used in the
treatment, a single bolus of 500 mg polypeptide of the invention or
control is delivered into the internal iliac artery of the ischemic
limb over a period of 1 min. through an infusion catheter. On day
30, various parameters are measured in these rabbits: (a) BP
ratio--The blood pressure ratio of systolic pressure of the
ischemic limb to that of normal limb; (b) Blood Flow and Flow
Reserve--Resting FL: the blood flow during undilated condition and
Max FL: the blood flow during fully dilated condition (also an
indirect measure of the blood vessel amount) and Flow Reserve is
reflected by the ratio of max FL:resting FL; (c) Angiographic
Score--This is measured by the angiogram of collateral vessels. A
score is determined by the percentage of circles in an overlaying
grid that with crossing opacified arteries divided by the total
number m the rabbit thigh; (d) Capillary density--The number of
collateral capillaries determined in light microscopic sections
taken from hindlimbs.
[2081] The studies described in this example tested activity of
polynucleotides and polypeptides of the invention. However, one
skilled in the art could easily modify the exemplified studies to
test the agonists, and/or antagonists of the invention.
Example 45
Effect of Polypeptides of the Invention on Vasodilation
[2082] Since dilation of vascular endothelium is important in
reducing blood pressure, the ability of polypeptides of the
invention to affect the blood pressure in spontaneously
hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30,
100, 300, and 900 mg/kg) of the polypeptides of the invention are
administered to 13-14 week old spontaneously hypertensive rats
(SHR). Data are expressed as the mean+/-SEM. Statistical analysis
are performed with a paired t-test and statistical significance is
defined as p<0.05 vs. the response to buffer alone.
[2083] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 46
Rat Ischemic Skin Flap Model
[2084] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. Expression of polypeptides of the
invention, during the skin ischemia, is studied using in situ
hybridization.
[2085] The study in this model is divided into three parts as
follows:
[2086] Ischemic skin
[2087] Ischemic skin wounds
[2088] Normal wounds
[2089] The experimental protocol includes:
[2090] Raising a 3.times.4 cm, single pedicle full-thickness random
skin flap (myocutaneous flap over the lower back of the
animal).
[2091] An excisional wounding (4-6 mm in diameter) in the ischemic
skin (skin-flap).
[2092] Topical treatment with a polypeptide of the invention of the
excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the
following various dosage ranges: 1 mg to 100 mg.
[2093] Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21
post-wounding for histological, immunohistochemical, and in situ
studies.
[2094] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 47
Peripheral Arterial Disease Model
[2095] Angiogenic therapy using a polypeptide of the invention is a
novel therapeutic strategy to obtain restoration of blood flow
around the ischemia in case of peripheral arterial diseases. The
experimental protocol includes: One side of the femoral artery is
ligated to create ischemic muscle of the hindlimb, the other side
of hindlimb serves as a control. A polypeptide of the invention, in
a dosage range of 20 mg-500 mg, is delivered intravenously and/or
intramuscularly 3 times (perhaps more) per week for 2-3 weeks. The
ischemic muscle tissue is collected after ligation of the femoral
artery at 1, 2, and 3 weeks for the analysis of expression of a
polypeptide of the invention and histology. Biopsy is also
performed on the other side of normal muscle of the contralateral
hindlimb. The studies described in this example tested activity of
a polypeptide of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 48
Ischemic Myocardial Disease Model
[2096] A polypeptide of the invention is evaluated as a potent
mitogen capable of stimulating the development of collateral
vessels, and restructuring new vessels after coronary artery
occlusion. Alteration of expression of the polypeptide is
investigated in situ. The experimental protocol includes: The heart
is exposed through a left-side thoracotomy in the rat Immediately,
the left coronary artery is occluded with a thin suture (6-0) and
the thorax is closed. A polypeptide of the invention, in a dosage
range of 20 mg-500 mg, is delivered intravenously and/or
intramuscularly 3 times (perhaps more) per week for 2-4 weeks.
Thirty days after the surgery, the heart is removed and
cross-sectioned for morphometric and in situ analyzes.
[2097] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 49
Rat Corneal Wound Healing Model
[2098] This animal model shows the effect of a polypeptide of the
invention on neovascularization. The
[2099] experimental protocol includes: Making a 1-1.5 mm long
incision from the center of cornea into the stromal layer.
Inserting a spatula below the lip of the incision facing the outer
corner of the eye. Making a pocket (its base is 1-1.5 mm form the
edge of the eye). Positioning a pellet, containing 50 ng-5 ug of a
polypeptide of the invention, within the pocket. Treatment with a
polypeptide of the invention can also be applied topically to the
corneal wounds in a dosage range of 20 mg-500 mg (daily treatment
for five days). The studies described in this example tested
activity of a polypeptide of the invention. However, one skilled in
the art could easily modify the exemplified studies to test the
activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the invention.
Example 50
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[2100] Diabetic db+/db+ Mouse Model. To demonstrate that a
polypeptide of the invention accelerates the healing process, the
genetically diabetic mouse model of wound healing is used. The full
thickness wound healing model in the db+/db+ mouse is a well
characterized, clinically relevant and reproducible model of
impaired wound healing. Healing of the diabetic wound is dependent
on formation of granulation tissue and re-epithelialization rather
than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389
(1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features
observed in Type II diabetes mellitus. Homozygous (db+/db+) mice
are obese in comparison to their normal heterozygous (db+/+m)
littermates. Mutant diabetic (db+/db+) mice have a single autosomal
recessive mutation on chromosome 4 (db+) (Coleman et al. Proc.
Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia,
polydipsia and polyuria. Mutant diabetic mice (db+/db+) have
elevated blood glucose, increased or normal insulin levels, and
suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)). The characteristics
observed in these animals suggests that healing in this model may
be similar to the healing observed in human diabetes (Greenhalgh,
et al., Am. J. of Pathol. 136:1235-1246 (1990)). Genetically
diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic
(db+/+m) heterozygous littermates are used in this study (Jackson
Laboratories). The animals are purchased at 6 weeks of age and are
8 weeks old at the beginning of the study. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. The experiments are
conducted according to the rules and guidelines of Human Genome
Sciences, Inc. Institutional Animal Care and Use Committee and the
Guidelines for the Care and Use of Laboratory Animals. Wounding
protocol is performed according to previously reported methods
(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).
Briefly, on the day of wounding, animals are anesthetized with an
intraperitoneal injection of Avertin (0.01 mg/mL),
2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized
water. The dorsal region of the animal is shaved and the skin
washed with 70% ethanol solution and iodine. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is then created using a Keyes tissue punch. Immediately
following wounding, the surrounding skin is gently stretched to
eliminate wound expansion. The wounds are left open for the
duration of the experiment. Application of the treatment is given
topically for 5 consecutive days commencing on the day of wounding.
Prior to treatment, wounds are gently cleansed with sterile saline
and gauze sponges.
[2101] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[2102] A polypeptide of the invention is administered using at a
range different doses, from 4 mg to 500 mg per wound per day for 8
days in vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[2103] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing. Three groups of 10 animals each (5 diabetic and
5 non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) untreated group, and 3) treated group.
[2104] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 is 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations are made using
the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[2105] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with a
polypeptide of the invention. This assessment included verification
of the presence of cell accumulation, inflammatory cells,
capillaries, fibroblasts, re-epithelialization and epidermal
maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235
(1990)). A calibrated lens micrometer is used by a blinded
observer. Tissue sections are also stained immunohistochemically
with a polyclonal rabbit anti-human keratin antibody using ABC
Elite detection system. Human skin is used as a positive tissue
control while non-immune IgG is used as a negative control.
Keratinocyte growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer. Proliferating cell nuclear antigen/cyclin (PCNA) in
skin specimens is demonstrated by using anti-PCNA antibody (1:50)
with an ABC Elite detection system. Human colon cancer can serve as
a positive tissue control and human brain tissue can be used as a
negative tissue control. Each specimen includes a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[2106] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[2107] Steroid Impaired Rat Model The inhibition of wound healing
by steroids has been well documented in various in vitro and in
vivo systems (Wahl, Glucocorticoids and Wound healing. In:
Anti-Inflammatory Steroid Action: Basic and Clinical Aspects.
280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975); Werb
et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard
wound healing by inhibiting angiogenesis, decreasing vascular
permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),
fibroblast proliferation, and collagen synthesis (Beck et al.,
Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest.
61: 703-797 (1978)) and producing a transient reduction of
circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797
(1978); Wahl, "Glucocorticoids and wound healing", In:
Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989)). The systemic
administration of steroids to impaired wound healing is a well
establish phenomenon in rats (Beck et al., Growth Factors. 5:
295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978);
Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory
Steroid Action: Basic and Clinical Aspects, Academic Press, New
York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA
86: 2229-2233 (1989)).
[2108] To demonstrate that a polypeptide of the invention can
accelerate the healing process, the effects of multiple topical
applications of the polypeptide on full thickness excisional skin
wounds in rats in which healing has been impaired by the systemic
administration of methylprednisolone is assessed. Young adult male
Sprague Dawley rats weighing 250-300 g (Charles River Laboratories)
are used in this example. The animals are purchased at 8 weeks of
age and are 9 weeks old at the beginning of the study. The healing
response of rats is impaired by the systemic administration of
methylprednisolone (17 mg/kg/rat intramuscularly) at the time of
wounding. Animals are individually housed and received food and
water ad libitum. All manipulations are performed using aseptic
techniques. This study is conducted according to the rules and
guidelines of Human Genome Sciences, Inc. Institutional Animal Care
and Use Committee and the Guidelines for the Care and Use of
Laboratory Animals. The wounding protocol is followed according to
section A, above. On the day of wounding, animals are anesthetized
with an intramuscular injection of ketamine (50 mg/kg) and xylazine
(5 mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges. Wounds are
visually examined and photographed at a fixed distance at the day
of wounding and at the end of treatment. Wound closure is
determined by daily measurement on days 1-5 and on day 8. Wounds
are measured horizontally and vertically using a calibrated Jameson
caliper. Wounds are considered healed if granulation tissue is no
longer visible and the wound is covered by a continuous epithelium.
The polypeptide of the invention is administered using at a range
different doses, from 4 mg to 500 mg per wound per day for 8 days
in vehicle. Vehicle control groups received 50 mL of vehicle
solution. Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing. Four groups of 10
animals each (5 with methylprednisolone and 5 without
glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle
placebo control 3) treated groups. Wound closure is analyzed by
measuring the area in the vertical and horizontal axis and
obtaining the total area of the wound. Closure is then estimated by
establishing the differences between the initial wound area (day 0)
and that of post treatment (day 8). The wound area on day 1 is 64
mm.sup.2, the corresponding size of the dermal punch. Calculations
are made using the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[2109] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining is performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin is improved by treatment with a polypeptide of the
invention. A calibrated lens micrometer is used by a blinded
observer to determine the distance of the wound gap. Experimental
data are analyzed using an unpaired t test. A p value of <0.05
is considered significant. The studies described in this example
tested activity of a polypeptide of the invention. However, one
skilled in the art could easily modify the exemplified studies to
test the activity of polynucleotides (e.g., gene therapy),
agonists, and/or antagonists of the invention.
Example 51
Lymphadema Animal Model
[2110] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of a polypeptide of the invention in
lymphangiogenesis and re-establishment of the lymphatic circulatory
system in the rat hind limb. Effectiveness is measured by swelling
volume of the affected limb, quantification of the amount of
lymphatic vasculature, total blood plasma protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps
more importantly, the chronic progress of the edema is followed for
up to 3-4 weeks.
[2111] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs are shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements are made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[2112] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated suture ligated.
[2113] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. The 2 proximal and 2 distal lymphatic
vessels and distal blood supply of the popliteal node are then and
ligated by suturing. The popliteal lymph node, and any accompanying
adipose tissue, is then removed by cutting connective tissues.
[2114] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[2115] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[2116] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people then those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[2117] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), both legs are shaved and
equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software (Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[2118] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[2119] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[2120] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics.
[2121] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 52
Suppression of TNF Alpha-Induced Adhesion Molecule Expression by a
Polypeptide of the Invention
[2122] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[2123] Tumor necrosis factor alpha (TNF-a), a potent
proinflammatory cytokine, is a stimulator of all three CAMs on
endothelial cells and may be involved in a wide variety of
inflammatory responses, often resulting in a pathological
outcome.
[2124] The potential of a polypeptide of the invention to mediate a
suppression of TNF-a induced CAM expression can be examined. A
modified ELISA assay which uses ECs as a solid phase absorbent is
employed to measure the amount of CAM expression on TNF-a treated
ECs when co-stimulated with a member of the FGF family of
proteins.
[2125] To perform the experiment, human umbilical vein endothelial
cell (HUVEC) cultures are obtained from pooled cord harvests and
maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.)
supplemented with 10% FCS and 1% penicillin/streptomycin in a 37
degree C. humidified incubator containing 5% CO.sub.2. HUVECs are
seeded in 96-well plates at concentrations of 1.times.10.sup.4
cells/well in EGM medium at 37 degree C. for 18-24 hrs or until
confluent. The monolayers are subsequently washed 3 times with a
serum-free solution of RPMI-1640 supplemented with 100 U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given
cytokine and/or growth factor(s) for 24 h at 37 degree C. Following
incubation, the cells are then evaluated for CAM expression.
[2126] Human Umbilical Vein Endothelial cells (HUVECs) are grown in
a standard 96 well plate to confluence. Growth medium is removed
from the cells and replaced with 90 ul of 199 Medium (10% FBS).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS (with Ca++ and
Mg++) is added to each well. Plates are held at 4.degree. C. for 30
min.
[2127] Fixative is then removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the
wells to dry. Add 10 .mu.l of diluted primary antibody to the test
and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and
Anti-E-selectin-Biotin are used at a concentration of 10 .mu.g/ml
(1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at
37.degree. C. for 30 min. in a humidified environment. Wells are
washed .times.3 with PBS(+Ca,Mg)+0.5% BSA.
[2128] Then add 20 .mu.l of diluted EXTRAVIDIN.TM.-Alkaline
Phosphotase (1:5,000 dilution) to each well and incubated at
37.degree. C. for 30 min. Wells are washed .times.3 with
PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is
dissolved in 5 ml of glycine buffer (pH 10.4). 100 .mu.l of pNPP
substrate in glycine buffer is added to each test well. Standard
wells in triplicate are prepared from the working dilution of the
EXTRAVIDIN.TM.-Alkaline Phosphotase in glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.5. 5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent must then be added to each of the standard
wells. The plate must be incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3M NaOH is added to all wells. The results
are quantified on a plate reader at 405 nm. The background
subtraction option is used on blank wells filled with glycine
buffer only. The template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
[2129] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 53
Assay for the Stimulation of Bone Marrow Cd34+ Cell
Proliferation
[2130] This assay is based on the ability of human CD34+ to
proliferate in the presence of hematopoietic growth factors and
evaluates the ability of isolated polypeptides expressed in
mammalian cells to stimulate proliferation of CD34+ cells.
[2131] It has been previously shown that most mature precursors
will respond to only a single signal. More immature precursors
require at least two signals to respond. Therefore, to test the
effect of polypeptides on hematopoietic activity of a wide range of
progenitor cells, the assay contains a given polypeptide in the
presence or absence of other hematopoietic growth factors. Isolated
cells are cultured for 5 days in the presence of Stem Cell Factor
(SCF) in combination with tested sample. SCF alone has a very
limited effect on the proliferation of bone marrow (BM) cells,
acting in such conditions only as a "survival" factor. However,
combined with any factor exhibiting stimulatory effect on these
cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore,
if the tested polypeptide has a stimulatory effect on a
hematopoietic progenitors, such activity can be easily detected.
Since normal BM cells have a low level of cycling cells, it is
likely that any inhibitory effect of a given polypeptide, or
agonists or antagonists thereof, might not be detected.
Accordingly, assays for an inhibitory effect on progenitors is
preferably tested in cells that are first subjected to in vitro
stimulation with SCF+IL+3, and then contacted with the compound
that is being evaluated for inhibition of such induced
proliferation.
[2132] Briefly, CD34+ cells are isolated using methods known in the
art. The cells are thawed and resuspended in medium (QBSF 60
serum-free medium with 1% L-glutamine (500 ml) Quality Biological,
Inc., Gaithersburg, Md. Cat #160-204-101). After several gentle
centrifugation steps at 200.times.g, cells are allowed to rest for
one hour. The cell count is adjusted to 2.5.times.10.sup.5
cells/ml. During this time, 100 .mu.l of sterile water is added to
the peripheral wells of a 96-well plate. The cytokines that can be
tested with a given polypeptide in this assay is rhSCF (R&D
Systems, Minneapolis, Minn., Cat #255-SC) at 50 ng/ml alone and in
combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis,
Minn., Cat #203-ML) at 30 ng/ml. After one hour, 10 .mu.l of
prepared cytokines, 50 .mu.l SID (supernatants at 1:2 dilution=50
.mu.l) and 20 .mu.l of diluted cells are added to the media which
is already present in the wells to allow for a final total volume
of 100 .mu.l. The plates are then placed in a 37.degree. C./5%
CO.sub.2 incubator for five days.
[2133] Eighteen hours before the assay is harvested, 0.5
.mu.Ci/well of [3H] Thymidine is added in a 10 .mu.l volume to each
well to determine the proliferation rate. The experiment is
terminated by harvesting the cells from each 96-well plate to a
filtermat using the Tomtec Harvester 96. After harvesting, the
filtermats are dried, trimmed and placed into OMNIFILTER.TM.
assemblies consisting of one OMNIFILTER.TM. plate and one
OMNIFILTER.TM. Tray. 60 .mu.l MICROSCINT.TM. is added to each well
and the plate sealed with TopSeal-A press-on sealing film A bar
code 15 sticker is affixed to the first plate for counting. The
sealed plates is then loaded and the level of radioactivity
determined via the Packard Top Count and the printed data collected
for analysis. The level of radioactivity reflects the amount of
cell proliferation.
[2134] The studies described in this example test the activity of a
given polypeptide to stimulate bone marrow CD34+ cell
proliferation. One skilled in the art could easily modify the
exemplified studies to test the activity of polynucleotides (e.g.,
gene therapy), antibodies, agonists, and/or antagonists and
fragments and variants thereof. As a nonlimiting example, potential
antagonists tested in this assay would be expected to inhibit cell
proliferation in the presence of cytokines and/or to increase the
inhibition of cell proliferation in the presence of cytokines and a
given polypeptide. In contrast, potential agonists tested in this
assay would be expected to enhance cell proliferation and/or to
decrease the inhibition of cell proliferation in the presence of
cytokines and a given polypeptide.
[2135] The ability of a gene to stimulate the proliferation of bone
marrow CD34+ cells indicates that polynucleotides and polypeptides
corresponding to the gene are useful for the diagnosis and
treatment of disorders affecting the immune system and
hematopoiesis. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections above, and elsewhere
herein.
Example 54
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
[2136] The objective of the Extracellular Matrix Enhanced Cell
Response (EMECR) assay is to identify gene products (e.g., isolated
polypeptides) that act on the hematopoietic stem cells in the
context of the extracellular matrix (ECM) induced signal.
[2137] Cells respond to the regulatory factors in the context of
signal(s) received from the surrounding microenvironment. For
example, fibroblasts, and endothelial and epithelial stem cells
fail to replicate in the absence of signals from the ECM.
Hematopoietic stem cells can undergo self-renewal in the bone
marrow, but not in in vitro suspension culture. The ability of stem
cells to undergo self-renewal in vitro is dependent upon their
interaction with the stromal cells and the ECM protein fibronectin
(fn). Adhesion of cells to fn is mediated by the
.alpha..sub.5..beta..sub.1 and .alpha..sub.4..beta..sub.1 integrin
receptors, which are expressed by human and mouse hematopoietic
stem cells. The factor(s) which integrate with the ECM environment
and responsible for stimulating stem cell self-renewal has not yet
been identified. Discovery of such factors should be of great
interest in gene therapy and bone marrow transplant
applications
[2138] Briefly, polystyrene, non tissue culture treated, 96-well
plates are coated with fn fragment at a coating concentration of
0.2 .mu.g/cm.sup.2. Mouse bone marrow cells are plated (1,000
cells/well) in 0.2 ml of serum-free medium. Cells cultured in the
presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the
positive control, conditions under which little self-renewal but
pronounced differentiation of the stem cells is to be expected.
Gene products are tested with appropriate negative controls in the
presence and absence of SCF (5.0 ng/ml), where test factor
supernates represent 10% of the total assay volume. The plated
cells are then allowed to grow by incubating in a low oxygen
environment (5% CO.sub.2, 7% O.sub.2, and 88% N.sub.2) tissue
culture incubator for 7 days. The number of proliferating cells
within the wells is then quantitated by measuring thymidine
incorporation into cellular DNA. Verification of the positive hits
in the assay will require phenotypic characterization of the cells,
which can be accomplished by scaling up of the culture system and
using appropriate antibody reagents against cell surface antigens
and FACScan.
[2139] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides (e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments
and variants thereof.
[2140] If a particular gene product is found to be a stimulator of
hematopoietic progenitors, polynucleotides and polypeptides
corresponding to the gene may be useful for the diagnosis and
treatment of disorders affecting the immune system and
hematopoiesis. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections above, and elsewhere
herein. The gene product may also be useful in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell
types.
[2141] Additionally, the polynucleotides and/or polypeptides of the
gene of interest and/or agonists and/or antagonists thereof, may
also be employed to inhibit the proliferation and differentiation
of hematopoietic cells and therefore may be employed to protect
bone marrow stem cells from chemotherapeutic agents during
chemotherapy. This antiproliferative effect may allow
administration of higher doses of chemotherapeutic agents and,
therefore, more effective chemotherapeutic treatment.
[2142] Moreover, polynucleotides and polypeptides corresponding to
the gene of interest may also be useful for the treatment and
diagnosis of hematopoietic related disorders such as, for example,
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex-vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia.
Example 55
Human Dermal Fibroblast and Aortic Smooth Muscle Cell
Proliferation
[2143] The polypeptide of interest is added to cultures of normal
human dermal fibroblasts (NHDF) and human aortic smooth muscle
cells (AoSMC) and two co-assays are performed with each sample. The
first assay examines the effect of the polypeptide of interest on
the proliferation of normal human dermal fibroblasts (NHDF) or
aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts
or smooth muscle cells is a part of several pathological processes,
including fibrosis, and restenosis. The second assay examines IL6
production by both NHDF and SMC. IL6 production is an indication of
functional activation. Activated cells will have increased
production of a number of cytokines and other factors, which can
result in a proinflammatory or immunomodulatory outcome. Assays are
run with and without co-TNFa stimulation, in order to check for
costimulatory or inhibitory activity.
[2144] Briefly, on day 1,96-well black plates are set up with 1000
cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 .mu.l culture
media. NHDF culture media contains: Clonetics FB basal media, 1
mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while
AoSMC culture media contains Clonetics SM basal media, 0.5 .mu.g/ml
hEGF, 5 mg/ml insulin, 1 .mu.g/ml hFGF, 50 mg/ml gentamycin, 50
.mu.g/ml Amphotericin B, 5% FBS. After incubation @ 37.degree. C.
for at least 4-5 hours culture media is aspirated and replaced with
growth arrest media. Growth arrest media for NHDF contains
fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth
arrest media for AoSMC contains SM basal media, 50 mg/ml
gentamycin, 50 .mu.g/ml Amphotericin B, 0.4% FBS. Incubate at 37 C
until day 2.
[2145] On day 2, serial dilutions and templates of the polypeptide
of interest are designed which should always include media controls
and known-protein controls. For both stimulation and inhibition
experiments, proteins are diluted in growth arrest media. For
inhibition experiments, TNFa is added to a final concentration of 2
ng/ml (NHDF) or 5 ng/ml (AoSMC). Then add 1/3 vol media containing
controls or supernatants and incubate at 37 C/5% CO.sub.2 until day
5.
[2146] Transfer 60 .mu.l from each well to another labeled 96-well
plate, cover with a plate-sealer, and store at 4 C until Day 6 (for
IL6 ELISA). To the remaining 100 .mu.l in the cell culture plate,
aseptically add ALAMAR BLUE.TM. in an amount equal to 10% of the
culture volume (100. Return plates to incubator for 3 to 4 hours.
Then measure fluorescence with excitation at 530 nm and emission at
590 nm using the CYTOFLUOR.TM.. This yields the growth
stimulation/inhibition data.
[2147] On day 5, the IL6 ELISA is performed by coating a 96 well
plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody
diluted in PBS, pH 7.4, incubate ON at room temperature.
[2148] On day 6, empty the plates into the sink and blot on paper
towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the
plates with 200 .mu.l/well of Pierce Super Block blocking buffer in
PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05%
Tween-20). Blot plates on paper towels. Then add 50 .mu.l/well of
diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50
mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0
ng/ml). Add duplicate samples to top row of plate. Cover the plates
and incubate for 2 hours at RT on shaker.
[2149] Wash plates with wash buffer and blot on paper towels.
Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100
.mu.l/well. Cover the plate and incubate 1 h at RT. Wash plates
with wash buffer. Blot on paper towels. Add 100 .mu.l/well of
Enhancement Solution. Shake for 5 minutes. Read the plate on the
Wallac DELFIA Fluorometer. Readings from triplicate samples in each
assay were tabulated and averaged. A positive result in this assay
suggests AoSMC cell proliferation and that the gene product of
interest may be involved in dermal fibroblast proliferation and/or
smooth muscle cell proliferation. A positive result also suggests
many potential uses of polypeptides, polynucleotides, agonists
and/or antagonists of the gene/gene product of interest. For
example, inflammation and immune responses, wound healing, and
angiogenesis, as detailed throughout this specification.
Particularly, polypeptides of the gene product and polynucleotides
of the gene may be used in wound healing and dermal regeneration,
as well as the promotion of vasculargenesis, both of the blood
vessels and lymphatics. The growth of vessels can be used in the
treatment of, for example, cardiovascular diseases. Additionally,
antagonists of polypeptides of the gene product and polynucleotides
of the gene may be useful in treating diseases, disorders, and/or
conditions which involve angiogenesis by acting as an anti-vascular
(e.g., anti-angiogenesis). These diseases, disorders, and/or
conditions are known in the art and/or are described herein, such
as, for example, malignancies, solid tumors, benign tumors, for
example hemangiomas, acoustic neuromas, neurofibromas, trachomas,
and pyogenic granulomas; artheroscleric plaques; ocular angiogenic
diseases, for example, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye; rheumatoid arthritis; psoriasis; delayed wound healing;
endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular
adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
Moreover, antagonists of polypeptides of the gene product and
polynucleotides of the gene may be useful in treating
anti-hyperproliferative diseases and/or anti-inflammatory known in
the art and/or described herein.
[2150] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides (e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments
and variants thereof.
Example 56
Cellular Adhesion Molecule (CAM) Expression on Endothelial
Cells
[2151] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs. Briefly, endothelial cells (e.g., Human
Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard
96 well plate to confluence, growth medium is removed from the
cells and replaced with 100 .mu.l of 199 Medium (10% fetal bovine
serum (FBS)). Samples for testing and positive or negative controls
are added to the plate in triplicate (in 10 .mu.l volumes). Plates
are then incubated at 37.degree. C. for either 5 h (selectin and
integrin expression) or 24 h (integrin expression only). Plates are
aspirated to remove medium and 100 .mu.l of 0.1%
paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well.
Plates are held at 4.degree. C. for 30 min. Fixative is removed
from the wells and wells are washed 1.times. with PBS(+Ca,Mg)+0.5%
BSA and drained. 10 .mu.l of diluted primary antibody is added to
the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin
and Anti-E-selectin-Biotin are used at a concentration of 10
.mu.g/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are
incubated at 37.degree. C. for 30 min. in a humidified environment.
Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 .mu.l of
diluted EXTRAVIDIN.TM.-Alkaline Phosphotase (1:5,000 dilution,
referred to herein as the working dilution) are added to each well
and incubated at 37.degree. C. for 30 min. Wells are washed three
times with PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol
Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 .mu.l of
pNPP substrate in glycine buffer is added to each test well.
Standard wells in triplicate are prepared from the working dilution
of the EXTRAVIDIN.TM.-Alkaline Phosphotase in glycine buffer:
1:5,000 (10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.5. 5
.mu.l of each dilution is added to triplicate wells and the
resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng,
0.18 ng. 100 .mu.l of pNNP reagent is then added to each of the
standard wells. The plate is incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3M NaOH is added to all wells. The plate is
read on a plate reader at 405 nm using the background subtraction
option on blank wells filled with glycine buffer only.
Additionally, the template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
Example 57
ALAMAR BLUE.TM. Endothelial Cells Proliferation Assay
[2152] This assay may be used to quantitatively determine protein
mediated inhibition of bFGF-induced proliferation of Bovine
Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells
(BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs).
This assay incorporates a fluorometric growth indicator based on
detection of metabolic activity. A standard ALAMAR BLUE.TM.
Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF
added as a source of endothelial cell stimulation. This assay may
be used with a variety of endothelial cells with slight changes in
growth medium and cell concentration. Dilutions of the protein
batches to be tested are diluted as appropriate. Serum-free medium
(GIBCO SFM) without bFGF is used as a non-stimulated control and
Angiostatin or TSP-1 are included as a known inhibitory controls.
Briefly, LEC, BAECs or UTMECs are seeded in growth media at a
density of 5000 to 2000 cells/well in a 96 well plate and placed at
37-C overnight. After the overnight incubation of the cells, the
growth media is removed and replaced with GIBCO EC-SFM. The cells
are treated with the appropriate dilutions of the protein of
interest or control protein sample(s) (prepared in SFM) in
triplicate wells with additional bFGF to a concentration of 10
ng/ml. Once the cells have been treated with the samples, the
plate(s) is/are placed back in the 37.degree. C. incubator for
three days. After three days 10 ml of stock ALAMAR BLUE.TM.
(Biosource Cat #DAL1100) is added to each well and the plate(s)
is/are placed back in the 37.degree. C. incubator for four hours.
The plate(s) are then read at 530 nm excitation and 590 nm emission
using the CYTOFLUOR.TM. fluorescence reader. Direct output is
recorded in relative fluorescence units. ALAMAR BLUE.TM. is an
oxidation-reduction indicator that both fluoresces and changes
color in response to chemical reduction of growth medium resulting
from cell growth. As cells grow in culture, innate metabolic
activity results in a chemical reduction of the immediate
surrounding environment. Reduction related to growth causes the
indicator to change from oxidized (non-fluorescent blue) form to
reduced (fluorescent red) form. i.e. stimulated proliferation will
produce a stronger signal and inhibited proliferation will produce
a weaker signal and the total signal is proportional to the total
number of cells as well as their metabolic activity. The background
level of activity is observed with the starvation medium alone.
This is compared to the output observed from the positive control
samples (bFGF in growth medium) and protein dilutions.
Example 58
Detection of Inhibition of a Mixed Lymphocyte Reaction
[2153] This assay can be used to detect and evaluate inhibition of
a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated
polypeptides) Inhibition of a MLR may be due to a direct effect on
cell proliferation and viability, modulation of costimulatory
molecules on interacting cells, modulation of adhesiveness between
lymphocytes and accessory cells, or modulation of cytokine
production by accessory cells. Multiple cells may be targeted by
these polypeptides since the peripheral blood mononuclear fraction
used in this assay includes T, B and natural killer lymphocytes, as
well as monocytes and dendritic cells. Polypeptides of interest
found to inhibit the MLR may find application in diseases
associated with lymphocyte and monocyte activation or
proliferation. These include, but are not limited to, diseases such
as asthma, arthritis, diabetes, inflammatory skin conditions,
psoriasis, eczema, systemic lupus erythematosus, multiple
sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's
disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs.
host disease, host vs. graft disease, hepatitis, leukemia and
lymphoma. Briefly, PBMCs from human donors are purified by density
gradient centrifugation using Lymphocyte Separation Medium
(LSM.RTM., density 1.0770 g/ml, Organon Teknika Corporation, West
Chester, Pa.). PBMCs from two donors are adjusted to
2.times.10.sup.6 cells/ml in RPMI-1640 (LIFE TECHNOLOGIES.TM.,
Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine.
PBMCs from a third donor is adjusted to 2.times.10.sup.5 cells/ml.
Fifty microliters of PBMCs from each donor is added to wells of a
96-well round bottom microtiter plate. Dilutions of test materials
(50 .mu.l) is added in triplicate to microtiter wells. Test samples
(of the protein of interest) are added for final dilution of 1:4;
rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number
202-IL) is added to a final concentration of 1 .mu.g/ml; anti-CD4
mAb (R&D Systems, clone 34930.11, catalog number MAB379) is
added to a final concentration of 10 .mu.g/ml. Cells are cultured
for 7-8 days at 37.degree. C. in 5% CO.sub.2, and 1 .mu.C of
[.sup.3H] thymidine is added to wells for the last 16 hrs of
culture. Cells are harvested and thymidine incorporation determined
using a Packard TopCount. Data is expressed as the mean and
standard deviation of triplicate determinations. Samples of the
protein of interest are screened in separate experiments and
compared to the negative control treatment, anti-CD4 mAb, which
inhibits proliferation of lymphocytes and the positive control
treatment, IL-2 (either as recombinant material or supernatant),
which enhances proliferation of lymphocytes. One skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), antibodies, agonists, and/or
antagonists and fragments and variants thereof. It will be clear
that the invention may be practiced otherwise than as particularly
described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible
in light of the above teachings and, therefore, are within the
scope of the appended claims.
[2154] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, Detailed Description, and Examples is hereby
incorporated herein by reference. Further, the hard copy of the
sequence listing submitted herewith and the corresponding computer
readable form are both incorporated herein by reference in their
entireties.
TABLE-US-00015 TABLE 3 Res Position I II III IV V VI VII VIII IX X
XI XII XIII XIV Met 1 A . . . . . . 1.30 -0.61 . . . 0.95 2.10 Arg
2 . . B . . . . 0.88 -1.04 * . . 0.95 3.21 Lys 3 . . B B . . . 0.98
-0.79 * . . 0.75 2.07 Thr 4 A . . B . . . 1.02 -0.30 . . . 0.45
2.20 Arg 5 . . B B . . . 0.60 -0.49 . . F 0.60 1.11 Leu 6 A . . B .
. . 0.39 0.20 * . . -0.30 0.46 Trp 7 . . B B . . . -0.01 0.89 * . .
-0.60 0.26 Gly 8 A . . B . . . -0.66 1.31 . * . -0.60 0.14 Leu 9 A
. . B . . . -1.16 1.93 * . . -0.60 0.17 Leu 10 . . B B . . . -1.97
1.93 * * . -0.60 0.13 Trp 11 . . B B . . . -2.01 1.80 . . . -0.60
0.12 Met 12 . . B B . . . -2.02 2.01 . . . -0.60 0.10 Leu 13 A . .
B . . . -1.68 1.71 . . . -0.60 0.17 Phe 14 A . . B . . . -1.68 1.03
* * . -0.60 0.28 Val 15 A . . B . . . -0.76 0.80 * * . -0.60 0.23
Ser 16 A A . . . . . -1.06 0.19 * * . -0.30 0.55 Glu 17 A A . . . .
. -1.04 0.00 . * . -0.30 0.64 Leu 18 A A . . . . . -0.54 -0.29 * *
. 0.30 0.88 Arg 19 A A . . . . . 0.20 -0.44 * * . 0.30 0.94 Ala 20
A A . . . . . 0.24 -0.83 * * . 0.75 1.09 Ala 21 A A . . . . . 0.23
-0.14 * * . 0.45 1.09 Thr 22 A A . . . . . 0.23 -0.34 * * F 0.45
0.80 Lys 23 A A . . . . . 1.04 -0.34 * * F 0.60 1.38 Leu 24 A A . .
. . . 0.98 -0.84 * * F 0.90 2.36 Thr 25 A A . . . . . 1.32 -1.34 .
. F 0.90 3.27 Glu 26 A A . . . . . 1.91 -1.07 . . F 0.90 2.56 Glu
27 A A . . . . . 1.41 -1.07 . * F 0.90 5.38 Lys 28 A A . . . . .
1.41 -1.07 . * F 0.90 3.08 Tyr 29 A A . . . . . 2.22 -1.56 . * F
0.90 3.55 Glu 30 A A . . . . . 2.19 -1.56 . * F 0.90 3.55 Leu 31 A
A . . . . . 2.19 -1.13 . * F 0.90 1.76 Lys 32 A A . . . . . 1.88
-0.73 * * F 0.90 1.94 Glu 33 A A . . . . . 1.02 -1.00 * * F 0.90
1.62 Gly 34 A A . . . . . 1.27 -0.31 . * F 0.60 1.62 Gln 35 A A . .
. . . 0.41 -1.00 . * F 0.90 1.35 Thr 36 A A . . . . . 1.27 -0.36 .
* F 0.45 0.58 Leu 37 A A . . . . . 0.56 -0.36 . * F 0.60 1.17 Asp
38 . A B . . . . 0.56 -0.21 . * . 0.30 0.36 Val 39 . A B . . . .
0.66 -0.61 . * . 0.60 0.42 Lys 40 . A B . . . . 0.34 -0.34 . * .
0.30 0.80 Cys 41 . . B . . T . -0.16 -0.54 . * . 1.00 0.69 Asp 42 A
. . . . T . 0.66 0.14 . * . 0.10 0.77 Tyr 43 A . . . . T . 0.70
-0.50 . * . 0.70 0.66 Thr 44 A . . . . T . 0.86 -0.50 * * . 0.85
2.47 Leu 45 A A . . . . . 0.22 -0.29 * * . 0.45 1.28 Glu 46 A A . .
. . . 0.59 0.21 * . . -0.30 0.83 Lys 47 A A . . . . . 0.29 -0.16 .
. . 0.30 0.77 Phe 48 A A . . . . . 0.53 -0.26 * . F 0.60 1.25 Ala
49 A . . . . T . 0.89 -0.54 . . F 1.30 1.25 Ser 50 A . . . . T .
1.11 -0.54 * . F 1.30 1.25 Ser 51 A . . . . T . 0.82 -0.04 . . F
1.00 1.46 Gln 52 A . . . . T . 0.78 0.09 . * F 0.40 1.51 Lys 53 A .
. . . . . 0.59 -0.01 * . F 0.80 1.96 Ala 54 A . . B . . . 0.29 0.29
* . . -0.15 1.02 Trp 55 A . . B . . . 0.70 0.59 * . . -0.60 0.41
Gln 56 . . B B . . . 1.00 0.19 * . . -0.30 0.41 Ile 57 . . B B . .
. 0.66 0.19 * . . 0.04 0.67 Ile 58 . . B . . T . 0.61 0.11 * . .
0.78 0.63 Arg 59 . . B . . T . 0.60 -0.80 * . . 2.02 0.63 Asp 60 .
. . . T T . 0.68 -0.59 * . F 2.91 0.89 Gly 61 . . . . T T . 0.72
-0.84 * . F 3.40 1.97 Glu 62 . . . . . . C 1.30 -1.53 * . F 2.66
2.01 Met 63 . . . . . T C 1.38 -1.04 . . F 2.52 1.74 Pro 64 . . . .
T T . 0.68 -0.36 * . F 2.08 1.45 Lys 65 . . . . T T . 0.01 -0.29 .
. F 1.59 0.84 Thr 66 A . . . . T . 0.04 0.29 . . F 0.25 0.46 Leu 67
A A . . . . . 0.04 0.16 * . . -0.30 0.43 Ala 68 A A . . . . . 0.76
-0.27 * . . 0.30 0.37 Cys 69 A A . . . . . 0.76 -0.27 * . . 0.30
0.50 Thr 70 A A . . . . . 0.41 -0.33 * . . 0.64 0.94 Glu 71 A . . .
. . . 0.77 -0.63 * . F 1.78 1.25 Arg 72 A . . . . . . 1.58 -1.13 *
. F 2.12 4.65 Pro 73 . . . . T . . 1.87 -1.30 . . F 2.86 5.18 Ser
74 . . . . T T . 2.50 -1.40 * . F 3.40 4.01 Lys 75 . . . . T T .
2.60 -0.90 * . F 3.06 2.79 Asn 76 . . . . T T . 1.74 -0.47 * . F
2.42 2.79 Ser 77 . . . . . T C 1.63 -0.26 . * F 1.88 1.54 His 78 .
. B B . . . 0.99 -0.24 . . F 0.94 1.34 Pro 79 . . B B . . . 0.94
0.40 . . . -0.60 0.62 Val 80 . . B B . . . 1.01 0.43 . * . -0.60
0.46 Gln 81 . . B B . . . 0.12 0.04 * . . -0.30 0.66 Val 82 . . B B
. . . -0.47 0.23 * . . -0.30 0.30 Gly 83 . . B B . . . -1.24 0.49 *
* . -0.60 0.28 Arg 84 . . B B . . . -1.03 0.53 * . . -0.60 0.13 Ile
85 . . B B . . . -0.18 0.13 * . . -0.30 0.31 Ile 86 . . B B . . .
-0.42 -0.51 * . . 0.60 0.53 Leu 87 . . B B . . . 0.40 -0.19 * * .
0.30 0.42 Glu 88 . . B B . . . 0.74 0.31 * * . -0.30 0.82 Asp 89 A
. . . . . . 0.60 -0.37 * * . 0.65 1.95 Tyr 90 A . . . . . . 1.14
-0.56 . . . 0.95 3.21 His 91 A . . . . T . 1.22 -0.81 . . . 1.15
1.84 Asp 92 A . . . . T . 1.22 -0.13 . * . 0.70 0.91 His 93 A . . .
. T . 1.33 0.56 * * . -0.20 0.48 Gly 94 A . . . . T . 0.48 -0.20 .
* . 0.70 0.69 Leu 95 A . . B . . . 0.83 -0.06 . * . 0.30 0.31 Leu
96 A . . B . . . 0.27 -0.06 . * . 0.30 0.44 Arg 97 . . B B . . .
-0.59 0.06 . * . -0.30 0.44 Val 98 . . B B . . . -0.56 0.27 . * .
-0.30 0.40 Arg 99 . . B B . . . -1.02 -0.01 . * . 0.30 0.77 Met 100
. . B B . . . -0.21 -0.01 . * . 0.30 0.32 Val 101 . . B B . . .
-0.26 0.39 . * . -0.30 0.76 Asn 102 . . B B . . . -0.37 0.39 . * .
-0.30 0.29 Leu 103 . . B B . . . 0.49 0.39 * * . -0.30 0.50 Gln 104
. . B B . . . 0.08 -0.23 * * . 0.73 1.13 Val 105 . . B B . . . 0.33
-0.49 . * . 0.86 0.94 Glu 106 . . B B . . . 0.38 -0.46 . * F 1.44
1.13 Asp 107 . . . . T T . 0.13 -0.46 . * F 2.37 0.54 Ser 108 . . .
. T T . 0.94 -0.10 . * F 2.80 1.14 Gly 109 . . . . T T . 0.28 -0.34
. . F 2.52 1.14 Leu 110 . . . . T T . 0.28 0.23 . . . 1.34 0.36 Tyr
111 . . B B . . . -0.61 0.87 . . . -0.04 0.20 Gln 112 . . B B . . .
-0.86 1.17 . . . -0.32 0.14 Cys 113 . . B B . . . -0.56 1.50 . . .
-0.60 0.27 Val 114 . . B B . . . -0.42 1.21 . . . -0.60 0.30 Ile
115 . . B B . . . 0.18 0.89 * . . -0.26 0.27 Tyr 116 . . B . . . .
0.47 0.91 * . . 0.28 0.77 Gln 117 . . . . . . C 0.47 0.34 * . .
1.27 2.09 Pro 118 . . . . . T C 0.92 -0.30 * . F 2.56 5.15 Pro 119
. . . . T T . 1.74 -0.56 * . F 3.40 5.08 Lys 120 . . . . . T C 2.03
-0.81 * . F 2.86 3.99 Glu 121 . . . . . T C 1.47 -0.60 * . F 2.52
2.56 Pro 122 A . . . . . . 0.77 -0.34 * . F 1.48 1.36 His 123 A . .
B . . . 0.98 0.01 * . . 0.04 0.59 Met 124 A . . B . . . 1.30 0.01 *
. . -0.30 0.57 Leu 125 A . . B . . . 0.37 0.01 * * . -0.30 0.72 Phe
126 A . . B . . . 0.48 0.27 * * . -0.30 0.37 Asp 127 A . . B . . .
-0.12 -0.23 * * . 0.30 0.74 Arg 128 A . . B . . . -0.94 -0.16 * * .
0.30 0.74 Ile 129 A . . B . . . -1.20 -0.20 * * . 0.30 0.63 Arg 130
. . B B . . . -0.70 -0.34 * * . 0.30 0.28 Leu 131 . . B B . . .
0.04 0.14 * * . -0.30 0.21 Val 132 . . B B . . . -0.30 0.14 * * .
-0.30 0.59 Val 133 . . B B . . . -1.11 -0.11 * * . 0.30 0.30 Thr
134 . . B B . . . -0.52 0.67 * * F -0.45 0.31 Lys 135 . . B B . . .
-0.98 0.37 . * F -0.15 0.56 Gly 136 . . B . . T . -0.48 0.16 . . F
0.25 0.75 Phe 137 . . B . . T . 0.17 0.00 . . F 0.25 0.75 Ser 138 .
. B . . T . 0.68 -0.06 . . F 0.85 0.58 Gly 139 . . . . . T C 0.69
0.37 . . F 0.45 0.58 Thr 140 . . . . . T C 0.64 0.33 . . F 0.45
0.90 Pro 141 . . . . . T C 0.99 -0.06 . . F 1.20 1.08 Gly 142 . . .
. . T C 1.69 -0.44 . . F 1.54 1.89 Ser 143 . . . . . T C 1.69 -0.47
. . F 1.88 2.11 Asn 144 . . . . . T C 1.72 -0.57 . . F 2.52 1.82
Glu 145 . . . . . T C 2.03 -0.51 . . F 2.86 2.66 Asn 146 . . . . T
T . 2.24 -0.54 . . F 3.40 3.44 Ser 147 . . . . T T . 1.73 -0.53 . .
F 3.06 3.44 Thr 148 . . . B T . . 1.79 -0.29 * . F 2.02 1.47 Gln
149 . . B B . . . 1.83 0.47 * . F 0.38 1.44 Asn 150 . . B B . . .
0.94 0.07 * . F 0.34 2.14 Val 151 . . B B . . . 0.73 0.37 * . F
0.00 1.04 Tyr 152 . . B B . . . 0.82 0.31 * . . -0.30 0.93 Lys 153
. . B B . . . 0.82 0.34 * . F -0.15 0.89 Ile 154 . . B B . . . 0.51
0.43 * . F -0.30 1.74 Pro 155 . . B . . T . 0.20 0.27 * . F 0.40
1.60 Pro 156 . . . . T T . 1.10 0.00 * . F 0.80 1.15 Thr 157 . . .
. T T . 0.76 0.00 * . F 0.80 3.29 Thr 158 . . B . . T . -0.10 -0.19
* . F 1.00 2.15 Thr 159 . A B . . . . 0.12 0.07 * . F 0.00 1.15 Lys
160 . A B . . . . 0.12 0.21 * . F -0.15 0.43 Ala 161 . A B . . . .
-0.48 0.16 * . . -0.30 0.46 Leu 162 . A B . . . . -0.41 0.36 * . .
-0.30 0.26 Cys 163 . . B . . T . -0.41 0.63 * . . -0.20 0.20 Pro
164 . . B . . T . -0.40 1.11 * . . -0.20 0.29 Leu 165 . . B . . T .
-0.66 1.00 * * . -0.20 0.47 Tyr 166 . . B . . T . 0.04 0.74 * * .
-0.05 1.37 Thr 167 . . B . . . . 0.54 0.17 * . F 0.20 1.74 Ser 168
. . . . . T C 0.36 0.23 * . F 0.60 3.04 Pro 169 . . B . . T . 0.26
0.19 * . F 0.40 1.44 Arg 170 . . B . . T . 1.07 -0.09 * * F 1.00
1.44 Thr 171 . . B . . T . 0.72 -0.17 * . F 1.00 1.86 Val 172 . . B
B . . . 0.82 -0.06 * . F 0.60 1.21 Thr 173 . . B B . . . 0.91 -0.06
* * F 0.73 0.96 Gln 174 . . B B . . . 1.17 0.37 * * F 0.56 1.03 Ala
175 . . B B . . . 0.76 -0.11 * * F 1.44 2.77 Pro 176 . . . . . T C
0.76 -0.37 . . F 2.32 2.57 Pro 177 . . . . T T . 1.02 -0.37 . * F
2.80 2.14 Lys 178 . . . . T T . 1.33 -0.27 . * F 2.52 2.14 Ser 179
. . B . . T . 0.48 -0.77 . * F 2.14 2.31 Thr 180 . . B B . . . 0.77
-0.56 . * F 1.46 1.11 Ala 181 . . B B . . . 0.67 -0.60 . * F 1.03
0.74 Asp 182 . . B B . . . 0.67 -0.11 . * F 0.45 0.80 Val 183 . . B
B . . . 0.62 -0.07 . * F 0.79 0.86 Ser 184 . . B . . . . 0.62 -0.56
. . F 1.78 1.42 Thr 185 . . B . . T . 0.93 -0.67 . * F 2.32 1.14
Pro 186 . . . . . T C 0.63 -0.67 . * F 2.86 2.66 Asp 187 . . . . T
T . 0.63 -0.63 . * F 3.40 1.39 Ser 188 . . B . . T . 0.68 -0.61 . *
F 2.66 1.55 Glu 189 . . B . . . . 0.67 -0.41 . * F 1.67 0.83 Ile
190 . . B . . . . 0.98 -0.36 . * F 1.33 0.71 Asn 191 . . B . . . .
0.33 0.04 . * . 0.24 0.86 Leu 192 . . B B . . . 0.02 0.30 . * .
-0.30 0.37 Thr 193 . . B B . . . 0.32 0.79 . * F -0.60 0.76 Asn 194
. . B B . . . -0.57 0.10 * * F -0.15 0.78 Val 195 . . B B . . .
-0.57 0.39 * * F -0.15 0.67 Thr 196 . . B B . . . -0.46 0.39 * * F
-0.15 0.32 Asp 197 . . B B . . . -0.50 -0.10 * * . 0.30 0.39 Ile
198 . . B B . . . -0.40 0.14 * * . -0.30 0.39 Ile 199 . . B B . . .
-1.26 -0.07 * * . 0.30 0.42 Arg 200 . . B B . . . -1.10 0.09 * * .
-0.30 0.19 Val 201 . . B B . . . -0.79 0.87 * . . -0.60 0.23 Pro
202 . . B B . . . -1.68 0.59 * . . -0.60 0.53 Val 203 . . B B . . .
-1.64 0.59 . * . -0.60 0.19 Phe 204 . . B B . . . -1.64 1.23 . * .
-0.60 0.19 Asn 205 . . B B . . . -2.57 1.27 . * . -0.60 0.09 Ile
206 . . B B . . . -2.52 1.53 . . . -0.60 0.10 Val 207 . . B B . . .
-2.90 1.57 . . . -0.60 0.09 Ile 208 . . B B . . . -2.39 1.29 . . .
-0.60 0.06 Leu 209 . . B B . . . -2.03 1.31 . * . -0.60 0.08 Leu
210 . . B . . T . -2.73 1.06 . . . -0.20 0.11 Ala 211 . . B . . T .
-2.66 1.20 . . . -0.20 0.13 Gly 212 A . . . . T . -2.10 1.20 . . .
-0.20 0.13 Gly 213 A . . . . T . -1.17 0.90 . . . -0.20 0.22 Phe
214 A . . . . . . -0.66 0.21 . . . -0.10 0.43 Leu 215 A . . . . . .
-0.66 0.10 . . F 0.05 0.58 Ser 216 . . B . . T . -0.92 0.36 . . F
0.25 0.48 Lys 217 . . B . . T . -1.28 0.57 . . F -0.05 0.42 Ser 218
. . B . . T . -1.23 0.57 . . F -0.05 0.44 Leu 219 . . B . . T .
-1.39 0.27 . . . 0.10 0.44 Val 220 . . B B . . . -1.39 0.53 . . .
-0.60 0.16 Phe 221 . . B B . . . -1.79 1.21 . . . -0.60 0.10 Ser
222 . . B B . . . -2.42 1.61 . . . -0.60 0.10 Val 223 . . B B . . .
-2.98 1.43 . . . -0.60 0.14 Leu 224 . . B B . . . -2.48 1.43 . * .
-0.60 0.12 Phe 225 A . . B . . . -2.43 1.13 * * . -0.60 0.13 Ala
226 A . . B . . . -1.62 1.43 * * . -0.60 0.15 Val 227 A . . B . . .
-1.62 0.79 * * . -0.60 0.35 Thr 228 . . B B . . . -1.47 0.49 . * .
-0.60 0.54 Leu 229 . . B B . . . -1.51 0.49 * * . -0.60 0.46 Arg
230 . . B B . . . -1.02 0.63 * * . -0.60 0.46 Ser 231 . . B B . . .
-0.82 0.41 . * . -0.60 0.49 Phe 232 . . B B . . . -0.36 0.36 . * .
-0.30 0.76 Val 233 . . B B . . . -0.43 0.10 * * . -0.30 0.50 Pro
234 . . B B . . . -0.01 0.53 * * . -0.60 0.48
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120122762A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120122762A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References