U.S. patent application number 12/036846 was filed with the patent office on 2009-08-06 for antibodies to hada2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Reiner L. Gentz, Jing-Shan Hu, Henry Hongjun Ji, Paul A. Moore, Jian Ni, Craig A. Rosen.
Application Number | 20090197278 12/036846 |
Document ID | / |
Family ID | 26671124 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090197278 |
Kind Code |
A9 |
Moore; Paul A. ; et
al. |
August 6, 2009 |
Antibodies to hADA2
Abstract
Human polypeptides and DNA (RNA) encoding such polypeptides and
a procedure for producing such polypeptides by recombinant
techniques is disclosed. Also disclosed are methods for utilizing
such polypeptides for therapeutic purposes. Antagonists against
such polypeptides and their use as a therapeutic are also
disclosed. Also disclosed are diagnostic methods for detecting
disease which utilize the sequences and polypeptides.
Inventors: |
Moore; Paul A.; (North
Bethesda, MD) ; Gentz; Reiner L.; (Belo Horizonte,
BR) ; Ji; Henry Hongjun; (San Diego, CA) ; Ni;
Jian; (Germantown, MD) ; Hu; Jing-Shan;
(Mountain View, CA) ; Rosen; Craig A.;
(Laytonsville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20080227110 A1 |
September 18, 2008 |
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|
Family ID: |
26671124 |
Appl. No.: |
12/036846 |
Filed: |
February 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10453478 |
Jun 4, 2003 |
7335489 |
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12036846 |
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09417540 |
Oct 14, 1999 |
6639052 |
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10453478 |
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08705771 |
Aug 30, 1996 |
6054289 |
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09417540 |
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60002993 |
Aug 30, 1995 |
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Current U.S.
Class: |
435/7.1 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/387.9; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/7.1 ;
536/23.5; 435/320.1; 435/325; 435/69.1; 530/350; 530/387.9 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07H 21/04 20060101 C07H021/04; C12N 15/00 20060101
C12N015/00; C12N 5/00 20060101 C12N005/00; C12P 21/04 20060101
C12P021/04; C07K 16/00 20060101 C07K016/00 |
Claims
1. An isolated polynucleotide comprising a polynucleotide having at
least a 95% identity to a member selected from the group consisting
of: (a) a polynucleotide encoding a polypeptide comprising the
amino acid according to SEQ ID NO:12; (b) a polynucleotide encoding
a polypeptide comprising the amino acid according to SEQ ID NO:13;
(c) a polynucleotide encoding a polypeptide comprising the amino
acid according to SEQ ID NO:14; (d) a polynucleotide encoding a
polypeptide comprising the amino acid according to SEQ ID NO:15;
(e) a polynucleotide encoding a polypeptide comprising the amino
acid according to SEQ ID NO:16; (f) a polynucleotide encoding a
polypeptide comprising the amino acid according to SEQ ID NO:17;
(g) a polynucleotide encoding a polypeptide comprising the amino
acid according to SEQ ID NO:18; (h) a polynucleotide encoding a
polypeptide comprising the amino acid according to SEQ ID NO:19;
(i) a polynucleotide encoding a polypeptide comprising the amino
acid according to SEQ ID NO:20; (j) a polynucleotide encoding a
polypeptide comprising the amino acid according to SEQ ID NO:21;
(k) a polynucleotide encoding a polypeptide comprising the amino
acid according to SEQ ID NO:22; and (l) the complement of (a), (b),
(c), (d), (e), (f), (g), (h), (i), (j), and (k).
2. The isolated polynucleotide of claim 1 wherein said member is
(a).
3. The isolated polynucleotide of claim 1 wherein said member is
(b).
4. The isolated polynucleotide of claim 1 wherein said member is
(c).
5. The isolated polynucleotide of claim 1 wherein said member is
(d).
6. The isolated polynucleotide of claim 1 wherein said member is
(e).
7. The isolated polynucleotide of claim 1 wherein said member is
(f).
8. The isolated polynucleotide of claim 1 wherein said member is
(g).
9. The isolated polynucleotide of claim 1 wherein said member is
(h).
10. The isolated polynucleotide of claim 1 wherein said member is
(i).
11. The isolated polynucleotide of claim 1 wherein said member is
(j).
12. The isolated polynucleotide of claim 1 wherein said member is
(k).
13. The isolated polynucleotide of claim 1, wherein the
polynucleotide is DNA.
14. A method of making a recombinant vector comprising inserting
the isolated polynucleotide of claim 1 into a vector, wherein said
polynucleotide is DNA.
15. A recombinant vector comprising the polynucleotide of claim 1,
wherein the polynucleotide is DNA.
16. A recombinant host cell comprising the polynucleotide of claim
1, wherein said polynucleotide is DNA.
17. A method for producing a polypeptide comprising expressing from
the recombinant cell of claim 16 the polypeptide encoded by said
polynucleotide.
18. The isolated polynucleotide of claim 1 comprising the
nucleotide sequence which is a member selected from the group
consisting of: (a) a polynucleotide comprising the sequence
according to SEQ ID NO:1; (b) a polynucleotide comprising the
sequence according to SEQ ID NO:2; (c) a polynucleotide comprising
the sequence according to SEQ ID NO:3; (d) a polynucleotide
comprising the sequence according to SEQ ID NO:4; (e) a
polynucleotide comprising the sequence according to SEQ ID NO:5;
(f) a polynucleotide comprising the sequence according to SEQ ID
NO:6; (g) a polynucleotide comprising the sequence according to SEQ
ID NO:7; (h) a polynucleotide comprising the sequence according to
SEQ ID NO:8; (i) a polynucleotide comprising the sequence according
to SEQ ID NO:9; (j) a polynucleotide comprising the sequence
according to SEQ ID NO: 10; and (k) a polynucleotide comprising the
sequence according to SEQ ID NO:11.
19. An isolated polypeptide comprising a polypeptide having at
least a 95% identity to a member selected form the group consisting
of: (a) a polypeptide comprising the amino acid according to SEQ ID
NO:12; (b) a polypeptide comprising the amino acid according to SEQ
ID NO:13; (c) a polypeptide comprising the amino acid according to
SEQ ID NO:14; (d) a polypeptide comprising the amino acid according
to SEQ ID NO:15; (e) a polypeptide comprising the amino acid
according to SEQ ID NO:16; (f) a polypeptide comprising the amino
acid according to SEQ ID NO:17; (g) a polypeptide comprising the
amino acid according to SEQ ID NO:18; (h) a polypeptide comprising
the amino acid according to SEQ ID NO:19; (i) a polypeptide
comprising the amino acid according to SEQ ID NO:20; (j) a
polypeptide comprising the amino acid according to SEQ ID NO:21;
and (k) a polypeptide comprising the amino acid according to SEQ ID
NO:22.
20. An antibody that specifically binds the polypeptide of claim
19.
21. A method for identifying an antagonist of the polypeptide of
claim 19, comprising: (a) contacting a cell expressing said
polypeptide with a compound to be screened; and (b) determining
whether said compound acts as an antagonist of said
polypeptide.
22. A method for identifying an agonist of the polypeptide of claim
19, comprising: (a) contacting a cell expressing said polypeptide
with a compound to be screened; and (b) determining whether said
compound acts as an agonist of said polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 10/453,478, filed Jun. 4, 2003, which is a Divisional of U.S.
application Ser. No. 09/417,540, filed Oct. 14, 1999 (now U.S. Pat.
No. 6,639,052, issued Oct. 28, 2003), which is a Divisional of U.S.
application Ser. No. 08/705,771, filed Aug. 30, 1996 (now U.S. Pat.
No. 6,054,289, issued Apr. 25, 2000), which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/002,993, filed Aug. 30, 1995.
BACKGROUND OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production of such
polynucleotides and polypeptides. The invention also relates to
inhibiting the action of such polypeptides.
[0003] Identification and sequencing of human genes is a major goal
of modern scientific research. For example, by identifying genes
and determining their sequences, scientists have been able to make
large quantities of valuable human "gene products." These include
human insulin, interferon, Factor VIII, tumor necrosis factor,
human growth hormone, tissue plasminogen activator, and numerous
other compounds. Additionally, knowledge of gene sequences can
provide the key to treatment or cure of genetic diseases (such as
muscular dystrophy and cystic fibrosis).
[0004] In accordance with one aspect of the present invention,
there are provided novel mature polypeptides, as well as
biologically active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof. The polypeptides of the
present invention are of human origin.
[0005] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding the
polypeptides, including mRNAs, DNAs, cDNAs, genomic DNAs as well as
analogs and biologically active and diagnostically or
therapeutically useful fragments thereof.
[0006] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptides by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
nucleic acid sequence of the present invention, under conditions
promoting expression of said proteins and subsequent recovery of
said proteins.
[0007] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptides, or polynucleotides encoding such polypeptide for
therapeutic and diagnostic purposes.
[0008] In accordance with yet a further aspect of the present
invention, there is also provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to specifically
hybridize to the nucleic acid sequences.
[0009] In accordance with yet a further aspect of the present
invention, there are provided antibodies against such
polypeptides.
[0010] In accordance with another aspect of the present invention,
there are provided agonists to the polypeptides.
[0011] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used to inhibit the action of such polypeptides, for
therapeutic and diagnostic purposes.
[0012] In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases related to the under-expression of the polypeptides of the
present invention and mutations in the nucleic acid sequences
encoding such polypeptides.
[0013] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptides, or polynucleotides encoding such polypeptides, for in
vitro purposes related to scientific research, synthesis of DNA and
manufacture of DNA vectors.
[0014] In the case where the polypeptides of the present invention
are receptors, there are provided processes for using the receptor
to screen for receptor antagonists and/or agonists and/or receptor
ligands.
[0015] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
[0016] Table 1 sets forth information regarding identifying
polynucleotide clone numbers, identification of the polynucleotide
sequence which corresponds to the putative identification of the
polypeptide encoded by the polynucleotide, and cross-referencing to
the SEQ ID NOS. as set forth in the sequence listing.
[0017] Table 2 includes information regarding identifying
polypeptide numbers, identification of the SEQ ID NOS. of the
polypeptides, and cross-reference to the SEQ ID NO. which sets
forth the amino acid sequence which corresponds to a given
polypeptide in the sequence listing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the full length polynucleotide sequence (SEQ ID
NO: 1) of the HBGBA67X clone and correlates the coding region with
the derived amino acids (127 amino acids whose entire sequence (SEQ
ID NO: 12) is also shown for the full length amyloid-like protein
present in breast tissue.
[0019] FIGS. 2A-2B show the complete nucleotide (SEQ ID NO:2) and
amino acid sequence (SEQ ID NO: 13) of the hADA2 gene and
protein.
[0020] FIGS. 3A-3B show the full length sequence of the TFIId
homolog clone (SEQ ID NO:3) including the full length sequence of
the polynucleotide coding for TATA related factor (TRF) (SEQ ID
NO:14).
[0021] FIG. 4 shows full length cDNA (SEQ ID NO:4) and deduced
amino acid sequence (SEQ ID NO:15) of hRPB 11.
[0022] FIGS. 5A-5B show the full nucleotide sequence (SEQ ID NO:5)
of the IRF3 gene and amino acid sequence (SEQ ID NO: 16) resulting
protein. The predicted molecular weight of IRF3 is 47,087; the
predicted isoelectric is 5.06; and the net charge equals -14.
[0023] FIG. 6 shows individually the full length sequence (SEQ ID
NO:6) of the TM4SF gene, the coding region sequence portion and the
amino acid sequence (SEQ ID NO: 17) of the translation product
TM4SF.
[0024] FIGS. 7A-7B show the full length nucleotide sequence (SEQ ID
NO:7) of TNFR AF1 C1, the complete coding sequence region of the
full length sequence and the derived amino acid sequence (SEQ ID
NO:18) of the resulting protein.
[0025] FIG. 8 shows the full length sequence (SEQ ID NO:8), the
coding region sequence and the derived amino acid sequence (SEQ ID
NO:19) of the expression product protein of TM4SF (transmembrane 4
super family) CD53.
[0026] FIG. 9 shows the full length cDNA (SEQ ID NO:9) and the
resulting expression of the product protein (SEQ ID NO:20) of its
coding region for retenoid receptor gamma.
[0027] FIG. 10 shows the full length nucleotide sequence (SEQ ID
NO:10) (1237 bp) and the translation product (412 amino acid, SEQ
ID NO:21) resulting from the nucleotide sequence for the cytosolic
resiniferatoxin binding protein RBP-26.
[0028] FIG. 11 shows the nucleotide sequence (SEQ ID NO:11) for the
human protein (SEQ ID NO:22) kinase C inhibitor protein.
[0029] In accordance with an aspect of the present invention, there
are provided isolated nucleic acids (polynucleotides) which code
for mature polypeptides having the deduced amino acid sequences
shown in the FIGS. 1-11 or for the mature polypeptides encoded by
the cDNA of the clone deposited as ATCC.TM. Deposit No. 97242 on
Aug. 15, 1995 with the ATCC.TM., 10801 University Boulevard,
Manassas, Va. 20110-2209.
[0030] The polynucleotides of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown in SEQ ID NOS:1-11) or that of the deposited clone or may be
a different coding sequence which coding sequence, as a result of
the redundancy or degeneracy of the genetic code, encodes the same
mature polypeptide as the DNA of SEQ ID NOS:1-11 or the deposited
cDNA.
[0031] The polynucleotides which code for the mature polypeptides
of FIGS. 1-11 or for the mature polypeptides encoded by the
deposited cDNA may include, but are not limited to: only the coding
sequence for the mature polypeptide; the coding sequence for the
mature polypeptide and additional coding sequence such as a leader
or secretory sequence or a proprotein sequence; the coding sequence
for the mature polypeptide (and optionally additional coding
sequence) and non-coding sequence, such as introns or non-coding
sequence (SEQ ID NO:1) 5' and/or 3' of the coding sequence (SEQ ID
NO:2) for the mature polypeptide.
[0032] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0033] The present invention further relates to variants of the
hereinabove described polynucleotides which code for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequences of FIGS. 1-11 or the polypeptides encoded by the
cDNA of the deposited clone. The variant of the polynucleotide may
be a naturally occurring allelic variant of the polynucleotide or a
non-naturally occurring variant of the polynucleotide.
[0034] Thus, the present invention includes polynucleotides
encoding the same mature polypeptides as shown in FIG. 1 or the
same mature polypeptides encoded by the cDNA of the deposited clone
as well as variants of such polynucleotides which variants code for
a fragment, derivative or analog of the polypeptides of FIGS. 1-11
or the polypeptides encoded by the cDNA of the deposited clone.
Such nucleotide variants include deletion variants, substitution
variants and addition or insertion variants.
[0035] As hereinabove indicated, the polynucleotides may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequences shown in FIGS. 1-11 or of the coding sequences
of the deposited clone. As known in the art, an allelic variant is
an alternate form of a polynucleotide sequence which may have a
substitution, deletion or addition of one or more nucleotides,
which does not substantially alter the function of the encoded
polypeptide.
[0036] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a leader sequence which functions as a secretory sequence
for controlling transport of a polypeptide from the cell. The
polypeptide having a leader sequence is a preprotein and may have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides may also code for a
proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein remains.
[0037] Thus, for example, the polynucleotide of the present
invention may code for a mature protein, or for a protein having a
prosequence or for a protein having both a prosequence and a
presequence (leader sequence).
[0038] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., Cell, 37:767 (1984)).
[0039] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0040] Fragments of the full length genes of the present invention
may be used as hybridization probes for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promotor regions, exons and introns. An
example of a screen comprises isolating the coding region of one of
the genes by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0041] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 70%, preferably at least 90%, and more preferably at least
95% identity between the sequences. The present invention
particularly relates to polynucleotides which hybridize under
stringent conditions to the hereinabove-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization
will occur only if there is at least 95% and preferably at least
97% identity between the sequences. The polynucleotides which
hybridize to the hereinabove described polynucleotides in a
preferred embodiment encode polypeptides which either retain
substantially the same biological function or activity as the
mature polypeptide encoded by the cDNAs of FIGS. 1-11 (SEQ ID
NOS:1-11) or the deposited cDNA(s).
[0042] Alternatively, the polynucleotides may have at least 20
bases, preferably 30 bases, and more preferably at least 50 bases
which hybridize to a polynucleotide of the present invention and
which have an identity thereto, as hereinabove described, and which
may or may not retain activity. For example, such polynucleotides
may be employed as probes for the polynucleotides any of SEQ ID
NOS:1-11, for example, for recovery of the polynucleotide or as a
diagnostic probe or as a PCR primer.
[0043] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90% and more
preferably at least a 95% identity to polynucleotides which encode
the polypeptides of SEQ ID NOS:12-22, as well as fragments thereof,
which fragments have at least 30 bases and preferably at least 50
bases and to polypeptides encoded by such polynucleotides.
[0044] The deposit(s) referred to herein will be maintained under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Micro-organisms for purposes of Patent Procedure.
These deposits are provided merely as convenience to those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn. 112. The sequence of the polynucleotides
contained in the deposited materials, as well as the amino acid
sequence of the polypeptides encoded thereby, are incorporated
herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such
license is hereby granted.
[0045] The present invention further relates to polypeptides which
have the deduced amino acid sequence of SEQ ID NOS. 12-22 or which
have the amino acid sequences encoded by the deposited cDNAs, as
well as fragments, analogs and derivatives of such
polypeptides.
[0046] The terms "fragment", "derivative" and "analog" when
referring to the polypeptides of SEQ ID NOS. 12-22 or those encoded
by the deposited cDNA, means polypeptides which retain essentially
the same biological function or activity as such polypeptide. Thus,
an analog and derivative includes a proprotein which can be
activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
[0047] The polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides or synthetic polypeptides,
preferably recombinant polypeptides.
[0048] The fragments, derivatives or analogs of the polypeptides of
SEQ ID NOS. 12-22 or those encoded by the deposited cDNAs may be
(i) those in which one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue) and such substituted
amino acid residue may or may not be one encoded by the genetic
code, (ii) those in which one or more of the amino acid residues
includes a substituent group, (iii) those in which the mature
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol) or (iv) those in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0049] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0050] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0051] The polypeptides of the present invention include the
polypeptides of SEQ ID NOS: 12-22 (in particular the mature
polypeptides) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptides of SEQ
ID NOS: 12-22 and more preferably a 90% similarity (more preferably
a 90% identity) to the polypeptides of SEQ ID NOS: 12-22 and still
more preferably a 95% similarity (still more preferably a 95%
identity) to the individual polypeptides of SEQ ID NOS: 12-22 and
also include portions of such polypeptides with such portion of the
polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0052] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide.
[0053] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0054] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0055] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the present invention. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0056] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotides may be included in any one of a
variety of expression vectors for expressing the corresponding
polypeptide. Such vectors include chromosomal, nonchromosomal and
synthetic DNA sequences, e.g., derivatives of SV40; bacterial
plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived
from combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies. However,
any other vector may be used as long as it is replicable and viable
in the host.
[0057] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0058] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0059] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0060] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0061] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0062] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, psiX174, pBluescript.TM. SK, pbsks, pNH8A, pNH16a,
pNH18A, pNH46A (STRATAGENE.TM.); ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 (PHARMACIA.TM.); Eukaryotic: pWLNEO, pSV2CAT, pOG44,
pXT1, pSG (STRATAGENE.TM.) pSVK3, pBPV, pMSG, pSVL (PHARMACIA.TM.).
However, any other plasmid or vector may be used as long as they
are replicable and viable in the host.
[0063] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0064] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, Dibner and Battey, Basic Methods in
Molecular Biology, (1986)).
[0065] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0066] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0067] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0068] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product.
[0069] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0070] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC.TM. 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0071] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0072] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0073] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0074] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0075] The polypeptides can be recovered and purified from
recombinant cell cultures by 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. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0076] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
[0077] The amyloid-like gene and gene product may be employed as
part of a diagnostic process for the early detection of
pre-cancerous growth or cancer in the breast. The amyloid protein
forms amyloid fibrils which in turn are capable of attracting
calcium molecules leading to calcium deposition and calcification.
Micro-fibrils and micro-calcification caused by microinjury in the
breast tissue result in densified breast tissue which is an early
symptom detectable by mammography. The amyloid-like protein gene
according to the invention was isolated and recovered as a full
length gene by computer-assisted analysis of expression sequence
tag data basis from a primary breast cancer library, a normal
breast library, an activated monocyte library and an embryonic
library. The assembly of ESTs represents a full length gene which
is illustrated in FIG. 1. Full length human ADA2 nucleotide
sequence was isolated from a 12 week old early stage human primary
testes lambda library.
[0078] The expression levels of the amyloid-like protein according
to the invention may be detectable in the serum and/or ductal fluid
of the breast due to its secretory nature, thus the amyloid-like
protein may be employed as a target for detection in such breast
fluids. Further, examination of tissue samples from the breast for
increased levels of the amyloid protein according to the invention
may be helpful as part of an overall diagnostic regimen to screen
for abnormal breast tissue growth or for breast cancer.
[0079] The amyloid-like protein according to the invention is toxic
to surrounding breast cells which leads to apoptosis. The
deposition of this protein in the breast tissue may be an early
lesion for cancerous growth in the breast. Thus, this gene may be a
target for breast cancer diagnosis.
[0080] Human transcriptional regulator hADA2 is the human homolog
of a yeast factor identified as being important for mediating the
transcriptional activation properties of the Herpes Simplex
transactivator VP16. It is possible that being able to control the
activity of this factor (perhaps through anti-sense or screened
antagonists) will allow the regulation of specific viral and human
genes whose expression is controlled by this factor. This could
lead to the controlled regulation of certain medically important
genes. Furthermore, it is possible that disruption of this gene
could result in unregulated transcription leading to cancer, in
which case gene therapy would be medically important.
Administration of HADA2 via gene therapy may be employed to treat
cancer since disruption of the HADA2 gene results in unregulated
transcription. We have recently mapped the chromosomal location of
this gene to 17q12-21. The gene encoding the HADA2 protein was
isolated from a 12 week old human primary testes library.
[0081] Modulating the activity of the human transcription regulator
HADA2 may be employed to enhance or reduce the amount of a
particular gene product produced. For example, in the case of an
elevated level of a polypeptide the gene responsible may be
down-regulated by inhibiting HADA2. Likewise, if an up-regulation
of a gene product is desired, e.g., growth hormone, HADA2 may be
stimulated.
[0082] Human transcription regulator factor (hTRF) is a homolog of
the TATA Box Binding protein which plays a pivotal role in the
expression of all genes. The full length cDNA of TRF was isolated
by screening a human testes library. The hRPB11 gene was isolated
from a subtracted human pituitary library. It is possible that lack
or overexpression of this gene could lead to unregulated
transcription leading to cancer. The human transcription factor
hTRF may play a pivotal role in the expression of nearly all human
genes since it is thought to bind to the "TATA box" upstream of all
translated genes. Accordingly, modulation of hTRF, via gene
therapy, stimulation and antagonism may be employed to control gene
expression. Lack of hTRF may cause unregulated transcription which
may lead to cancer. Accordingly administration of hTRF protein, or
administration of the hTRF gene via gene therapy may be employed to
treat cancer.
[0083] The human RNA polymerase subunits hRPB8, hRPB10 and hRPB11
play vital roles in mRNA synthesis since they possess the catalytic
machinery for the formation of the 3'-5' phosphodiester bonds
between ribonucleoside triphosphates and respond to signals from
the multiple factors involved in regulating their function during
initiation and elongation of mRNA synthesis. These subunits are
able to support normal yeast cell growth in vivo. The coding region
in some flanking 5' and 3' UTR have been sequenced. The protein has
a predicted molecular weight of 13,293; an isoelectric point of
5.73 and is 117 residues long.
[0084] Accordingly, since the subunits are vital to mRNA synthesis,
their administration may be employed to up-regulate the expression
of certain genes and to down-regulate others as needed.
Administration may be via gene therapy. Abnormal cellular
proliferation, e.g., cancer, may be treated with the subunits since
lack of expression of these genes may lead to unregulated
transcription.
[0085] The human interferon regulatory factor IRF3 gene shows
strong homology to a group of transcription factors including IRF1
(Interferon Regulatory factor 1) and IRF2 (interferon Regulatory
factor 2) which are important in mediating the transcriptional
activation of interferon-alpha and -beta induced genes. It is
possible that this gene also is important in mediating the
transcriptional activation properties of interferon and that this
factor may have some of the properties associated with interferon
such as anti-viral activity. The human interferon regulatory factor
IRF3 is potentially important in regulating the transcriptional
activation of interferon-.alpha. and -.beta. genes. IRF3 may also
be important in mediating the transcriptional activation properties
of interferon. The IRF3 polypeptide may be employed as an
anti-viral agent. The administration of the IRF3 gene and its gene
product may be employed to enhance the expression of interferon
which has many medically important uses. The IRF3 gene was isolated
from a human adult retina library.
[0086] The TM4SF gene may be employed as a target for the
development of compounds to treat human T-cell leukemia virus type
I since several monoclonal-antibodies inhibitory to syncytium
formation targeted this TM4SF molecule.
[0087] The TM4SF gene may also be employed in the regulation of
cell growth. This gene may also be employed as an immunogen or
target to implement active and passive immunotherapy in patients
with cancer. The gene encoding TM4SF was isolated from a human
T-cell lymphoma library.
[0088] The TNFR-AF1, C1 gene and gene product may be employed to
regulate B-lymphocyte proliferation, immunoglobulin class-switching
and apoptosis. The TNFR-AF1 may also be employed to up-regulate the
biological activity of TNF which is known to regress tumors. The
gene encoding TNFR-AF1 C1 was isolated from an activated human
nutrophil library.
[0089] The TM4SF, CD53 gene and gene product may be employed to
regulate lymphoma cell growth and may also be employed to regulate
cell growth. The gene encoding TM4SF (transmembrane 4 super
family), CD53 was isolated from a human tumor pancreas library.
[0090] The retinoid X receptor .gamma. may be employed to treat
psoriasis and recalcitrincystic acne and cancer. This retinoid X
receptor y may also be employed to prevent a variety of
pre-malignant lesions of skin and mucous membranes. The receptor
may also be employed as a tumor suppressor. The receptor may also
be employed to stimulate cell proliferation, differentiation and
keratinization. The receptor may also be employed to treat X linked
adrenal hypoplasia and hypogonatropic hypoglonatism. The gene
encoding retinoid X receptor gamma was isolated from a human fetal
lung III library.
[0091] The cytosolic resiniferatoxin binding protein (RBP-26) may
be employed to reduce pain sensation due to its ability to
selectively block mechanoheat nociceptors and warm receptors of the
skin that are known to play a significant role in sensation of
pain. The gene encoding RBP-26 was isolated from a human
osteoclastoma stromal cell library.
[0092] The protein kinase C inhibitor protein has significant
medical application uses such as inhibiting tumor cell growth and
in regulating the many physiological functions that are mediated by
the activation of protein kinase C. The gene encoding the protein
kinase C inhibitor protein was isolated from a human corpus colosum
library.
[0093] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to human disease.
[0094] This invention provides a method for identification of the
receptors for the polypeptides listed in Table 1. The gene encoding
the receptor 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)). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the
respective polypeptide, 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 proteins. Transfected cells which
are grown on glass slides are exposed to labeled protein. The
protein can be labeled by a variety of means including iodination
or inclusion of a recognition site for a site-specific protein
kinase. 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
clone that encodes the putative receptor.
[0095] As an alternative approach for receptor identification,
labeled protein can be photoaffinity linked with cell membrane or
extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and exposed to X-ray
film. The labeled complex containing the protein-receptor 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
gene encoding the putative receptor.
[0096] This invention provides a method of screening compounds to
identify those which enhance (agonists) or block (antagonists)
interaction of protein to receptor. An agonist is a compound which
increases the natural biological functions, while antagonists
eliminate such functions. As an example, a mammalian cell or
membrane preparation expressing the receptor would be incubated
with labeled protein in the presence of the drug. The ability of
the drug to enhance or block this interaction could then be
measured.
[0097] Alternatively, the response of a known second messenger
system following interaction of protein and receptor would be
measured and compared in the presence or absence of the drug. Such
second messenger systems include but are not limited to, cAMP
guanylate cyclase, ion channels or phosphoinositide hydrolysis.
[0098] In the case where the polypeptides of the present invention
are receptor polypeptides, the present invention also relates to
methods for determining whether a ligand can bind to the receptor
which comprises transfecting a cell population (one presumed not to
contain a receptor) with the appropriate vector expressing the
receptor, such that the cell will now express the receptor. A
suitable response system is obtained by transfection of the DNA
into a suitable host containing the desired second messenger
pathways including cAMP, ion channels, phosphoinositide kinase, or
calcium response. Such a transfection system provides a response
system to analyze the activity of various ligands exposed to the
cell. Ligands chosen could be identified through a rational
approach by taking known ligands that interact with similar types
of receptors or using small molecules, cell supernatants or
extracts or natural products.
[0099] The present invention also relates to an assay for
identifying potential antagonists. An example of such an assay
combines the protein and a potential antagonist with membrane-bound
receptors or recombinant receptors under appropriate conditions for
a competitive inhibition assay. The protein can be labeled, such as
by radio activity, such that the number of molecules bound to the
receptor can determine the effectiveness of the potential
antagonist.
[0100] The polypeptides listed in Table 1 of the present invention
which have putatively been identified as receptors may be employed
in a process for screening for antagonists and/or agonists for the
receptor.
[0101] In general, such screening procedures involve providing
appropriate cells which express the receptor on the surface
thereof. In particular, a polynucleotide encoding the receptor of
the present invention is employed to transfect cells to thereby
express the receptor. Such transfection may be accomplished by
procedures as hereinabove described.
[0102] One such screening procedure involves the use of
melanophores which are transfected to express the receptor of the
present invention. Such a screening technique is described in PCT
WO 92/01810 published Feb. 6, 1992.
[0103] Thus, for example, such assay may be employed for screening
for a receptor antagonist by contacting the melanophore cells which
encode the receptor with both the receptor ligand and a compound to
be screened. Inhibition of the signal generated by the ligand
indicates that a compound is a potential antagonist for the
receptor, i.e., inhibits activation of the receptor.
[0104] The screen may be employed for determining an agonist by
contacting such cells with compounds to be screened and determining
whether such compound generates a signal, i.e., activates the
receptor.
[0105] Other screening techniques include the use of cells which
express the receptor (for example, transfected CHO cells) in a
system which measures extracellular pH changes caused by receptor
activation, for example, as described in Science, volume 246, pages
181-296 (October 1989). For example, potential agonists or
antagonists may be contacted with a cell which expresses the
receptor and a second messenger response, e.g. signal transduction
or pH changes, may be measured to determine whether the potential
agonist or antagonist is effective.
[0106] Another such screening technique involves introducing RNA
encoding the receptor into xenopus oocytes to transiently express
the receptor. The receptor oocytes may then be contacted in the
case of antagonist screening with the receptor ligand and a
compound to be screened, followed by detection of inhibition of a
calcium signal.
[0107] Another screening technique involves expressing the receptor
in which the receptor is linked to a phospholipase C or D. As
representative examples of such cells, there may be mentioned
endothelial cells, smooth muscle cells, embryonic kidney cells,
etc. The screening for an antagonist or agonist may be accomplished
as hereinabove described by detecting activation of the receptor or
inhibition of activation of the receptor from the phospholipase
second signal.
[0108] Another method involves screening for antagonists by
determining inhibition of binding of labeled ligand to cells which
have the receptor on the surface thereof. Such a method involves
transfecting a eukaryotic cell with DNA encoding the receptor such
that the cell expresses the receptor on its surface and contacting
the cell with a potential antagonist in the presence of a labeled
form of a known ligand. The ligand can be labeled, e.g., by
radioactivity. The amount of labeled ligand bound to the receptors
is measured, e.g., by measuring radioactivity of the receptors. If
the potential antagonist binds to the receptor as determined by a
reduction of labeled ligand which binds to the receptors, the
binding of labeled ligand to the receptor is inhibited.
[0109] The present invention also provides a method for determining
whether a ligand not known to be capable of binding to a receptor
can bind to such receptor which comprises contacting a mammalian
cell which expresses a receptor with the ligand under conditions
permitting binding of ligands to the receptor, detecting the
presence of a ligand which binds to the receptor and thereby
determining whether the ligand binds to the receptor. The systems
hereinabove described for determining agonists and/or antagonists
may also be employed for determining ligands which bind to the
receptor.
[0110] In general, antagonists for receptors which are determined
by screening procedures may be employed for a variety of
therapeutic purposes. For example, such antagonists have been
employed for treatment of hypertension, angina pectoris, myocardial
infarction, ulcers, asthma, allergies, psychoses, depression,
migraine, vomiting, and benign prostatic hypertrophy.
[0111] Agonists for receptors are also useful for therapeutic
purposes, such as the treatment of asthma, Parkinson's disease,
acute heart failure, hypotension, urinary retention, and
osteoporosis.
[0112] Potential antagonists against the polypeptides of the
present invention include an antibody, or in some cases, an
oligopeptide, which binds to the polypeptide. Alternatively, a
potential antagonist may be a closely related protein which binds
to the receptors of the polypeptide, however, they are inactive
forms of the polypeptide and thereby inhibit the action of the
polypeptides.
[0113] Another potential antagonist is an antisense construct
prepared using antisense technology. Antisense technology can be
used to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to DNA or RNA. For example, the 5' coding portion
of the polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is 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 (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)),
thereby preventing transcription and the production of the
polypeptide. The antisense RNA oligonucleotide hybridizes to the
mRNA in vivo and blocks translation of the mRNA molecule into the
polypeptide (Antisense--Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the
protein.
[0114] Potential antagonists include a small molecule which binds
to and occupies the active site of the polypeptide or to the
receptor where the polypeptide of the present invention is a
receptor, thereby making it inaccessible to substrate such that
normal biological activity is prevented. Examples of small
molecules include but are not limited to small peptides or
peptide-like molecules.
[0115] Another potential antagonist includes a soluble form of the
receptor polypeptides, e.g. a fragment of the receptor, which binds
to the ligand and prevents the ligand from interacting with
membrane bound receptors.
[0116] Antagonists to the human transcription regulator hADA2 may
be employed to regulate the expression of Herpes simplex
transactivator VP 16, since hADA2 mediates its transcriptional
activation properties. Many medically important genes may also be
regulated by the antagonism of hADA2.
[0117] Antagonists to TATA related factor (TRF) may be employed to
control general protein expression and for the regulation of the
expression of specific important gene groups.
[0118] Antagonists to RNA polymerase subunits HRPB8, HRPB10 and
HRPB11 may be employed to treat cancer since over expression of
these subunits may lead to unregulated transcription.
[0119] Antagonists to interferon related factor-3 (IRF-3) may be
employed to down regulate the overexpression of interferon with its
adverse effects.
[0120] Antagonists to TM4SF may be employed to inhibit tumor
growth.
[0121] Antagnoists to TNFR AF 1, C1 may be employed to inhibit
inflammation and apoptosis.
[0122] Antagnoists to TM4SF (transmembrane 4 super family) CD53 may
be employed to inhibit certain leukemias.
[0123] Antagonists to the retinoid X receptor .gamma. may be
employed to treat psoriasis and inflammation. The antagonists may
also be employed to prevent and/or treat hyperplasia and tumors in
the lung, breast and other tissues.
[0124] Antagonists to protein kinase C inhibitor protein may be
employed to inhibit the activation function of protein kinase
C.
[0125] The antagonists may be employed therapeutically in a
composition with a pharmaceutically acceptable carrier, e.g., as
hereinafter described.
[0126] The polypeptides of the present invention and agonists and
antagonists may be employed in combination with a suitable
pharmaceutical carrier. Such compositions comprise a
therapeutically effective amount of the polypeptide or agonist or
antagonist, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0127] 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.
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 polypeptides of the present
invention or agonists or antagonists may be employed in conjunction
with other therapeutic compounds.
[0128] The pharmaceutical compositions may be administered in a
convenient manner such as by the oral, topical, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes. The pharmaceutical compositions are
administered in an amount which is effective for treating and/or
prophylaxis of the specific indication. In general, they are
administered in an amount of at least about 10 .mu.g/kg body weight
and in most cases they will be administered in an amount not in
excess of about 8 mg/kg body weight per day. In most cases, the
dosage is from about 10 .mu.g/kg to about 1 mg/kg body weight
daily, taking into account the routes of administration, symptoms,
etc.
[0129] The polypeptides and agonists and antagonists which are
polypeptides may also be employed in accordance with the present
invention by expression of such polypeptides in vivo, which is
often referred to as "gene therapy."
[0130] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) encoding a polypeptide 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 and are apparent from the teachings herein. For example, cells
may be engineered by the use of a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention.
[0131] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by, for example, procedures known in the art.
For example, a packaging cell is transduced with a retroviral
plasmid vector containing RNA encoding a polypeptide of the present
invention such that the packaging cell now produces infectious
viral particles containing the gene of interest. These producer
cells may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention.
[0132] Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0133] The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller et al.,
Biotechniques, 7(9) .delta. 980-990 (1989), or any other promoter
(e.g., cellular promoters such as eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and
.beta.-actin promoters). Other viral promoters which may be
employed include, but are not limited to, adenovirus promoters,
thymidine kinase (TK) promoters, and B19 parvovirus promoters. The
selection of a suitable promoter will be apparent to those skilled
in the art from the teachings contained herein.
[0134] The nucleic acid sequence encoding the polypeptide of the
present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not
limited to, 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 (including the modified retroviral LTRs hereinabove
described); the .beta.-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter which
controls the gene encoding the polypeptide.
[0135] 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 PES01, PA317, .psi.-2, .psi.-AM, PA12, T19-14X,
VT-19-17-H2, .psi.CRE, .psi.CRIP, 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.
[0136] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0137] This invention is also related to the use of the DNA (RNA)
sequences diagnostically. Detection of a mutated form of sequences
will allow a diagnosis of a disease or a susceptibility to a
disease which results from under-expression of the protein.
[0138] Individuals carrying mutations in a human gene of the
present invention may be detected at the DNA level by a variety of
techniques. Nucleic acids for diagnosis may be obtained from a
patient's cells, such as from blood, urine, saliva, tissue biopsy
and autopsy material. The genomic DNA may be used directly for
detection or may be amplified enzymatically by using PCR (Saiki et
al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may
also be used for the same purpose. As an example, PCR primers
complementary to the nucleic acid encoding the protein can be used
to identify and analyze mutations. For example, deletions and
insertions can be detected by a change in size of the amplified
product in comparison to the normal genotype. Point mutations can
be identified by hybridizing amplified DNA to radiolabeled RNA or
alternatively, radiolabeled antisense DNA sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase A digestion or by differences in melting temperatures.
[0139] Sequence differences between the reference gene and genes
having mutations may be revealed by the direct DNA sequencing
method. In addition, cloned DNA segments may be employed as probes
to detect specific DNA segments. The sensitivity of this method is
greatly enhanced when combined with PCR. For example, a sequencing
primer is used with double-stranded PCR product or a
single-stranded template molecule generated by a modified PCR. The
sequence determination is performed by conventional procedures with
radiolabeled nucleotide or by automatic sequencing procedures with
fluorescent-tags.
[0140] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230:1242 (1985)).
[0141] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., PNAS, USA,
85:4397-4401 (1985)).
[0142] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting of genomic DNA.
[0143] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0144] The present invention also relates to a diagnostic assay for
detecting altered levels of the polypeptides of the present
invention and soluble form of the receptor polypeptides of the
present invention, in various tissues since an over-expression of
the proteins compared to normal control tissue samples can detect
the presence of a disease. Assays used to detect levels of protein
in a sample derived from a host are well-known to those of skill in
the art and include radioimmunoassays, competitive-binding assays,
Western Blot analysis and preferably an ELISA assay. An ELISA assay
initially comprises preparing an antibody specific to the antigen,
preferably a monoclonal antibody. In addition a reporter antibody
is prepared against the monoclonal antibody. To the reporter
antibody is attached a detectable reagent such as radioactivity,
fluorescence or in this example a horseradish peroxidase enzyme. A
sample is now removed from a host and incubated on a solid support,
e.g. a polystyrene dish, that binds the proteins in the sample. Any
free protein binding sites on the dish are then covered by
incubating with a non-specific protein such as bovine serum
albumin. Next, the monoclonal antibody is incubated in the dish
during which time the monoclonal antibodies attach to any proteins
attached to the polystyrene dish. All unbound monoclonal antibody
is washed out with buffer. The reporter antibody linked to
horseradish peroxidase is now placed in the dish resulting in
binding of the reporter antibody to any monoclonal antibody bound
to the protein of interest. Unattached reporter antibody is then
washed out. Peroxidase substrates are then added to the dish and
the amount of color developed in a given time period is a
measurement of the amount of protein present in a given volume of
patient sample when compared against a standard curve.
[0145] A competition assay may be employed wherein antibodies
specific to the protein is attached to a solid support and labeled
protein and a sample derived from the host are passed over the
solid support and the amount of label detected attached to the
solid support can be correlated to a quantity of the protein in the
sample.
[0146] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0147] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region of the gene is used to rapidly select
primers that do not span more than one exon in the genomic DNA,
thus complicating the amplification process. 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 primer will yield an amplified
fragment.
[0148] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0149] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA having at least 50 or 60 bases. For a review of this
technique, see Verma et al., Human Chromosomes: a Manual of Basic
Techniques, Pergamon Press, New York (1988).
[0150] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0151] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0152] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0153] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0154] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0155] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72, 1983), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0156] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0157] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0158] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0159] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted bases, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0160] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0161] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8:4057 (1980).
[0162] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0163] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units of
T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0164] Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
EXAMPLE 1
Bacterial Expression and Purification of the Proteins
[0165] The DNA sequence encoding any of the proteins, is initially
amplified using PCR oligonucleotide primers corresponding to the 5'
sequences of the processed protein (minus the signal peptide
sequence) and the vector sequences 3' to the gene. Additional
nucleotides corresponding to the DNA sequence are added to the 5'
and 3' sequences respectively. The 5' oligonucleotide primer may
contain, for example, a restriction enzyme site followed by
nucleotides of coding sequence starting from the presumed terminal
amino acid of the processed protein. The 3' sequence may, for
example, contain complementary sequences to a restriction enzyme
site and also be followed by nucleotides of the nucleic acid
sequence encoding the protein of interest. The restriction enzyme
sites correspond to the restriction enzyme sites on a bacterial
expression vector, for example, pQE-9 (Qiagen, Inc. Chatsworth,
Calif.). pQE-9 encodes antibiotic resistance (Amp.sup.r), a
bacterial origin of replication (ori), an IPTG-regulatable promoter
operator (P/O), a ribosome binding site (RBS), a 6-His tag and
restriction enzyme sites. pQE-9 is then digested with the
restriction enzymes corresponding to restriction enzyme sites
contained in he primer sequences. The amplified sequences are
ligated into pQE-9 and inserted in frame with the sequence encoding
for the histidine tag and the RBS. The ligation mixture is then
used to transform an E. coli strain, for example, M15/rep 4
(Qiagen) by the procedure described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).
M15/rep4 contains multiple copies of the plasmid pREP4, which
expresses the lacI 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. Clones containing the desired constructs are grown
overnight (O/N) in liquid culture in LB media supplemented with
both Amp (100 ug/ml) and Kan (25 ug/ml). The O/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 lacI repressor, clearing the P/0 leading to
increased gene expression. Cells are grown an extra 3 to 4 hours.
Cells are then harvested by centrifugation. The cell pellet is
solubilized in the chaotropic agent 6 Molar Guanidine HCl. After
clarification, solubilized protein is purified from this solution
by chromatography on a Nickel-Chelate column under conditions that
allow for tight binding by proteins containing the 6-His tag
(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). The
protein is eluted from the column in 6 molar guanidine HCl pH 5.0
and for the purpose of renaturation adjusted to 3 molar guanidine
HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2
mmolar glutathione (oxidized). After incubation in this solution
for 12 hours the protein is dialyzed to 10 mmolar sodium
phosphate.
EXAMPLE 2
Cloning and Expression of the Proteins Using the Baculovirus
Expression System
[0166] The DNA sequence encoding one of the full length proteins,
is amplified using PCR oligonucleotide primers corresponding to the
5' and 3' sequences of the gene.
[0167] The 5' primer may contain a restriction enzyme site and be
followed by a number of nucleotides resembling an efficient signal
for the initiation of translation in eukaryotic cells (Kozak, J.
Mol. Biol., 196:947-950 (1987) which is just behind the first few
nucleotides of the gene of interest.
[0168] The 3' primer may also contain a restriction endonuclease
and have extra nucleotides which are complementary to the 3'
non-translated sequence of the gene. The amplified sequences are
isolated from a 1% agarose gel using a commercially available kit
("GENECLEAN.TM.," BIO 101 Inc., La Jolla, Calif.). The fragment is
then digested with the endonucleases and purified again on a 1%
agarose gel. This fragment is designated F2.
[0169] A vector, for example, pA2 or pRG1 (modification of pVL941
vector, discussed below) may be used for the expression of the
protein using the baculovirus expression system (for review see:
Summers, M. D. and Smith, G. E. 1987, A manual of methods for
baculovirus vectors and insect cell culture procedures, Texas
Agricultural Experimental Station Bulletin No. 1555). These vectors
contain the strong polyhedrin promoter of the Autographa
californica nuclear polyhedrosis virus (AcMNPV) followed by the
recognition sites for the respective restriction endonucleases. The
polyadenylation site of the simian virus (SV)40 is used for
efficient polyadenylation. For an easy selection of recombinant
virus the beta-galactosidase gene from E. coli is inserted in the
same orientation as the polyhedrin promoter followed by the
polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the
cell-mediated homologous recombination of co-transfected wild-type
viral DNA. Many other baculovirus vectors could be used in place of
pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers,
M. D., Virology, 170:31-39).
[0170] The plasmid is digested with the restriction enzymes and
dephosphorylated using calf intestinal phosphatase by procedures
known in the art. The DNA is then isolated from a 1% agarose gel
using the commercially available kit ("GENECLEAN.TM." BIO 101 Inc.,
La Jolla, Calif.). This vector DNA is designated V2.
[0171] Fragment F2 and the dephosphorylated plasmid V2 are ligated
with T4 DNA ligase. An E. coli strain, for example, HB101 cells are
then transformed and bacteria which contain the recombinant plasmid
are identified using the restriction enzymes. The sequence of the
cloned fragment is confirmed by DNA sequencing.
[0172] 5 .mu.g of the plasmid is co-transfected with 1.0 .mu.g of a
commercially available linearized baculovirus ("BaculoGold.TM.
baculovirus DNA", Pharmingen, San Diego, Calif.) using the
lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,
84:7413-7417 (1987)).
[0173] 1 .mu.g of BaculoGold.TM. virus DNA and 5 .mu.g of the
plasmid are mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 .mu.l LIPOFECTIN.TM. plus
90 .mu.l Grace's medium are added, mixed and incubated for 15
minutes at room temperature. Then the transfection mixture is added
drop-wise to the 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 rocked back and forth to mix the newly added solution.
The plate was then incubated for 5 hours at 27.degree. C. After 5
hours the transfection solution is removed from the plate and 1 ml
of Grace's insect medium supplemented with 10% fetal calf serum is
added. The plate is put back into an incubator and cultivation
continued at 27.degree. C. for four days.
[0174] After four days the supernatant is collected and a plaque
assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies
Inc., Gaithersburg) is used which allows an easy isolation of blue
stained plaques. (A detailed description of a "plaque assay" can
also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0175] Four days after the serial dilution the virus is added to
the cells and blue stained plaques are picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses is
then resuspended in an Eppendorf tube containing 200 .mu.l of
Grace's medium. The agar is removed by a brief centrifugation and
the supernatant 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 stored
at 4.degree. C.
[0176] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus at a multiplicity of infection (MOI) of 2. Six hours
later the medium is removed and replaced with SF900 II medium minus
methionine and cysteine (Life Technologies Inc., Gaithersburg). 42
hours later 5 .mu.Ci of 35 S-methionine and 5 .mu.Ci.sup.35 S
cysteine (Amersham) are added. The cells are further incubated for
16 hours before they are harvested by centrifugation and the
labelled proteins visualized by SDS-PAGE and autoradiography.
EXAMPLE 3
Expression of Recombinant Protein in COS Cells
[0177] The expression of plasmid, protein-HA is derived from a
vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of
replication, 2) ampicillin resistance gene, 3) E. coli replication
origin, 4) CMV promoter followed by a polylinker region, an SV40
intron and polyadenylation site. A DNA fragment encoding the entire
precursor and a HA tag fused in frame to its 3' end is cloned into
the polylinker region of the vector, therefore, the recombinant
protein expression is directed under the CMV promoter. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein as previously described (I. Wilson, H. Niman, R. Heighten,
A Cherenson, Connolly, and Lerner, Cell 37:767, (1984)). The
infusion of HA tag to the target protein allows easy detection of
the recombinant protein with an antibody that recognizes the HA
epitope.
[0178] The DNA sequence encoding the protein is constructed by PCR
using two primers, .delta. 5' primer containing a restriction
enzyme site followed by a number of nucleotides of the coding
sequence starting from the initiation codon, and a 3' primer also
containing complementary sequences to a restriction site,
translation stop codon, HA tag and the last few nucleotides of the
coding sequence (not including the stop codon). Therefore, the PCR
product contains restriction enzyme sites, coding sequence followed
by HA tag fused in frame, a translation termination stop codon next
to the HA tag, and the other restriction enzyme site. The PCR
amplified DNA fragment and the vector, pcDNAI/Amp, are digested
with appropriate restriction enzymes and ligated. The ligation
mixture is transformed into an E. coli strain, for example, SURE
(Stratagene Cloning Systems, La Jolla, Calif.) and the transformed
culture is plated on ampicillin media plates and resistant colonies
are selected. Plasmid DNA is isolated from transformants and
examined by restriction analysis for the presence of the correct
fragment. For expression of the recombinant protein, COS cells are
transfected with the expression vector by DEAE-DEXTRAN method (J.
Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory Press, (1989)). The expression of
the HA protein is detected by radiolabelling and
immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)).
Cells are labelled for 8 hours with 35 S-cysteine two days post
transfection. Culture media is then collected and cells are lysed
with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1%
NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767
(1984)). Both cell lysate and culture media are precipitated with
an HA specific monoclonal antibody. Proteins precipitated are
analyzed on 15% SDS-PAGE gels.
EXAMPLE 4
.beta. Isolation of a Selected Clone From the Deposited cDNA
Library
[0179] Two approaches are used to isolate a particular gene out of
the deposited cDNA library.
[0180] In the first, a clone is isolated directly by screening the
library using an oligonucleotide probe. To isolate a particular
gene, a specific oligonucleotide with 30-40 nucleotides is
synthesized using an Applied Biosystems DNA synthesizer according
to a fragment of the gene sequence. The oligonucleotide is labeled
with 32 P-ATP using T4 polynucleotide kinase and purified according
to the standard protocol (Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y.,
1982). The Lambda cDNA library deposited is plated on 1.5% agar
plate to the density of 20,000-50,000 pfu/150 mm plate. These
plates are screened using Nylon membranes according to the standard
phage screening protocol (STRATAGENE.TM., 1993). Specifically, the
Nylon membrane with denatured and fixed phage DNA is prehybridized
in 6.times.SSC, 20 mM NaH.sub.2 PO.sub.4, 0.4% SDS,
5.times.Denhardt's 500 .mu.g/ml denatured, sonicated salmon sperm
DNA; and 6.times.SSC, 0.1% SDS. After one hour of prehybridization,
the membrane is hybridized with hybridization buffer 6.times.SSC,
20 mM NaH.sub.2 PO.sub.4, 0.4% SDS, 500 ug/ml denatured, sonicated
salmon sperm DNA with 1.times.10.sup.6 cpm/ml 32 P-probe overnight
at 42.degree. C. The membrane is washed at 45-50.degree. C. with
washing buffer 6.times.SSC, 0.1% SDS for 20-30 minutes dried and
exposed to Kodak X-ray film overnight. Positive clones are isolated
and purified by secondary and tertiary screening. The purified
clone is sequenced to verify its identity to the fragment
sequence.
[0181] An alternative approach to screen the deposited cDNA library
is to prepare a DNA probe corresponding to the entire sequence. To
prepare a probe, two oligonucleotide primers of 17-20 nucleotides
derived from both ends of the sequence are synthesized and
purified. These two oligonucleotide are used to amplify the probe
using the cDNA library template. The DNA template is prepared from
the phage lysate of the deposited cDNA library according to the
standard phage DNA preparation protocol (Maniatis et al.). The
polymerase chain reaction is carried out in 25 .mu.l of reaction
mixture with 0.5 ug of the above cDNA template. The reaction
mixture is 1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each
of dATP, dCTP, dGTP, dTTP, 25 .mu.mol 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 the
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 probe by subcloning and sequencing the DNA
product. The probe is labeled with the Multiprime DNA Labelling
System (Amersham) at a specific activity <1.times.10.sup.9
dpm/.mu.g. This probe is used to screen the deposited lambda cDNA
library according to Stratagene's protocol. Hybridization is
carried out with 5.times.TEN (20.times.TEN:0.3M Tris-HCl pH 8.0,
0.02M EDTA and 3M NaCl), 5.times.Denhardts, 0.5% sodium
pyrophosphate, 0.1% SDS, 0.2 mg/ml heat denatured salmon sperm DNA
and 1.times.10.sup.6 cpm/ml of [32 P]-labeled probe at 55.degree.
C. for 12 hours. The filters are washed in 0.5.times.TEN at room
temperature for 20-30 min., then at 55.degree. C. for 15 min. The
filters are dried and autoradiographed at -70.degree. C. using
Kodak XAR-5 film. The positive clones are purified by secondary and
tertiary screening. The sequence of the isolated clone are verified
by DNA sequencing.
[0182] General procedures for obtaining complete sequences from
probes are summarized as follows:
[0183] Procedure
[0184] Selected human DNA from a probe corresponding to part of the
human gene is purified e.g., by endonuclease digestion using EcoRI,
gel electrophoresis, and isolation of the probe sequence by removal
from low melting agarose gel. The isolated insert DNA, is
radiolabeled e.g., with .sup.32P labels, preferably by nick
translation or random primer labeling. The labeled probe insert is
used as a probe to screen a lambda phage cDNA library or a plasmid
cDNA library. Colonies containing genes related to the probe cDNA
are identified and purified by known purification methods. The ends
of the newly purified genes are nucleotide sequenced to identify
full length sequences. Complete sequencing of full length genes is
then performed by Exonuclease III digestion or primer walking.
Northern blots of the mRNA from various tissues using at least part
of the EST clone as a probe can optionally be performed to check
the size of the mRNA against that of the purported full length
cDNA.
EXAMPLE 5
Expression Via Gene Therapy
[0185] 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. This is
then incubated at 37.degree. C. for approximately one week. 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.
[0186] pMV-7 (Kirschmeier 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.
[0187] The cDNA encoding a polypeptide of the present invention is
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively. The 5' primer containing an EcoRI site and
the 3' primer further 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 used to transform bacteria HB101, which are then plated onto
agar-containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
[0188] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagle's 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 are 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).
[0189] 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.
[0190] 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.
[0191] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
TABLE-US-00001 TABLE 1 PUTATIVE CLONE NO. IDENTIFICATION SEQ ID NO.
HBGBA67 amyloid-like protein 1 present in breast HE2CB95 hADA2 2
HTEAZ96 TRF 3 HPTIK55 hRPB11 4 HARA063 IRF3 5 HLTAH80 TM4SF 6
HNFBT92 TNFR AF1, C1 7 HTPBA27 TM4SF, CD53 8 HLHAR55 Retinoid X
Receptor 9 HSRDG78 RBP-26 10 HCCAA03 Protein kinase C 11 inhibitor
protein
TABLE-US-00002 TABLE 2 PUTATIVE CLONE NO. IDENTIFICATION SEQ ID NO.
HBGBA67 amyloid-like protein 12 present in breast HE2CB95 hADA2 13
HTEAZ96 TRF 14 HPTIK55 hRPB11 15 HARA063 IRF3 16 HLTAH80 TM4SF 17
HNFBT92 TNFR AF1, C1 18 HTPBA27 TM4SF, CD53 19 HLHAR55 Retinoid X
Receptor 20 HSRDG78 RBP-26 21 HCCAA03 Protein kinase C 22 inhibitor
protein
Sequence CWU 1
1
* * * * *