U.S. patent application number 11/985824 was filed with the patent office on 2008-10-02 for methods and materials relating to novel stem cell growth factor-like polypeptides and polynucleotides.
Invention is credited to Cheng-Chi Chao, Radoje T. Drmanac, Ivan Labat, Nancy Mize, Mitsuo Nishikawa, Y. Tom Tang.
Application Number | 20080241882 11/985824 |
Document ID | / |
Family ID | 46280000 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241882 |
Kind Code |
A1 |
Tang; Y. Tom ; et
al. |
October 2, 2008 |
Methods and materials relating to novel stem cell growth
factor-like polypeptides and polynucleotides
Abstract
The invention provides novel polynucleotides and polypeptides
encoded by such polynucleotides and mutants or variants thereof
that correspond to a novel human stem cell growth factor-like
protein. These polynucleotides comprise nucleic acid sequences
isolated from cDNA libraries from human testis cells (Hyseq clone
identification numbers 2880984 and 2881695), from human fetal skin
(Hyseq clone identification number 15375176), adult spleen (Hyseq
clone identification number 14856094), and human endothelial cells
(Hyseq clone identification numbers 13804756, 13687487, 13804756).
Other aspects of the invention include vectors containing processes
fro producing novel human stem cell growth factor-like
polypeptides, and antibodies specific for such polypeptides.
Inventors: |
Tang; Y. Tom; (San Jose,
CA) ; Labat; Ivan; (Mountain View, CA) ;
Drmanac; Radoje T.; (Palo Alto, CA) ; Mize;
Nancy; (Mountain View, CA) ; Nishikawa; Mitsuo;
(Gunma, JP) ; Chao; Cheng-Chi; (Cupertino,
CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD, SUITE 230
PALO ALTO
CA
94303
US
|
Family ID: |
46280000 |
Appl. No.: |
11/985824 |
Filed: |
November 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10968674 |
Oct 19, 2004 |
7319141 |
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11985824 |
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09894912 |
Jun 28, 2001 |
6824973 |
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10968674 |
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09757562 |
Jan 9, 2001 |
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09894912 |
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09543774 |
Apr 5, 2000 |
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09757562 |
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09496914 |
Feb 3, 2000 |
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09543774 |
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60282397 |
Apr 5, 2001 |
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60215733 |
Jun 28, 2000 |
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60266614 |
Feb 5, 2001 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 48/00 20130101; C12N 9/16 20130101; A01K 2217/05 20130101;
C07K 14/475 20130101; C07K 16/00 20130101; A61P 43/00 20180101;
Y10S 435/81 20130101; C12Y 304/21006 20130101; C12N 9/6432
20130101; A61K 38/00 20130101; Y10S 435/975 20130101 |
Class at
Publication: |
435/69.1 ;
536/23.5; 435/320.1; 435/325 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 5/06 20060101 C12N005/06 |
Claims
1. An isolated polynucleotide encoding a polypeptide with stem cell
growth factor activity, wherein said polypeptide comprises the
amino acid sequence of SEQ ID NO:13, 16, 32 or 34, or the mature
protein portion thereof, or a polypeptide with at least 98%
sequence identity thereto.
2. The polynucleotide of claim 1, wherein the polynucleotide
encodes a polypeptide that does not consist of the amino acid
sequence of SEQ ID NO:48.
3. The polynucleotide of claim 1, wherein the polypeptide encoded
by the polynucleotide comprises of the mature protein portion of
SEQ ID NO:13, 16, 32 or 34. The polynucleotide of claim 1, wherein
the polypeptide encoded by the polynucleotide consists of the
mature protein portion of SEQ ID NO:13, 16, 32 or 34.
4. The polynucleotide of claim 1, wherein the polynucleotide
comprises the nucleotide sequence of SEQ ID NO:11, 12, 31 or
33.
5. The polynucleotide of claim 1, wherein the polynucleotide is
DNA.
6. The polynucleotide of claim 1, wherein the polypeptide encoded
by the polynucleotide consists of the amino acid sequence of amino
acid residues 22 to 279 of SEQ ID NO:32, or an amino acid sequence
with at least 98% sequence identity thereto.
7. The polynucleotide of claim 1, wherein the polypeptide encoded
by the polynucleotide consists of the amino acid sequence of amino
acid residues 22 to 272 of SEQ ID NO:34, or an amino acid sequence
with at least 98% sequence identity thereto.
8. The polynucleotide of claim 1, wherein the polypeptide encoded
by the polynucleotide comprises at least 272 amino acids and has at
least 98% identity with SEQ ID NO:10.
9. An isolated polynucleotide encoding a polypeptide with stem cell
growth factor activity, wherein the polypeptide has at least 98%
sequence identity with SEQ ID NO:10, 13, or 16 and lacks the amino
acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein
A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine.
10. An isolated polynucleotide encoding a polypeptide with stem
cell growth factor activity, wherein the polypeptide has at least
98% sequence identity with SEQ ID NO:10, 13, or 16 and lacks any 10
consecutive amino acids from an amino acid sequence
GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine,
C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,
G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine,
M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine,
S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
11. An isolated polynucleotide which comprises the complement of
the polynucleotide of claim 1.
12. A vector comprising the polynucleotide of claim 1.
13. An expression vector comprising the polynucleotide of claim 1
in operative association with a regulatory sequence capable of
effecting the expression of the polynucleotide in a host cell.
14. A host cell genetically engineered to express the
polynucleotide of claim 1.
15. A host cell genetically engineered to contain the
polynucleotide of claim 1 in operative association with a
regulatory sequence capable of effecting the expression of the
polynucleotide in the host cell.
16. The host cell of claim 15 which has been genetically engineered
to contain a heterologous regulatory sequence that increases
expression of an endogenous polynucleotide.
17. A method of producing a polypeptide with stem cell growth
factor activity comprising culturing the host cell of claim 14
under conditions that permit expression of said polypeptide and
isolating said polypeptide.
18. An isolated polynucleotide comprising the protein coding cDNA
insert of the plasmid deposited with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology Zip code 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki,
Japan) on Jun. 26, 2000 under accession number FERM BP-7198.
19. An isolated polynucleotide comprising the protein coding cDNA
insert of the plasmid deposited with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology Zip code 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki,
Japan) on Jun. 26, 2000 under accession number FERM BP-7197.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/968,674, filed Oct. 19, 2004, which is a continuation
of U.S. patent application Ser. No. 09/894,912, filed Jun. 28,
2001, now U.S. Pat. No. 6,824,973, from which applications priority
is claimed pursuant to 35 U.S.C. .sctn.120. U.S. patent application
Ser. No. 09/894,912 claims the benefit under 35 U.S.C.
.sctn.119(e)(1) of U.S. Provisional Application No. 60/282,397,
filed Apr. 5, 2001, 60/215,733, filed Jun. 28, 2000, and
60/266,614, filed Feb. 5, 2001. U.S. patent application Ser. No.
09/894,912 is also a continuation-in-part application of U.S.
patent application Ser. No. 09/757,562, filed Jan. 9, 2001, now
abandoned, which is a continuation application of U.S. patent
application Ser. No. 09/543,774, filed Apr. 5, 2000, now abandoned,
which is a continuation-in-part application of U.S. patent
application Ser. No. 09/496,914, filed Feb. 3, 2000, now abandoned,
from which applications priority is claimed pursuant to 35 U.S.C.
.sctn.120. All of the foregoing applications are incorporated
herein by reference in their entireties.
2. TECHNICAL FIELD
[0002] The present invention provides novel polynucleotides and
proteins encoded by such polynucleotides, along with uses for these
polynucleotides and proteins, for example in therapeutic,
diagnostic and research methods. In particular, the invention
relates to a novel human stem cell growth factor-like protein.
2.1 BACKGROUND ART
[0003] Technology aimed at the discovery of protein factors
(including e.g., cytokines, such as lymphokines, interferons,
circulating soluble factors, chemokines, and interleukins) has
matured rapidly over the past decade. The now routine hybridization
cloning and expression cloning techniques clone novel
polynucleotides "directly" in the sense that they rely on
information directly related to the discovered protein (i.e.,
partial. DNA/amino acid sequence of the protein in the case of
hybridization cloning; activity of the protein in the case of
expression cloning). More recent "indirect" cloning techniques such
as signal sequence cloning, which isolates DNA sequences based on
the presence of a now well-recognized secretory leader sequence
motif, as well as various PCR-based or low stringency
hybridization-based cloning techniques, have advanced the state of
the art by making available large numbers of DNA/amino acid
sequences for proteins that are known to have biological activity,
for example, by virtue of their secreted nature in the case of
leader sequence cloning, by virtue of their cell or tissue source
in the case of PCR-based techniques, or by virtue of structural
similarity to other genes of known biological activity.
[0004] Identified polynucleotide and polypeptide sequences have
numerous applications in, for example, diagnostics, forensics, gene
mapping; identification of mutations responsible for genetic
disorders or other traits, to assess biodiversity, and to produce
many other types of data and products dependent on DNA and amino
acid sequences.
3. SUMMARY OF THE INVENTION
[0005] The present invention provides an isolated polynucleotide
encoding a polypeptide having stem cell growth factor activity,
said polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 9, 11, 12, 31 or 33 or the mature protein coding portion
thereof, or a fragment, analog, variant or derivative thereof that
encodes a polypeptide retaining stem cell growth factor activity.
These polypeptides include those which hybridize to the complement
of the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under
stringent hybridization conditions, those which comprise a
nucleotide sequence having greater than about 85% sequence identity
with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33,
those which comprise a nucleotide sequence having greater
than-about 90% sequence identity with the nucleotide sequence of
SEQ ID NO:9, 11, 12, 31 or 33 and those polypeptides which comprise
a nucleotide sequence having greater than about 92% sequence
identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or
33. 14. The polynucleotides may be a DNA. The present invention
also encompasses polynucleotides which comprise the complement of
these polynucleotides.
[0006] The present invention provides for isolated polynucleotide
encoding a polypeptide having stem cell growth factor activity,
said polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 9, 11, 12, 31 or 33 or the mature protein coding portion
thereof, or a fragment, analog, variant or derivative thereof that
encodes a polypeptide retaining stem cell growth factor activity
with the proviso that said polynucleotide sequence does not consist
of the nucleotide sequence of SEQ ID NO: 47. These polypeptides
include those which hybridize to the complement of the nucleotide
sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under stringent
hybridization conditions and with the proviso that said
polynucleotide sequence does not consist of the nucleotide sequence
of SEQ ID NO: 47, those which comprise a nucleotide sequence having
greater than about 85% sequence identity with the nucleotide
sequence of SEQ ID NO:9, 11, 12, 31 or 33 and with the proviso that
said polynucleotide sequence does consist of the nucleotide
sequence of SEQ ID NO: 47, those which comprise a nucleotide
sequence having greater than about 90% sequence identity with the
nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33 and with the
proviso that said polynucleotide sequence does not consist of the
nucleotide sequence of SEQ ID NO: 47, and those polypeptides which
comprise a nucleotide sequence having greater than about 92%
sequence identity with the nucleotide sequence of SEQ ID NO:9, 11,
12, 31 or 33 and with the proviso that said polynucleotide sequence
does not consist of the nucleotide sequence of SEQ ID NO: 47.
[0007] The present invention provides for an isolated
polynucleotide that comprises the mature protein coding sequence of
SEQ ID NO: 9, 11, 12, 31 or 33. The invention also provides for an
isolated polynucleotide that comprises the nucleotide sequence of
SEQ ID NO: 9, 11, 12, 31 or 33.
[0008] The invention provides for a DNA encoding a polypeptide
having stem cell growth factor activity, said polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or
33 or the mature protein coding portion thereof, or a fragment,
analog, variant or derivative thereof that encodes a polypeptide
retaining stem cell growth factor activity wherein the encoded
polypeptide has an amino acid sequence comprising at least amino
acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence
comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34;
or the encoded polypeptide has an amino acid sequence including
deletion, substitution or insertion of one or several amino acids
in the amino acid sequence comprising at least amino acid residues
22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at
least amino acid residues 22 to 272 of SEQ ID NO: 34, and which has
an activity to support proliferation or survival of hematopoietic
stem cell or hematopoietic progenitor cell, with a proviso that
C-terminal amino acid sequence does not comprise the amino acid
sequence of SEQ ID NO: 46.
[0009] The invention provides for a DNA encoding a polypeptide
having stem cell growth factor activity, said polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or
33 or the mature protein coding portion thereof, or a fragment,
analog, variant or derivative thereof that encodes a polypeptide
retaining stem cell growth factor activity, which is a DNA which
comprises at least nucleotides 574 to 1347 of SEQ ID NO: 31; or a
DNA which is hybridizable with the nucleotide sequence of SEQ ID
NO: 31 or a probe prepared from said sequence, under stringent
conditions, and which has an activity to support proliferation or
survival of hematopoietic stem cell or hematopoietic progenitor
cell. These include DNAs which hybridize under the following
stringent conditions: 6.times.SSC/5.times. Denhardt, 0.5% SDS and
68.degree. C. (SSC 3M NaCl, 0.3M sodium citrate, 50.times.
Denhardt/1% BSA/1% polyvinyl pyrrolidone, 1% Ficoll 400/, or
6.times.SSC, 5.times. Denhardt, 0.5% SDS, 50% formamide and
42.degree. C.
[0010] The invention provides for a DNA encoding a polynucleotide
encoding a polypeptide having stem cell growth factor activity,
said polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 9, 11, 12, 31 or 33 or the mature protein coding portion
thereof, or a fragment, analog, variant or derivative thereof that
encodes a polypeptide retaining stem cell growth factor activity,
which is a DNA which comprises at least nucleotides 321 to 1074 of
SEQ ID NO: 33; or DNA which is hybridizable with the nucleotide
sequence of SEQ ID NO: 33 or a prove prepared from said sequence,
under stringent conditions, and which has an activity to support
proliferation or survival of hematopoietic stem cell or
hematopoietic progenitor cell. These include DNAs which hybridize
under the following stringent conditions: 6.times.SSC/5.times.
Denhardt, 0.5% SDS and 68.degree. C. (SSC 3M NaCl, 0.3M sodium
citrate, 50.times. Denhardt/1% BSA/1% polyvinyl pyrrolidone, 1%
Ficoll 400, or 6.times.SSC, 5.times. Deanhardt, 0.5% SDS, 50%
Formamide and 42.degree. C.
[0011] The invention also provides for vectors, including
expression vectors, comprising the polynucleotide of the present
invention. The invention futher provides for host cells genetically
engineered to express a polynucleotide of the present invention.
The invention provides for host cells genetically engineered to
contain a polynucleotide of the present invention in operative
association with a regulatory sequence that controls expression of
the polynucleotide in the host cell. These host cells include those
which have been genetically engineered to contain a heterologous
regulatory sequence that increases expression of an endogenous
polynucleotide.
[0012] The invention provides for a method of producing a
polypeptide having stem cell growth factor activity comprising
growing these host cells in a culture medium under conditions that
permit expression of said polypeptide and isolating said
polypeptide from said host cell or said culture medium The
invention also encompasses a polypeptide produced by this
method.
[0013] The invention provides for an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity. These polypeptides include polypeptides which are encoded
by an isolated polynucleotide encoding a polypeptide having stem
cell growth factor activity, said polynucleotide comprising the
nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature
protein coding portion thereof, or a fragment, analog, variant or
derivative thereof that encodes a polypeptide retaining stem cell
growth factor activity and which hybridizes to the complement of
the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under
stringent hybridization conditions.
[0014] The invention provides for an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity which comprises an amino acid sequence having greater than
about 85% sequence identity with the nucleotide sequence of SEQ ID
NO: 10, 13, 16, 32 or 34, an amino acid sequence having greater
than about 92% sequence identity with the nucleotide sequence of
SEQ ID NO: 10, 13, 16, 32 or 34, with the proviso that said
polypeptide sequence does not consist of the amino acid sequence of
SEQ ID NO: 48.
[0015] The invention also provides for an isolated polypeptide
comprising the mature protein portion of SEQ ID NO: 10, 13, 16, 32
or 34.
[0016] The invention provides for an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity, wherein the polypeptide comprises one or more motifs
selected from the group of a laminin-type EGF-like domain, a
membrane attack complex component/perforin domain, and
neurohypophysial hormone signature.
[0017] The invention provides for polypeptides which are an
expression product of a DNA of the present invention, where these
polypeptide which have an activity to support proliferation or
survival of hematopoietic stem cell or hematopoietic progenitor
cell, with the proviso that the C-terminal amino acid sequence does
not comprise the amino acid sequence of SEQ ID NO: 46.
[0018] The invention provides for an isolated polynucleotide that
comprises the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or
33, which has an amino acid sequence comprising at least amino acid
residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence
including deletion, substitution or insertion of one or several
amino acids in the amino acid sequence comprising at least amino
acid residues 22 to 279 of SEQ ID NO: 32
[0019] The invention provides an isolated polypeptide comprising
the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the
mature protein portion thereof, or a fragment, analog, variant or
derivative thereof that retains stem cell growth factor activity
polypeptide, which has an amino acid sequence comprising at least
amino acid residues 22 to 272 of SEQ ID NO: 34, or an amino acid
sequence including deletion, substitution or insertion of one or
several amino acids in the amino acid sequence comprising at least
amino acid residues 22 to 272 of SEQ ID NO: 34.
[0020] The invention also provides for an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity, which is modified with one or more modifying agent
selected from the group consisting of polyethylene glycol (PEG),
dextran, poly(N-vinyl-pyrrolidone), polypropylene glycol
homopolymer, copolymer of polypropylene oxide/ethylene oxide,
polyoxyethylated polyol and polyvinyl alcohol.
[0021] The invention provides for an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity which comprises at least ten consecutive amino acids from
SEQ ID NO: 10 or 13. The invention also provides for an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
13, 16, 32 or 34, or the mature protein portion thereof, or a
fragment, analog, variant or derivative thereof that retains stem
cell growth factor activity, which comprises at least ten
consecutive amino acids from the C-terminal seventeen amino acids
of SEQ ID NO: 10 or 13.
[0022] The invention provides for a polypeptide with biological
activity, said polypeptide comprising at least 272 amino acids and
having at least 98% identity with SEQ ID NO: 10. The invention also
provides for an isolated polypeptide with stem cell growth factor
activity having at least 90% identity with SEQ ID NO: 10, 13, or 16
and lacking amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID
NO: 29) wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic
Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,
K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline,
Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine,
W=Tryptophan, Y=Tyrosine.
[0023] The invention also provides for an isolated polypeptide with
stem cell growth factor activity having at least 90% identity with
SEQ ID NO: 10, 13, or 16 and lacking any 10 consecutive amino acids
from a amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO:
29), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic
Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,
K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline,
Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine,
W=Tryptophan, Y=Tyrosine.
[0024] The invention provides for an isolated polypeptide with stem
cell growth factor activity having at least an amino acid sequence
SVSVSTVH (SEQ ID NO: 27) or VSVSTVH (SEQ ID NO: 28), wherein
A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine.
[0025] The invention encompasses a polynucleotide which encodes any
of the polypeptides of the present invention.
[0026] The invention provides for a kit comprising an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
13, 16, 32 or 34, or the mature protein portion thereof, or a
fragment, analog, variant or derivative thereof that retains stem
cell growth factor activity.
[0027] The invention further provides for a culture medium
comprising an amount of an isolated polypeptide comprising the
amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the
mature protein portion thereof, or a fragment, analog, variant-or
derivative thereof that retains stem cell growth factor activity
polypeptide, wherein the amount is effective to maintain survival
of or promote proliferation of a stem cell or germ cell.
[0028] The composition comprising an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or
34, or the mature protein portion thereof, or a fragment, analog,
variant or derivative thereof that retains stem cell growth factor
activity and a pharmaceutically acceptable carrier or diluent.
These compositions can be pharmaceutical compositions including
those having an effect to support proliferation or survival of
hematopoietic stem cell or hematopoietic progenitor cell, which
comprises:a polypeptide which has an amino acid sequence comprising
at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an
amino acid sequence comprising at least amino acid residues 22 to
272 of SEQ ID NO: 34; or a polypeptide which has an amino acid
sequence including deletion, substitution or insertion of one or
several amino acids in the amino acid sequence comprising at least
amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid
sequence comprising at least amino acid residues 22 to 272 of SEQ
ID NO: 34, and which has an activity to support proliferation or
survival of hematopoietic stem cell or hematopoietic progenitor
cell.
[0029] The invention provides for an antibody that binds to an
isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or
a fragment, analog, variant or derivative thereof that retains stem
cell growth factor activity. The antibodies of the present
invention may specifically binds to a polypeptide having the amino
acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34including those
which do not bind to a polypeptide having the amino acid sequence
of SEQ ID NO: 48. The antibodies of the present invention include
polyclonal antibodies, monoclonal antibodies, antibody fragments,
chimeric antibodies, and humanized antibodies. Further, the
invention encompasses kits comprising the antibodies of the present
invention.
[0030] The invention provides for a method for detecting a
polynucleotide of the present invention in a sample, comprising: a)
contacting the sample with a compound that binds to and forms a
complex with the polynucleotide for a period sufficient to form the
complex; and b) detecting the complex, so that if a complex is
detected, the polynucleotide is detected. The invention also
provides for methods for detecting a polynucleotide of the present
invention in a sample, comprising: a) contacting the sample under
stringent hybridization conditions with nucleic acid primers that
anneal to the polynucleotide under such conditions; b) amplifying a
product comprising at least a portion of the polynucleotide; and c)
detecting said product and thereby the polynucleotide in the
sample. These methods include a method wherein the polynucleotide
detected encodes a polypeptide comprising the amino acid sequence
of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion
thereof, or a fragment, analog, variant or derivative thereof that
retains stem cell growth factor activitya polypeptide of claim 23,
and the method further comprises reverse transcribing an annealed
RNA molecule into a cDNA polynucleotide.
[0031] The invention also provides for a method for detecting a
polypeptide of the present invnetion in a sample, comprising: a)
contacting the sample with a compound that binds to and forms a
complex with the polypeptide under conditions and for a period
sufficient to form the complex; and b) detecting formation of the
complex, so that if a complex formation is detected, the
polypeptide is detected.
[0032] The invention also provides for a method for identifying a
compound that binds to a polypeptide of the invention, comprising:
a) contacting the compound with the polypeptide under conditions
and for a time sufficient to form a polypeptide/compound complex;
and b) detecting the complex, so that if the polypeptide/compound
complex is detected, a compound that binds to the polypeptide is
identified.
[0033] The invention also provides for a method for identifying a
compound that binds to the polypeptide of the present invention,
comprising: a) contacting the compound with the polypeptide, in a
cell, for a time sufficient to form a polypeptide/compound complex,
wherein the complex drives expression of a reporter gene sequence
in the cell; and b) detecting the complex by detecting reporter
gene sequence expression, so that if the polypeptide/compound
complex is detected, a compound that binds to the polypeptide is
identified.
[0034] The invention provides for a nucleic acid array comprising a
polynucleotide of the present invention or a unique segment of a
polynucleotide of the present invnetion attached to a surface.
These arrays include those which full-matches to the polynucleotide
or a unique segment of the polynucleotide of the present
inventions, those which detect mismatches to the polynucleotide or
a unique segment of the polynucleotide of the present
invention.
[0035] The invention provides for a method of treatment of a
subject in need of enhanced activity or expression of stem cell
growth factor-like polypeptide of the present invention comprising
administering to the subject: (a) a composition comprising a
therapeutic amount of an agonist of said polypeptide; (b) a
composition comprising a therapeutic amount of the polypeptide; or
(c) a composition comprising a therapeutic amount of a
polynucleotide encoding the polypeptide in form and under
conditions such that the polypeptide is produced; said composition
comprising a pharmaceutically acceptable carrier or diluent.
[0036] The invention also provdies for a method of treatment of a
subject having need of decreased activity or expression of stem
cell growth factor-like polypeptide of the present invention
comprising administering to the subject: (a) a composition
comprising a therapeutic amount of an antagonist of said
polypeptide; (b) a composition comprising a therapeutic amount of
the polynucleotide that inhibits the expression of the nucleotide
sequence encoding said polypeptide; and (c) a composition
comprising a therapeutic amount of a polypeptide that competes with
the stem cell growth factor-like polypeptide for its ligand; said
composition comprising a pharmaceutically acceptable carrier or
diluent.
[0037] The invention also provides for a method of supporting
proliferation or survival of a stem cell or germ cell comprising
contacting said cell with an amount of a polypeptide of the present
invention effective to maintain survival of or promote
proliferation of said cell. These methods include those wherein
said cell is a primordial germ cell, germ line stem cell, embryonic
stem cell, hematopoietic stem cell, hematopoietic progenitor cell,
pluripotent cell, or totipotent cell. These methods also include
those wherein the polypeptide comprises an amino acid sequence of
SEQ ID NO: 10, 13, or 16, or comprises an amino acid sequence 90%
identical to SEQ ID NO. 10, 13, or 16. These methods further
include those wherein the stem cell growth factor-like polypeptide
is encoded by a polynucleotide that hybridizes to the complement of
a polynucleotide encoding SEQ ID NO: 10, 13, or 16 under stringent
hybridization conditions.
[0038] The invention encompasses a stromal cell genetically
engineered to express a polypeptide of the invention in an amount
effective to support proliferation or survival of a stem cell or
germ cell. These cells include primordial germ cells germ line stem
cells embryonic stem cells hematopoietic stem cells hematopoietic
progenitor cells pluripotent cells or totipotent cells
[0039] The invention provides for an implant comprising a cell
genetically engineered to express a polypeptide of the present
invention in an amount effective to support proliferation or
survival of a stem cell or germ cell. These implants of the present
invention include those wherein the cell is a primordial germ cell,
germ line stem cell, embryonic stem cell, hematopoietic stem cell,
hematopoietic progenitor cell, pluripotent cell, or totipotent
cell.
[0040] The invention provides for an isolated polynucleotide
comprising the protein coding cDNA insert of the plasmid deposited
with the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology (Zip code 305-8566;
Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 under
accession number FERM BP-7198 and the mature polypeptide expressed
by this polynucleotide of in a suitable host cell.
[0041] The invention also provides for an isolated polynucleotide
comprising the protein coding cDNA insert of the plasmid deposited
with the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology (Zip code 305-8566;
Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 under
accession number FERM BP-7197 and the mature polypeptide expression
product expressed by this polynucleotide in a suitable host
cell.
[0042] Optionally preferred are polynucleotides and polypeptides
other than the nucleotide sequence set forth as SEQ ID NO: 3284
(and the polypeptide sequence encoded therein) in U.S. application
Ser. No. 09/496,914 filed Feb. 3, 2000 and the protein set out in
Genbank Accession No. BAB28811.
[0043] The compositions of the present invention include novel
isolated polypeptides, novel isolated polynucleotides encoding such
polypeptides, including recombinant DNA molecules, cloned genes or
degenerate variants thereof, especially naturally occurring
variants such as allelic variants, antisense polynucleotide
molecules, and antibodies that specifically recognize one or more
epitopes present on such polypeptides, as well as hybridomas
producing such antibodies. Specifically, the polynucleotides of the
present invention are based on polynucleotides isolated from cDNA
libraries prepared from human testis cells (Hyseq clone
identification numbers 2880984 and 2881695), from human fetal skin
(Hyseq clone identification number 15375176), adult spleen (Hyseq
clone identification number 14856094), and human endothelial cells
(Hyseq clone identification numbers 13804756, 13687487,
13804756).
[0044] In one aspect, the invention involves an isolated
polynucleotide with stem cell growth factor activity comprising a
nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 30, the mature
protein coding portion thereof, the extracellular coding portion
thereof, or the active domain coding portion thereof.
[0045] In one embodiment, the invention involves an isolated
polynucleotide encoding a polypeptide with biological activity, and
said polynucleotide hybridizes to the complement of the
polynucleotide with stem cell growth factor activity under
stringent hybridization conditions.
[0046] In a further embodiment, the invention involves an isolated
polynucleotide encoding a polypeptide with biological activity,
said polynucleotide having at least about 92% sequence identity
with the polynucleotide with stem cell growth factor activity.
[0047] In a further embodiment, the invention involves an isolated
polynucleotide encoding a polypeptide with biological activity,
said polypeptide having greater than about 95% sequence identity
with the polynucleotide with stem cell growth factor activity.
[0048] In a still further embodiment, the polynucleotide with stem
cell growth factor activity is a DNA.
[0049] In another embodiment, the invention involves an isolated
polynucleotide which comprises the complement of the polynucleotide
with stem cell growth factor activity.
[0050] The invention also involves a vector comprising the
polynucleotide with stem cell growth factor activity.
Alternatively, the invention involves an expression vector
comprising the polynucleotide with stem cell growth factor
activity. A host cell genetically engineered to express the
polynucleotide with stem cell growth factor activity is also
provided. The host cell genetically engineered to contain the
polynucleotide with stem cell growth factor activity in operative
association with a regulatory sequence that controls expression of
the polynucleotide in the host cell.
[0051] In another aspect, the invention involves an isolated
polypeptide comprising an amino acid sequence consisting of SEQ ID
NO: 10, 13, 16, 32 or 34, the mature protein portion thereof, the
extracellular portion thereof, or active domain thereof.
[0052] Also provided is a composition comprising the polypeptide
and a carrier. In another embodiment, the invention involves an
antibody directed against the polypeptide.
[0053] In another aspect, the invention involves a method for
detecting the polynucleotide with stem cell growth factor activity
in a sample, comprising contacting the sample with a compound that
binds to and forms a complex with the polynucleotide for a period
sufficient to form the complex; and detecting the complex, so that
if a complex is detected, the polynucleotide is detected.
[0054] The invention also involves a method for detecting the
polynucleotide with stem cell growth factor activity in a sample,
comprising contacting the sample under stringent hybridization
conditions with nucleic acid primers that anneal to the
polynucleotide under such conditions; amplifying a product
comprising at least a portion of the polynucleotide; and detecting
said product and thereby the polynucleotide in the sample.
[0055] In a further embodiment, the polynucleotide is an RNA
molecule that encodes the polypeptide, and the method further
comprises reverse transcribing an annealed RNA molecule into a cDNA
polynucleotide.
[0056] Also provided is a method for detecting the polypeptide in a
sample, comprising contacting the sample with a compound that binds
to and forms a complex with the polypeptide under conditions and
for a period sufficient to form the complex; and detecting
formation of the complex, so that if a complex formation is
detected, the polypeptide is detected.
[0057] In another embodiment, the invention provides a method for
identifying a compound that binds to the polypeptide, comprising
contacting the compound with the polypeptide of under conditions
and for a time sufficient to form a polypeptide/compound complex;
and detecting the complex, so that if the polypeptide/compound
complex is detected, a compound that binds to the polypeptide is
identified.
[0058] In a further embodiment, the invention involves a method for
identifying a compound that binds to the polypeptide, comprising
contacting the compound with the polypeptide in a cell, for a time
sufficient to form a polypeptide/compound complex, wherein the
complex drives expression of a reporter gene sequence in the cell;
and detecting the complex by detecting reporter gene sequence
expression, so that if the polypeptide/compound complex is
detected, a compound that binds to the polypeptide is
identified.
[0059] In another embodiment, the invention involves a method of
producing the polypeptide, comprising, culturing the host cell for
a period of time sufficient to express the polypeptide in said
cell; and isolating the polypeptide from the cell culture or
cells.
[0060] In another aspect, the invention involves a kit comprising
the polypeptide. Also provided is a nucleic acid array comprising
the polynucleotide or a segment of the polynucleotide attached to a
surface. In a further embodiment, the array detects full-matches to
the polynucleotide or a unique segment of the polynucleotide. In
another embodiment, the array detects mismatches to the
polynucleotide or a unique segment of the polynucleotide.
[0061] The invention also provides for a method of treatment of a
subject in need thereof enhanced activity or expression of stem
cell growth factor-like polypeptide comprising administering to the
subject a composition selected from the group consisting of a)
therapeutic amount of an agonist of said polypeptide; b) a
therapeutic amount of the polypeptide; and c) a therapeutic amount
of a polynucleotide encoding the polypeptide in form and under
conditions such that the polypeptide is produced,
and a pharmaceutically acceptable carrier. The invention also
provides for a method of treatment of a subject having need to
inhibit activity or expression of stem cell growth factor-like
polypeptide comprising administering to the subject a composition
selected from the group consisting of a) a therapeutic amount of an
antagonist of said polypeptide; b) a therapeutic amount of the
polynucleotide that inhibits the expression of the nucleotide
sequence encoding said polypeptide; and c) a therapeutic amount of
a polypeptide that competes with the stem cell growth factor-like
polypeptide for its ligand, and a pharmaceutically acceptable
carrier.
[0062] In another embodiment, the invention involves a polypeptide
having stem cell growth factor activity comprising at least ten
consecutive amino acids from SEQ ID NO: 10, 13, 16, 32 or 34. In
still another embodiment, the invention involves this polypeptide
comprising at least ten consecutive amino acids from the C-terminal
seventeen amino acids of SEQ ID NO: 10, 13, 16, 32 or 34.
[0063] Also provided is a polypeptide with biological activity,
said polypeptide comprising at least 272 amino acids and having at
least 98% identity with SEQ ID NO: 10 or 34 or said polypeptide
comprising at least 273 amino acids and having at least 98%
identity with SEQ ID NO: 13.
[0064] In a further embodiment, the invention involves an isolated
polypeptide with stem cell growth factor activity having at least
90% identity with SEQ ID NO: 10, 13, 16, 32 or 34 and lacking amino
acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein
A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine.
[0065] In yet another embodiment, the invention involves an
isolated polypeptide with stem cell growth factor activity having
at least 90% identity with SEQ ID NO: 10, 13, 16, 32 or 34 and
lacking any 10 consecutive amino acids from amino acid sequence
GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine,
C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,
G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine,
M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine,
S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
[0066] In another embodiment, the invention concerns a method of
maintaining or promoting proliferation of a cell selected from the
group consisting of primordial germ cells, germ line stem cells,
embryonic stem cells, pluripotent cell, and totipotent cells,
comprising contacting the cell with an effective amount of a stem
cell growth factor-like polypeptide. In a further embodiment, the
polypeptide comprises an amino acid sequence of SEQ ID NO: 10, 13,
16, 32 or 34, or comprises an amino acid sequence 90% identical to
SEQ ID NO. 10, 13, 16, 32 or 34. In still a further embodiment, the
stem cell growth factor-like polypeptide is encoded by a
polynucleotide that hybridizes to the complement of a
polynucleotide encoding SEQ ID NO: 10, 13, 16, 32 or 34 under
stringent hybridization conditions.
[0067] The invention also involves an isolated polypeptide with
stem cell growth factor activity having at least an amino acid
sequence SVSVSTVH (SEQ ID NO: 27) or VSVSTVH (SEQ ID NO: 28),
wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine.
[0068] In an additional embodiment, the invention concerns the
polypeptide according to this invention, wherein the polypeptide
comprises one or more motifs selected from the group of a
laminin-type EGF-like domain, a membrane attack complex
component/perforin domain, and neurohypophysial hormone
signature.
[0069] The invention also encompasses any polynucleotides encoding
a polypeptide according to this invention.
[0070] The compositions of the present invention additionally
include vectors, including expression vectors, containing the
polynucleotides of the invention, cells genetically engineered to
contain such polynucleotides and cells genetically engineered to
express such polynucleotides.
[0071] The isolated polynucleotides of the invention include, but
are not limited to, a polynucleotide comprising any one of the
nucleotide sequences set forth in the SEQ ID NO: 9, 11, 12, 31 or
33; a polynucleotide comprising any of the full length protein
coding sequences of the SEQ ID NO: 9, 11, 12, 31 or 33; and a
polynucleotide comprising any of the nucleotide sequences of the
mature protein coding sequences of the SEQ ID NO: 9, 11, 12, 31 or
33. The polynucleotides of the present invention also include, but
are not limited to, a polynucleotide that hybridizes under
stringent hybridization conditions to (a) the complement of any one
of the nucleotide sequences set forth in the SEQ ID NO: 9, 11, 12,
31 or 33; (b) a nucleotide sequence encoding SEQ ID NO: 10, 13-24,
32 or 34; a polynucleotide which is an allelic variant of any
polynucleotides recited above; a polynucleotide which encodes a
species homolog (e.g. orthologs) of any of the proteins recited
above; or a polynucleotide that encodes a polypeptide comprising a
specific domain or truncation of any of the polypeptides comprising
SEQ ID NO: 10, 13-24, 32 or 34.
[0072] The nucleic acid sequences of the present invention also
include the sequence information from the nucleic acid sequences of
SEQ ID NO: 11, 12, 31 or 33. The sequence information can be a
segment of any one of SEQ ID NO: 1-7 that uniquely identifies or
represents the sequence information of SEQ ID NO: 11, 12, 31 or 33.
One such segment can be a twenty-mer nucleic acid sequence because
the probability that a twenty-mer is fully matched in the human
genome is 1 in 300. In the human genome, there are three billion
base pairs in one set of chromosome. Because there are 4.sup.20
possible twenty-mers exist, there are 300 times more twenty-mers
than there are base pairs in a set of human chromosome. Using the
same analysis, the probability for a seventeen-mer to be fully
matched in the human genome is approximately 1 in 5. When these
segments are used in arrays for expression studies, fifteen-mer
segment can be used. The probability that the fifteen-mer is fully
matched in the expressed sequences is also approximately one in
five because expressed sequences in one tissue comprise
approximately 5% of the entire genome sequence. Preferably, the
nucleic acid fragment or subsequence comprise the twenty-one 3'
coding nucleotides.
[0073] Similarly, when using sequence information for detecting a
single mismatch, a segment can be a twenty-five mer. The
probability that the twenty-five mer would appear in a human genome
with a single mismatch is calculated by multiplying the probability
for a full match (1/4.sup.25) times the increased probability for
mismatch at each nucleotide position (3.times.25). The probability
that an eighteen mer with a single mismatch can be detected in an
array for expression studies is approximately one in five. The
probability that a twenty-mer with a single mismatch can be
detected in a human genome is approximately one in five.
[0074] A collection as used in this application can be a collection
of only one polynucleotide. The collection of sequence information
or unique identifying information of each sequence can be provided
on a nucleic acid array. In one embodiment, segments of sequence
information are provided on a nucleic acid array to detect the
polynucleotide that contains the segment. The array can be designed
to detect full-match or mismatch to the polynucleotide that
contains the segment. The collection can also be provided in a
computer-readable format.
[0075] This invention also includes the reverse or direct
complement of any of the nucleic acid sequences recited above;
cloning or expression vectors containing the nucleic acid
sequences; and host cells or organisms transformed with these
expression vectors.
[0076] Human stem cell growth factor-like polypeptide (SEQ ID NO:
10 or 34) is approximately a 272-amino acid protein with a
predicted molecular mass of approximately 30 kDa unglycosylated.
The mouse homolog is set out in SEQ ID NO: 32. Protein database
searches with the BLAST algorithm indicate that SEQ ID NO: 10 is
homologous to Mus musculus thrombospondin type 1 domain. FIG. 1
shows the alignment of polynucleotide SEQ ID NO: 9 and EST
sequences SEQ ID NO: 1-7. The sequences of the present invention
(SEQ ID NO: 1-12) are expected to have stem cell growth factor
activity, including hematopoietic stem cell growth factor activity,
as described herein.
[0077] Stem cell growth factor-like polypeptide (SEQ ID NO: 10)
also has the following motifs at the designated amino acid sequence
corresponding to SEQ ID NO: 10 wherein A=Alanine, C=Cysteine,
D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,
H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,
N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine,
T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:
TABLE-US-00001 Laminin-type EGF-like (LE) domain proteins at 100
ADCDTCFNKNFCTKCKSGFYLIIL 122 (SEQ ID NO: 17) Vertebrate
metallothioneins proteins at 92
INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANN 137 (SEQ ID NO: 18)
Endogenous opioids neuropeptides precursors proteins at 33
MHPNVSQGCQGGCATCSDYN 52 (SEQ ID NO: 19) Membrane attack complex
components/perform proteins at 145
IVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQ 181 (SEQ ID NO: 20) HMG-I and
HMG-Y DNA-binding domain proteins (Ahook) at 213 KKGRERKRKK 222
(SEQ ID NO: 21) HMG1/2 proteins at 198
KCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLE 239 (SEQ ID NO: 22)
VERTEBRATE METALLOTHIONEIN SIGNATURE at 104 TCFNKNFCTKCKSG 117 (SEQ
ID NO: 23) NEUROHYPOPHYSIAL HORMONE SIGNATURE at 148
CEVSEWNPWSPCTKKGKTCG 167 (SEQ ID NO: 24)
[0078] Motif 100-122, a laminin-type EGF-like domain, is a
component of extracellular matrix which promotes cell growth. The
membrane attack complex component/perforin domain (145-185) is
postulated to mediate cell-cell interaction and thus cell growth
and differentiation. Neurohypophysial hormone is itself regulated
by many other factors including Interleukin-1 beta and
Interleukin-6. The presence of these motifs are expected in stem
cell growth factor activity.
[0079] Stem cell growth factor-like protein and/or fragments or
derivatives would have similar activity to stem cell growth factors
and anabolic growth factors and receptors.
[0080] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising SEQ ID NO: 10, 13-24, 32
or 34; or the corresponding full length or mature protein.
Polypeptides of the invention also include polypeptides with
biological activity that are encoded by (a) any of the
polynucleotides having a nucleotide sequence set forth in the SEQ
ID NO: 1-9; 11, 12, 31 or 33 or (b) polynucleotides that hybridize
to the complement of the polynucleotides of (a) under stringent
hybridization conditions. Biologically or immunologically active
variants of any of the protein sequences listed as SEQ ID NO: 10,
13-24, 32 or 34, and "substantial equivalents" thereof (e.g., with
at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% amino acid sequence identity) that
preferably retain biological activity are also contemplated. The
polypeptides of the invention may be wholly or partially chemically
synthesized but are preferably produced by recombinant means using
the genetically engineered cells (e.g. host cells) of the
invention.
[0081] The invention also provides compositions comprising a
polypeptide of the invention. Polypeptide compositions of the
invention may further comprise an acceptable carrier, such as a
hydrophilic, e.g., pharmaceutically acceptable, carrier.
[0082] The invention also provides host cells transformed or
transfected with a polynucleotide of the invention.
[0083] The invention also relates to methods for producing a
polypeptide of the invention comprising growing a culture of the
host cells of the invention in a suitable culture medium under
conditions permitting expression of the desired polypeptide, and
purifying the protein from the culture or from the host cells.
Preferred embodiments include those in which the protein produced
by such process is a mature form of the protein.
[0084] Polynucleotides according to the invention have numerous
applications in a variety of techniques known to those skilled in
the art of molecular biology. These techniques include use as
hybridization probes, use as oligomers, or primers, for PCR, use in
an array, use in computer-readable media, use for chromosome and
gene mapping, use in the recombinant production of protein, and use
in generation of anti-sense DNA or RNA, their chemical analogs and
the like. For example, when the expression of an mRNA is largely
restricted to a particular cell or tissue type, polynucleotides of
the invention can be used as hybridization probes to detect the
presence of the particular cell or tissue mRNA in a sample using,
e.g., in situ hybridization.
[0085] In other exemplary embodiments, the polynucleotides are used
in diagnostics as expressed sequence tags for identifying expressed
genes or, as well known in the art and exemplified by Vollrath et
al., Science 258:52-59 (1992), as expressed sequence tags for
physical mapping of the human genome.
[0086] A polynucleotide according to the invention can be joined to
any of a variety of other nucleotide sequences by well-established
recombinant DNA techniques (see Sambrook, J., et al. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, NY). Useful nucleotide sequences for joining to
polypeptides include an assortment of vectors, e.g., plasmids,
cosmids, lambda phage derivatives, phagemids, and the like, that
are well known in the art. Accordingly, the invention also provides
a vector including a polynucleotide of the invention and a host
cell containing the polynucleotide. In general, the vector contains
an origin of replication functional in at least one organism,
convenient restriction endonuclease sites, and a selectable marker
for the host cell. Vectors according to the invention include
expression vectors, replication vectors, probe generation vectors,
and sequencing vectors. A host cell according to the invention can
be a prokaryotic or eukaryotic cell and can be a unicellular
organism or part of a multicellular organism.
[0087] The polypeptides according to the invention can be used in a
variety of conventional procedures and methods that are currently
applied to other proteins. For example, a polypeptide of the
invention can be used to generate an antibody that specifically
binds the polypeptide. Such antibodies, particularly monoclonal
antibodies, are useful for detecting or quantitating the
polypeptide in tissue. The polypeptides of the invention can also
be used as molecular weight markers, and as a food supplement.
[0088] Methods are also provided for preventing, treating, or
ameliorating a medical condition which comprises the step of
administering to a mammalian subject a therapeutically effective
amount of a composition comprising a protein of the present
invention and a pharmaceutically acceptable carrier.
[0089] In particular, the polypeptides and polynucleotides of the
invention can be utilized, for example, as part of methods for the
prevention and/or treatment of disorders involving aberrant protein
expression or biological activity.
[0090] The methods of the invention also provides methods for the
treatment of disorders as recited herein which may involve the
administration of the polynucleotides or polypeptides of the
invention to individuals exhibiting symptoms or tendencies related
to disorders as recited herein. In addition, the invention
encompasses methods for treating diseases or disorders as recited
herein comprising the step of administering compounds and other
substances that modulate the overall activity of the target gene
products. Compounds and other substances can effect such modulation
either on the level of target gene/protein expression or target
protein activity. Specifically, methods are provided for
preventing, treating or ameliorating a medical condition, including
neurological diseases, which comprises administering to a mammalian
subject, including but not limited to humans, a therapeutically
effective amount of a composition comprising a polypeptide of the
invention or a therapeutically effective amount of a composition
comprising a binding partner of (e.g., antibody specifically
reactive for) stem cell growth factor-like polypeptides of the
invention. The mechanics of the particular condition or pathology
will dictate whether the polypeptides of the invention or binding
partners (or inhibitors) of these would be beneficial to the
individual in need of treatment.
[0091] The invention also provides a method of promoting wound
healing comprising administering a stem cell growth factor-like
polypeptide of the present invention to the site of a wound or
injury. The invention provides a method of promoting cell growth
and morphogenesis comprising administering a stem cell growth
factor-like polypeptide of the present invention to a medium of
nerve cells. According to this method, polypeptides of the
invention can be administered to produce an in vitro or in vivo
promotion of cellular function. A polypeptide of the invention can
be administered in vivo as a stem cell growth factor alone or as an
adjunct to other therapies.
[0092] The invention further provides methods for manufacturing
medicaments useful in the above described methods.
[0093] The present invention further relates to methods for
detecting the presence of the polynucleotides or polypeptides of
the invention in a sample (e.g., tissue or sample). Such methods
can, for example, be utilized as part of prognostic and diagnostic
evaluation of disorders as recited herein and for the
identification of subjects exhibiting a predisposition to such
conditions. The invention also provides kits comprising
polynucleotide probes and/or monoclonal antibodies, and optionally
quantitative standards, for carrying out methods of the invention.
Furthermore, the invention provides methods for evaluating the
efficacy of drugs, and monitoring the progress of patients,
involved in clinical trials for the treatment of disorders as
recited above.
[0094] The invention also provides methods for the identification
of compounds that modulate (i.e., increase or decrease) the
expression or activity of the polynucleotides and/or polypeptides
of the invention. Such methods can be utilized, for example, for
the identification of compounds that can ameliorate symptoms of
disorders as recited herein. Such methods can include, but are not
limited to, assays for identifying compounds and other substances
that interact with (e.g., bind to) the polypeptides of the
invention.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1 shows the alignment of SEQ ID NO. 9 with SEQ ID NO.
1-7.
[0096] FIG. 2 shows the BLASTP amino acid sequence alignment
between the SEQ ID NO: 10, stem cell growth factor-like polypeptide
and mouse thrombospondin type 1 domain protein SEQ ID NO: 25,
indicating that the two sequences share 64% similarity over amino
acid residues 19-254 of SEQ ID NO: 10 and 47% identity over the
same amino acid residues 19-254 of SEQ ID NO: 10, wherein
A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine. Gaps are presented as dashes.
[0097] FIG. 3 shows the BLASTP amino acid sequence alignment
between the SEQ ID NO: 10, stem cell growth factor-like polypeptide
and human secreted protein clone da228.sub.--6 protein (Patent
Application No. WO98/49302), SEQ ID NO: 26, indicating that the two
sequences share 100% similarity over amino acid residues 1-265 of
SEQ ID NO: 10 and 100% identity over the same amino acid residues
1-265 of SEQ ID NO: 10, wherein A=Alanine, C=Cysteine, D=Aspartic
Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine,
I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine,
P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,
V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as
dashes.
[0098] FIG. 4 shows proliferation statuses of hematopoietic stem
cells and hematopoietic progenitor cells determined by a clonogenic
assay after co-culture of CD34 positive hematopoietic stem cells
with AGM-s3 subclone A9, A7, or D11 cells for two weeks;
[0099] FIG. 5 shows proliferation statuses of hematopoietic stem
cells and hematopoietic progenitor cells determined by a clonogenic
assay after co-culture of CD34 positive hematopoietic stem cells
with AGM-s3 subclone A9, A7, or OP9 cells for two weeks;
[0100] FIG. 6 shows time course of donor derived lymphoid lineage
cells or myeloid lineage cells reconstitution in irradiated
recipient mice that received the hematopoietic stem cells
co-cultured with stromal cells; and
[0101] FIG. 7 shows time course of donor derived lymphoid lineage
cells or myeloid lineage cells reconstitution in irradiated
recipient mice that received the hematopoietic stem cells
co-cultured with AGM-s3-A7 cell lines (A7/pMXIG-SCR-1 and A7/pMXIG)
transfected with a vector including SCR-1 (pMXIG-SCR-1) or a vector
which does not include SCR-1 (pMXIG).
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 DEFINITIONS
[0102] The term "primordial germ cells (PGCs)" refers to a small
population of cells set aside from other cell lineages particularly
from the yolk sac, mesenteries, or gonadal ridges during
embryogenesis that have the potential to differentiate in to germ
cells and other cells. PGCs are the source from which GSCs and ES
cells are derived
[0103] The term "germ line stem cells (GSCs)" refers to stem cells
derived from primordial stem cells that provide a steady and
continuous source of germ cells for the production of gametes.
[0104] The term "embryonic stem cells (ES)" refers to a cell which
can give rise to many differentiated cell types in an embryo or an
adult, including the germ cells. The PGCs, the GSCs and the ES
cells are capable of self-renewal. Thus these cells not only
populate the germ line and give rise to a plurality of terminally
differentiated cells which comprise the adult specialized organs,
but are able to regenerate themselves.
[0105] The term "totipotent" refers to the capability of a cell to
differentiate into all of the cell types of an adult organism.
[0106] The term "pluripotent" refers to the capability of a cell to
differentiate into a number of differentiated cell types that are
present in an adult organism. A pluripotent cell is restricted in
its differentiation capability in comparison to a totipotent
cell.
[0107] The term "nucleotide sequence" refers to a heteropolymer of
nucleotides or the sequence of these nucleotides. The terms
"nucleic acid" and "polynucleotide" are also used interchangeably
herein to refer to a heteropolymer of nucleotides. Generally,
nucleic acid segments provided by this invention may be assembled
from fragments of the genome and short oligonucleotide linkers, or
from a series of oligonucleotides, or from individual nucleotides,
to provide a synthetic nucleic acid which is capable of being
expressed in a recombinant transcriptional unit comprising
regulatory elements derived from a microbial or viral operon, or a
eukaryotic gene.
[0108] The terms "oligonucleotide fragment" or a "polynucleotide
fragment", "portion," or "segment" is a sequence of nucleotide
residues which is long enough to use in polymerase chain reaction
(PCR) or various hybridization procedures to identify or amplify
identical or related parts of mRNA or DNA molecules. A fragment or
segment may uniquely identify each polynucleotide sequence of the
present invention.
[0109] The terms "oligonucleotides" or "nucleic acid probes" are
prepared based on the polynucleotide sequences provided in the
present invention. Oligonucleotides comprise portions of such a
polynucleotide sequence having at least about 15 nucleotides and
usually at least about 20 nucleotides. Nucleic acid probes comprise
portions of such a polynucleotide sequence having fewer nucleotides
than about 6 kb, usually fewer than about 1 kb. After appropriate
testing to eliminate false positives, these probes may, for
example, be used to determine whether specific mRNA molecules are
present in a cell or tissue or to isolate similar nucleic acid
sequences from chromosomal DNA as described by Walsh et al. (Walsh,
P. S. et al., 1992, PCR Methods Appl 1:241-250).
[0110] The term "probes" includes naturally occurring or
recombinant or chemically synthesized single- or double-stranded
nucleic acids. They may be labeled by nick translation, Klenow
fill-in reaction, PCR, or other methods well known in the art.
Probes of the present invention, their preparation and/or labeling
are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel,
F. M. et al., 1989, Current Protocols in Molecular Biology, John
Wiley & Sons. New York N.Y. both of which are incorporated
herein by reference in their entirety.
[0111] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Stringent conditions
can include highly stringent conditions (i.e., hybridization to
filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C.), and moderately stringent
conditions (i.e., washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C.). Other exemplary hybridization conditions are described herein
in the examples.
[0112] In instances of hybridization of deoxyoligonucleotides,
additional exemplary stringent hybridization conditions include
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C.
(for 14-base oligos), 48.degree. C. (for 17-base oligos),
55.degree. C. (for 20-base oligos), and 60.degree. C. (for 23-base
oligos).
[0113] The term "recombinant," when used herein to refer to a
polypeptide or protein, means that a polypeptide or protein is
derived from recombinant (e.g., microbial, insect, or mammalian)
expression systems. "Microbial" refers to recombinant polypeptides
or proteins made in bacterial or fungal (e.g., yeast) expression
systems. As a product, "recombinant microbial" defines a
polypeptide or protein essentially free of native endogenous
substances and unaccompanied by associated native glycosylation.
Polypeptides or proteins expressed in most bacterial cultures,
e.g., E. coli, will be free of glycosylation modifications;
polypeptides or proteins expressed in yeast will have a
glycosylation pattern in general different from those expressed in
mammalian cells.
[0114] The term "recombinant expression vehicle or vector" refers
to a plasmid or phage or virus or vector, for expressing a
polypeptide from a DNA (RNA) sequence. An expression vehicle can
comprise a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, promoters or enhancers, (2) a structural
or coding sequence which is transcribed into mRNA and translated
into protein, and (3) appropriate transcription initiation and
termination sequences. Structural units intended for use in yeast
or eukaryotic expression systems preferably include a leader
sequence enabling extracellular secretion of translated protein by
a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it may include an amino
terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein to
provide a final product.
[0115] The term "recombinant expression system" means host cells
which have stably integrated a recombinant transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit
extrachromosomally. Recombinant expression systems as defined
herein will express heterologous polypeptides or proteins upon
induction of the regulatory elements linked to the DNA segment or
synthetic gene to be expressed. This term also means host cells
which have stably integrated a recombinant genetic element or
elements having a regulatory role in gene expression, for example,
promoters or enhancers. Recombinant expression systems as defined
herein will express polypeptides or proteins endogenous to the cell
upon induction of the regulatory elements linked to the endogenous
DNA segment or gene to be expressed. The cells can be prokaryotic
or eukaryotic.
[0116] The term "open reading frame," ORF, means a series of
nucleotide triplets coding for amino acids without any termination
codons and is a sequence translatable into protein.
[0117] The term "expression modulating fragment," EMF, means a
series of nucleotides which modulates the expression of an operably
linked ORF or another EMF.
[0118] As used herein, a sequence is said to "modulate the
expression of an operably linked sequence" when the expression of
the sequence is altered by the presence of the EMF. EMFs include,
but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of EMFs are fragments
which induce the expression or an operably linked ORF in response
to a specific regulatory factor or physiological event.
[0119] As used herein, an "uptake modulating fragment," UMF, means
a series of nucleotides which mediate the uptake of a linked DNA
fragment into a cell. UMFs can be readily identified using known
UMFs as a target sequence or target motif with the computer-based
systems described below.
[0120] The presence and activity of a UMF can be confirmed by
attaching the suspected UMF to a marker sequence. The resulting
nucleic acid molecule is then incubated with an appropriate host
under appropriate conditions and the uptake of the marker sequence
is determined. As described above, a UMF will increase the
frequency of uptake of a linked marker sequence.
[0121] The term "active" refers to those forms of the polypeptide
which retain the biologic and/or immunologic activities of any
naturally occurring polypeptide. According to the invention, the
term "biologically active" means that the polypeptide retains at
least one of the biological activities of the polypeptide of the
invention. The term "stem cell growth factor activity" or "stem
cell growth factor-like activity" refers to biological activity
that is similar to the biological activity of stem cell growth
factor polypeptide, such as cell growth or morphogenesis
activity.
[0122] The term "naturally occurring polypeptide" refers to
polypeptides produced by cells that have not been genetically
engineered and specifically contemplates various polypeptides
arising from post-translational modifications of the polypeptide
including, but not limited to, acetylation, carboxylation,
glycosylation, phosphorylation, lipidation and acylation.
[0123] The term "derivative" refers to polypeptides chemically
modified by such techniques as ubiquitination, labeling (e.g., with
radionuclides or various enzymes), covalent polymer attachment such
as pegylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of amino acids such
as ornithine, which do not normally occur in human proteins.
[0124] The term "variant" (or "analog") refers to any polypeptide
differing from naturally occurring polypeptides by amino acid
insertions, deletions, and substitutions, created using, e g.,
recombinant DNA techniques. Guidance in determining which amino
acid residues may be replaced, added or deleted without abolishing
activities of interest, may be found by comparing the sequence of
the particular polypeptide with that of homologous peptides and
minimizing the number of amino acid sequence changes made in
regions of high homology (conserved regions) or by replacing amino
acids with consensus sequence.
[0125] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. "Conservative" amino acid substitutions may be
made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
"Insertions" or "deletions" are typically in the range of about 1
to 5 amino acids. The variation allowed may be experimentally
determined by systematically making insertions, deletions, or
substitutions of amino acids in a polypeptide molecule using
recombinant DNA techniques and assaying the resulting recombinant
variants for activity.
[0126] Alternatively, where alteration of function is desired,
insertions, deletions or non-conservative alterations can be
engineered to produce altered polypeptides. Such alterations can,
for example, alter one or more of the biological functions or
biochemical characteristics of the polypeptides of the invention.
For example, such alterations may change polypeptide
characteristics such as ligand-binding affinities, interchain
affinities, or degradation/turnover rate. Further, such alterations
can be selected so as to generate polypeptides that are better
suited for expression, scale up and the like in the host cells
chosen for expression. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0127] As used herein, "substantially equivalent" or "substantially
similar" can refer both to nucleotide and amino acid sequences, for
example a mutant sequence, that varies from a reference sequence by
one or more substitutions, deletions, or additions, the net effect
of which does not result in an adverse functional dissimilarity
between the reference and subject sequences. Typically, such a
substantially equivalent sequence varies from one of those listed
herein by no more than about 35% (i.e., the number of individual
residue substitutions, additions, and/or deletions in a
substantially equivalent sequence, as compared to the corresponding
reference sequence, divided by the total number of residues in the
substantially equivalent sequence is about 0.35 or less). Such a
sequence is said to have 65% sequence identity to the listed
sequence. In one embodiment, a substantially equivalent, e.g.,
mutant, sequence of the invention varies from a listed sequence by
no more than 30% (70% sequence identity); in a variation of this
embodiment, by no more than 25% (75% sequence identity); and in a
further variation of this embodiment, by no more than 20% (80%
sequence identity) and in a further variation of this embodiment,
by no more than 10% (90% sequence identity) and in a further
variation of this embodiment, by no more that 5% (95% sequence
identity). Substantially equivalent, e.g., mutant, amino acid
sequences according to the invention preferably have at least 80%
sequence identity with a listed amino acid sequence, more
preferably at least 85% sequence identity, more preferably at least
90% sequence identity, more preferably at least 95% sequence
identity, more preferably at least 98% sequence identity, and most
preferably at least 99% sequence identity. Substantially equivalent
nucleotide sequence of the invention can have lower percent.
sequence identities, taking into account, for example, the
redundancy or degeneracy of the genetic code. Preferably, the
nucleotide sequence has at least about 65% identity, more
preferably at least about 75% identity, more preferably at least
about 80% sequence identity, more preferably at least 85% sequence
identity, more preferably at least 90% sequence identity, more
preferably at least about 95% sequence identity, more preferably at
least 98% sequence identity, and most preferably at least 99%
sequence identity. For the purposes of the present invention,
sequences having substantially equivalent biological activity and
substantially equivalent expression characteristics are considered
substantially equivalent. For the purposes of determining
equivalence, truncation of the mature sequence (e.g., via a
mutation which creates a spurious stop codon) should be
disregarded. Sequence identity may be determined, e.g., using the
Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645).
Identity between sequences can also be determined by other methods
known in the art, e.g. by varying hybridization conditions.
[0128] Nucleic acid sequences encoding such substantially
equivalent sequences, e.g., sequences of the recited percent
identities can routinely be isolated and identified via standard
hybridization procedures well known to those of skill in the
art.
[0129] Where desired, an expression vector may be designed to
contain a "signal or leader sequence" which will direct the
polypeptide through the membrane of a cell. Such a sequence may be
naturally present on the polypeptides of the present invention or
provided from heterologous protein sources by recombinant DNA
techniques.
[0130] A polypeptide "fragment," "portion," or "segment" is a
stretch of amino acid residues of at least about 5 amino acids,
often at least about 7 amino acids, typically at least about 9 to
13 amino acids, and, in various embodiments, at least about 17 or
more amino acids. To be active, any polypeptide must have
sufficient length to display biological and/or immunological
activity.
[0131] Alternatively, recombinant variants encoding these same or
similar polypeptides may be synthesized or selected by making use
of the "redundancy" in the genetic code. Various codon
substitutions, such as the silent changes which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0132] The term "activated" cells as used in this application are
those which are engaged in extracellular or intracellular membrane
trafficking, including the export of secretory or enzymatic
molecules as part of a normal or disease process.
[0133] The term "purified" as used herein denotes that the
indicated nucleic acid or polypeptide is present in the substantial
absence of other biological macromolecules, e.g., polynucleotides,
proteins, and the like. In one embodiment, the polynucleotide or
polypeptide is purified such that it constitutes at least 95% by
weight, more preferably at least 99.8% by weight, of the indicated
biological macromolecules present (but water, buffers, and other
small molecules, especially molecules, having a molecular weight of
less than 1000 daltons, can be present).
[0134] The term "isolated" as used herein refers to a nucleic acid
or polypeptide separated from at least one other component (e.g.,
nucleic acid or polypeptide) present with the nucleic acid or
polypeptide in its natural source. In one embodiment, the nucleic
acid or polypeptide is found in the presence of (if anything) only
a solvent, buffer, ion, or other component normally present in a
solution of the same. The terms "isolated" and "purified" do not
encompass nucleic acids or polypeptides present in their natural
source.
[0135] The term "infection" refers to the introduction of nucleic
acids into a suitable host cell by use of a virus or viral
vector.
[0136] The term "transformation" means introducing DNA into a
suitable host cell so that the DNA is replicable, either as an
extrachromosomal element, or by chromosomal integration.
[0137] The term "transfection" refers to the taking up of an
expression vector by a suitable host cell, whether or not any
coding sequences are in fact expressed.
[0138] The term "intermediate fragment" means a nucleic acid
between 5 and 1000 bases in length, and preferably between 10 and
40 bp in length.
[0139] The term "secreted" includes a protein that is transported
across or through a membrane, including transport as a result of
signal sequences in its amino acid sequence when it is expressed in
a suitable host cell. "Secreted" proteins include without
limitation proteins secreted wholly (e.g., soluble proteins) or
partially (e.g., receptors) from the cell in which they are
expressed. "Secreted" proteins also include without limitation
proteins which are transported across the membrane of the
endoplasmic reticulum. "Secreted" proteins are also intended to
include proteins containing non-typical signal sequences (e.g.
Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992)
Cytokine 4(2):134-143) and factors released from damaged cells
(e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.
(1998) Annu. Rev. Immunol. 16:27-55)
[0140] Each of the above terms is meant to encompasses all that is
described for each, unless the context dictates otherwise.
5.1.1 DESCRIPTION OF THE INVENTION
[0141] Since stromal cells can support the proliferation or the
survival of hematopoietic stem cells or hematopoietic progenitor
cells ex vivo, stromal cells are expected to produce factors
mediating support proliferation or survival of hematopoietic stem
cells or hematopoietic progenitor cells, as defined herein.
[0142] An object of the present invention is to provide a factors
supporting proliferation or survival of hematopoietic stem cells or
hematopoietic progenitor cells and these factors are/can be derived
from the stromal cells.
[0143] Mouse stromal cells produce factors supporting the
proliferation or the survival of hematopoietic stem cells or
hematopoietic progenitor cells, as mentioned above. Attention is
given that there are two kinds of stromal cells. One has an ability
to support the proliferation or survival of hematopoietic stem
cells or hematopoietic progenitor cells (hereafter sometimes
referred to as "activity to support hematopoietic stem cells"). The
other does not have the activity to support hematopoietic stem
cells. This difference in the abilities maybe due to differential
expression of the factors that facilitate supporting hematopoietic
stem cells or progenitor cells at the transcription level. That is
to say it is speculated that the supportive stromal cells express
at high levels of mRNAs coding the factors and that non-supportive
stromal cells express less mRNAs. Thus mRNAs that code for the
factors maybe among the genes expressed higher in the supportive
cells compared to in the non-supportive cells. In this context, the
inventors confirmed the hematopoietic stem and/or progenitor cell
supporting ability of AGM-s3-A9, AGM-s3-D11, OP9, and SWISS3T3 cell
lines and the non-supportive ability of AGM-s3-A7, AGM-s3-G1, and
NIH3T3 cell lines (AGM-s3-A9, AGM-s3-D11, AGM-s3-A7, and AGM-s3-G1
cell lines are obtained by subcloning the stromal cell strain
AGM-s3 derived from AGM disclosed in the prior application
WO99/03980). Next, the genes that are highly expressed in
AGM-s3-A9, AGM-s3-D11, OP9, and 3T3Swiss cell lines and show low
expression or are undetected in AGM-s3-A7, AGM-s3-G1, and NIH3T3
cell lines were identified. After the assessment of the abilities
of supporting the proliferation or the survival of the
hematopoietic stem cells or the hematopoietic progenitor cells of
these gene groups and careful examinations, the present invention
has been completed.
[0144] That is, the present invention provides the followings.
[0145] (1) A DNA coding for a polypeptide as defined in the
following (A) or (B):
[0146] (A) a polypeptide which has an amino acid sequence
comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32,
or an amino acid sequence comprising at least amino acid residues
22 to 272 of SEQ ID NO: 34; or
[0147] (B) a polypeptide which has an amino acid sequence including
deletion, substitution or insertion of one or several amino acids
in the amino acid sequence comprising at least amino acid residues
22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at
least amino acid residues 22 to 272 of SEQ ID NO: 34, and which has
an activity to support proliferation or survival of hematopoietic
stem cell or hematopoietic progenitor cell, with a proviso that
C-terminal amino acid sequence dose not comprise the amino acid
sequence of SEQ ID NO: 45. [0148] (2) The DNA according to (1),
which is a DNA as defined in the following (a) or (b):
[0149] (a) a DNA which comprises at least nucleotides 574 to 1347
of SEQ ID NO: 31; or
[0150] (b) a DNA which is hybridizable with the nucleotide sequence
of SEQ ID NO: 31 or a probe or fragment prepared from the sequence,
under the stringent condition, and which has an activity to support
proliferation or survival of hematopoietic stem cell or
hematopoietic progenitor cell. [0151] (3) The DNA According to (2),
the Stringent Condition is 6.times.SSC5.times. Denhardt, 0.5% SDS
and 68.degree. C. (SSC 3M NaCl, 0.3M sodium citrate, 50.times.
Denhardt 1% BSA 1% polyvinyl pyrrolidone, 1% Ficoll 400, or
6.times.SSC, 5.times. Denhardt, 0.5% SDS, 50% formamide and
42.degree. C. [0152] (4) The DNA according to (1), which is a DNA
as defined in the following (a) or (b):
[0153] (a) a DNA which comprises at least nucleotides 321 to 1074
of SEQ ID NO: 33; or
[0154] (b) a DNA which is hybridizable with the nucleotide sequence
of SEQ ID NO: 33 or a prove prepared from the sequence, under the
stringent condition, and which has an activity to support
proliferation or survival of hematopoietic stem cell or
hematopoietic progenitor cell. [0155] (5) The DNA according to (4),
the stringent condition is 6.times.SSC 5.times. Denhardt 0.5% SDS
and 68.degree. C. (SSC 3M NaCl, 0.3M sodium citrate, 50.times.
Denhardt 1% BSA 1% polyvinyl pyrrolidone, 1% Ficoll 400, or
6.times.SSC, 5.times. Deanhardt, 0.5% SDS, 50% Formamide and
42.degree. C. [0156] (6) A expression vector which comprises a DNA
of any one of (1) to (5) or other polynucleotides of the invention
in such a manner that the DNA can be expressed. [0157] (7) A cell
which is introduced (i.e., transformed or transfected) with a DNA
of any one of (1) to (5) or other polynucleotides of the invention
in such a manner that the DNA can be expressed. [0158] (8) A
polypeptide (An isolated polypeptide) which is an expression
product of a DNA according to any one of (1) to (5) or other
polynucleotides of the invention, the polypeptide having an
activity to support proliferation or survival of hematopoietic stem
cells or hematopoietic progenitor cells, with a proviso that
C-terminal amino acid sequence dose not comprise the amino acid
sequence of SEQ ID NO: 14. [0159] (9) The polypeptide according to
(8), which has an amino acid sequence comprising at least amino
acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence
including deletion, substitution or insertion of one or several
amino acids in the amino acid sequence comprising at least amino
acid residues 22 to 279 of SEQ ID NO: 32. [0160] (10) The
polypeptide according to (8), which has an amino acid sequence
comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34,
or an amino acid sequence including deletion, substitution or
insertion of one or several amino acids in the amino acid sequence
comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34.
[0161] (11) The polypeptide according to (8) or other polypeptides
of the invention, which is modified with one or more modifying
agent selected from the group consisting of polyethylene glycol
(PEG), dextran, poly(N-vinyl-pyrrolidone), polypropylene glycol
homopoymer, copolymer of polypropylene oxide/ethylene oxide,
polyoxyethylated polyol and polyvinyl alcohol. [0162] (12)
Pharmaceutical composition having an effect to support
proliferation or survival of hematopoietic stem cells or
hematopoietic progenitor cells, which comprises the polypeptide as
defined in the following (A) (B) or (C): (A) a polypeptide which
has an amino acid sequence comprising at least amino acid residues
22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at
least amino acid residues 22 to 272 of SEQ ID NO: 34; or
[0163] (B) a polypeptide which has an amino acid sequence including
deletion, substitution or insertion of one or several amino acids
in the amino acid sequence comprising at least amino acid residues
22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at
least amino acid residues 22 to 272 of SEQ ID NO: 34, and which has
an activity to support proliferation or survival of hematopoietic
stem cell or hematopoietic progenitor cell, or
[0164] (C) any of the other polypeptides of the invention described
therein. [0165] (13) A monoclonal antibody which binds to the
polypeptide of (9) to (11).
[0166] Terms used in this specification are defined as follows.
[0167] A hematopoietic stem cell is defined as a cell having
totipotency, that is, a capacity to differentiate into all the cell
lineages of the hematopoietic cells, and simultaneously having a
potency of self-renew with retaining the totipotency. Erythrocyte
precursor cells hardly survive and proliferate in vitro culture
circumstances and rapidly disappear. If the survival and the
proliferation of the erythrocyte precursor cells are confirmed,
continuous production of the erythrocyte precursor cells seems to
occur due to the survival and/or the proliferation of the more
immature hematopoietic stem cells or the hematopoietic progenitor
cells. Therefore, to assess the survival and/or proliferation of
the human hematopoietic stem cells, to enumerate the erythrocyte
precursor cells ((BFU-E, CFU-E, and CFU-Emix) in cultures is an
appropriate way.
[0168] A hematopoietic progenitor cell is defined as a cell which
can differentiate a single cell lineage of the hematopoietic
lineage or a plural cell lineages but cannot differentiate into all
of the cell lineages. A stromal cell is defined as a cell which can
be co-cultured together with the hematopoietic stem cells in vitro
to simulate in vivo hematopoietic environment. Cells derived from
any origin can be used as long as the cells can be co-cultured with
the hematopoietic cells in vitro.
[0169] Polypeptides in accordance with the present invention have
an activity to support proliferation or survival of hematopoietic
stem cells or hematopoietic progenitor cells. The concrete
embodiment of the polypeptides in accordance with the present
invention are an expressed product (hereafter sometimes referred to
as a mouse "supporting factor for the proliferation of stem cells")
of a gene named SCR-1 isolated from a mouse stromal cell (hereafter
sometimes referred to as "mouse SCR-1") and an expressed product
(hereafter sometimes referred to as a human "supporting factor for
the proliferation of stem cells") of a human orthologous gene
thereof (hereafter sometimes referred to as "human SCR-1"). The
term SCR-1 may be used herein to refer to the polypeptide sequences
set out in SEQ ID NO: 10, 13, 16, 32 and 34 which are respectively
encoded by the polnucleotide sequences set out in SEQ ID NOS: 9,
11, 12, 31 and 33.
[0170] Although an amino acid sequence of the expressed product of
human SCR-1 (SEQ ID NO: 34) has homology at 97.4% with the known
polypeptide (WO98/49302) whose function has not been clear, the
amino acid sequence at the C-terminal region thereof differs, so
that it is a novel polypeptide. A part of the amino acid sequence
in the above described polypeptide having unknown functions which
is different from that in SEQ ID NO: 34 is shown in SEQ ID NO: 45.
On the other hand, mouse SCR-1 has homology at 84.6% with the
above-described polypeptide.
[0171] The above described homologies are calculated as percentage
of the number of same amino acids to the total number of amino
acids using a comparison manually (266/273 and 237/280,
respectively).
[0172] The supporting factor for the proliferation of stem cells,
that is, the polypeptides in accordance with the present invention
can be produced by preparing transformed cells by transducing mouse
or human SCR-1 into appropriate host cells and by expressing the
DNAs in the transformed cells. When DNA including a nucleotide
sequence shown in SEQ ID NO: 31 is used as SCR-1, a mouse
supporting factor for the proliferation of stem cells is obtained.
When DNA including a nucleotide sequence shown in SEQ ID NO: 33 is
used as SCR-1, a human supporting factor for the proliferation of
stem cells is obtained. The mouse supporting factor for the
proliferation of stem cells and the human supporting factor for the
proliferation of stem cells comprise amino acid sequences
represented by SEQ ID NO: 32 and SEQ ID NO: 34, respectively. These
supporting factors for the proliferation of stem cells are
precursors including signal peptides, and are assumed to be
processed to mature supporting factors for the proliferation of
stem cells in mouse or human cells. As based on the results of
Signal P test which searches breakage sites of the signal peptides
in these amino acid sequences (Nielsen H., protein Engineering, 10:
1-6, 1997; Nielsen H., Int. J. Neural Sys., 8: 581-599, 1997), the
breakage or cleavage sites seem to exist between the amino acid 21
and the amino acid 22 in the amino acid sequences of SEQ ID NO: 32
and SEQ ID NO: 34.
[0173] The mouse mature supporting factor for the proliferation of
stem cells comprises the amino acid sequence represented by amino
acids 22 to 279 of SEQ ID NO: 32. The human mature supporting
factor for the proliferation of stem cells comprises the amino acid
sequence represented by amino acids 22 to 272 of SEQ ID NO: 34.
[0174] When supporting factors for the proliferation of stem cells
are prepared, SCR-1 which is transferred into host cells may be DNA
coding precursor polypeptide or DNA coding mature polypeptide. An
example of the DNA coding the mouse mature supporting factor for
the proliferation of stem cells comprises the DNA comprising at
least a nucleotide sequence consisting of nucleotide numbers 574 to
1347 of SEQ ID NO: 31. An example of the DNA coding the human
mature supporting factor for the proliferation of stem cells
comprises the DNA comprising at least a nucleotide sequence
consisting of nucleotide numbers 321 to 1074 of SEQ ID NO: 33.
[0175] DNA in accordance with the present invention may code the
above described factors which have amino acid sequences including
substitution, deletion or insertion of one or several amino acids,
as long as the activity of the supporting factor for the
proliferation of stem cells to be coded is not lost. DNAs coding
substantially identical polypeptides to this supporting factor for
the proliferation of stem cells are obtained by modifying the
nucleotide sequences so as to include substitution, deletions
insertion, addition, or inversion of amino acid residues in a
specific region using site-directed mutagenesis.
[0176] The DNAs including the above described mutation can be
expressed in appropriate cells and the activity to support the
hematopoietic stem cells of the expressed products can be examined,
so that the DNAs coding the polypeptide having functions which are
substantially identical to this supporting factor for the
proliferation of stem cells are obtained. In addition, the DNAs
coding substantially identically active protein as this supporting
factor for the proliferation of stem cells can be obtained by
hybridization with DNAs including, for example, the nucleotide
sequence as described in SEQ ID NO: 1 or SEQ ID NO: 3 from the
cells having thereof, or probes prepared from these DNAs under the
stringent condition; and by isolating the DNAs coding the protein
possessing the activity to support the hematopoietic stem cells.
The stringent condition is, for example, one in which DNAs having
homology at not less than 70%, preferably at not less than 80%, are
hybridized each other and DNAs having less homology than those are
not hybridized each other. The above described stringent condition
is 6.times.SSC, 5.times. Denhardt, 0.5% SDS, 68.degree. C. (SSC; 3M
NaCl, 0.3M sodium citrate) (50.times. Denhardt; 1% BSA, 1%
polyvinyl pyrrolidone, 1% Ficoll 400) or 6.times.SSC, 5.times.
Deanhardt, 0.5% SDS, 50% Formamide, 42.degree. C., or the like.
Strategy of hybridization is further defined by fmal wash
conditions as set out herein.
[0177] Microorganisms such as Escherichia coli and yeast, culture
cells derived from animals or plants, and the like are used for
host cells for expressing SCR-1. Preferably, culture cells derived
from mammals are used as the host cells. In the case that
prokaryotic cells are used as the host cells, the expression is
preferably performed in a condition in which a signal peptide
region is replaced with a leader sequence suitable for the
prokaryotic cells such as--lactamase (bla), alkaline phosphatase
(phoA), and outer membrane protein A (ompA) and the like, or in a
form in which a methionine residue is added to the N-terminal site
of the mature protein.
[0178] The supporting factor for the proliferation of stem cells
obtained as above may be added with sugar chains at any of
positions 23, 36 and 137, alone, or a plurality of positions
thereof in mouse SCR-1. The supporting factor for the proliferation
of stem cells obtained as above may be added with sugar chains at
any of positions 23, 36, 137 or 194, alone, or a plurality of
positions thereof in human SCR-1.
[0179] For example, SCR-1 is integrated into a vector corresponding
to the host in a form capable of expression and the obtained
recombinant vector is transferred into the host cells, so that the
transfer of SCR-1 into the host cells is completed.
[0180] Examples of the culture cells derived from mammals are CHO
cells, 293 cells, COS7 cells, and the like. Gene expression
regulatory sequence such as a promoter to express SCR-1 may be
originated from SCR-1 itself, or may be derived from other genes
such as cytomegalovirus promoter and elongation factor 1 promoter
and the like.
[0181] Examples of a vector for animal culture cells are plasmid
vectors, retrovirus vectors, adenovirus vectors (Neering, S. J.,
Blood, 88: 1147, 1996), herpes virus vectors (Dilloo, D., Blood,
89: 119, 1997), HIV vectors, and the like.
[0182] In order to transfer the recombinant vector into culture
cells, the conventional methods which are usually employed for
transformation of culture cells such as calcium phosphate
transfection, liposome method, DEAE dextran method, electroporation
and microinjection method are employed.
[0183] The polypeptides in accordance with the present invention
also comprise polypeptides having amino acid sequences in which one
or several amino acids are substituted, deleted or inserted in the
amino acid sequence represented in SEQ ID NO: 32 or SEQ ID NO: 34
or other polynucleotides of the invention, and having activity to
support the hematopoietic stem cells in addition to the
polypeptides having the amino acid sequence represented in SEQ ID
NO: 32 or SEQ ID NO: 34 or other polynucleotides of the invention.
That is, even if a mouse and a human supporting factor for the
proliferation of stem cells is modified by substitution, deletion,
insertion or the like, polypeptides holding essential functions as
a supporting factor for the proliferation of stem cells can be
considered to be substantially identical with the supporting factor
for the proliferation of stem cells. The above described "several"
denotes ranging from two to 110, and preferably ranging from two to
55 as a total number depending on the region of polypeptide in
accordance with the present invention.
[0184] These modified supporting factors for the proliferation of
stem cells can be obtained by treating DNA coding the supporting
factors for the proliferation of stem cells or host cells including
the above mentioned DNA with mutagens, or by mutating the above
mentioned DNA so as to substitute, delete, or insert an amino acid
at a specific site using site-directed mutagenesis. The residual of
the activity to support hematopoietic stem cells in the obtained
mutant polypeptides is confirmed by the examples described below.
That is, after the cultured hematopoietic stem cells which express
the mutant polypeptides are transferred into irradiated mice,
peripheral hematological cellularity after the transfer may be
observed over time.
[0185] Since the nucleotide sequences of the invention have been
described, the modified supporting factor for the proliferation of
stem cells can be also obtained by isolating the corresponding DNAs
from mouse or human cDNA or chromosome DNA libraries using PCR in
which the oligonucleotides prepared based on these nucleotide
sequences are used as primers or using hybridization in which the
oligonucleotides prepared based on these nucleotide sequences are
used as probes.
[0186] In one aspect, the DNAs in accordance with the present
invention was isolated from cDNA library of AGM-s3-A9 cells which
are a mouse stromal cell strain having the activity to support
hematopoietic stem cells using SBH (Sequencing By Hybridization)
method (Drmanac, S., Nat. Biotechnol., 16. 54, 1998; Drmanac, R.,
Methods. Enzymol., 303, 165, 1999) as described below. The mouse
stromal cell lines having the activity to support hematopoietic
stem cells can be obtained using the method disclosed in WO99/03980
or from Cell Development Bank of Institute of Physical and Chemical
Research (RIKEN) or ATCC.
[0187] An outline of SBH method will be described below. Probes
including eight or nine nucleotides whose sequences are different
from each other are prepared. When the nucleotide sequences
corresponding to those of the probe exist in targeted gene, the
probes can hybridize with the gene. The hybridization can be easily
detected with utilization of radio isotope or fluorescence
conjugated probes. Each clone in the library is picked up, and
blotted on a membrane. Then, repeated hybridizations are performed
with the above described probes, so that one can identify the
combination of probes that hybridize to each clone. Since the
combination of probes that hybridize to each gene depend on the
sequences of clones, identical genes have identical signature
hybridization patterns with the probes. That is, the same gene can
be identified as a one group (cluster) according to the signature
of the hybridized probes. The number of clones derived from each
gene in the library can be determined by clustering and counting
the members of the clusters based on the hybridization profiles of
the probes. Thus, incidence of expression of each gene in the
library can be determined.
[0188] Clustering analysis was performed for cDNA libraries derived
from supportive and non-supportive stromal cell lines. Thus,
incidences of expressed genes among cells were compared, so that
the genes specifically highly expressed in the supportive stromal
cell lines were selected. The incidences of these genes in each
cell were further examined by Northern blot analysis, so that genes
which highly expressed in the cells having activity to support the
hematopoietic stem cells were obtained.
[0189] SCR-1 is one of the genes which was highly expressed with
specificity in the supporting cells obtained through the above
described process. After clustering and analyzing using Northern
blot analysis, the gene comprising nucleotide numbers 1032 to 1484
of SEQ ID NO: 31 was identified. The complete gene encoding SCR-1
was cloned from the cDNA library derived from AGM-s3-A9 cells.
[0190] Further, in order to assess supporting ability for
hematopoiesis of SCR-1, a gene fragment including ORF (nucleotide
numbers 511 to 1350 of SEQ ID NO: 31) in SCR-1 gene was transferred
into stromal cells (AGM-s3-A7 cell) which cannot support the
hematopoietic stem cells using a retrovirus vector, and assessed
the change in the activity to support the hematopoietic stem cells
of the stromal cells. Substantially, after the stromal cells which
were not transferred with the gene and those which were transferred
with the gene were independently co-cultured with the mouse
hematopoietic stem cells, the hematopoietic cells were transplanted
into irradiated mice. Engraftment of the co-cultured hematopoietic
cells in recipient mice were examined. As a result, the mice
transplanted with the hematopoietic stem cells which were
co-cultured with the AGM-s3-A7 cell line transferred with SCR-1
showed increased chimerism after the transplantation compared with
the AGM-s3-A7 cell line which were not transferred with SCR-1 gene.
This result shows that the AGM-s3-A7 stromal cells that express
SCR-1 have obtained supporting activity for the proliferation or
survival of the hematopoietic stem cells or the hematopoietic
progenitor cells. As a result, it has become evident that SCR-1 has
a function to add the above described activity to the stromal cells
that originally do not posses the activities for supporting
proliferation or survival of hematopoietic stem cells or
hematopoietic progenitor cells. Therefore, it was revealed that
SCR-1 has an activity to support the survival or the proliferation
of the hematopoietic stem cell or the hematopoietic progenitor
cell, or has an activity to add an activity to support the
hematopoietic stem cells to stromal cells.
[0191] The polypeptides in accordance with the present invention
can be used as a medicine to proliferate or support human
hematopoietic stem cell or human hematopoietic progenitor cell.
This pharmaceutical composition can be used for supporting
proliferation or survival of human hematopoietic stem cells or
human hematopoietic progenitor cells ex vivo. It is of problem for
hematopoietic stem cell transplantation therapies such as
peripheral blood stem cell transplantation and cord blood stem cell
transplantation that sometimes sufficient amount of the
hematopoietic stem cells cannot be collected and the
transplantation may not be performed. Even if enough stem cells
could not be collected, a sufficient amount of the hematopoietic
stem cells could be obtained (and transplanted) by amplification of
the hematopoietic stem cells in vitro using polypeptides of the
invention. That is, hematopoietic stem cells can be amplified
without differentiation by culturing the hematopoietic stem cells
in culture medium including polypeptides of the invention. It may
be considered the hematopoietic stem cells are able to be amplified
more efficiently with addition of a variety of cytokines to the
medium.
[0192] When hematopoietic stem cells or hematopoietic progenitor
cells are cultured in the medium including the polypeptides in
accordance with the present invention, the hematopoietic stem cells
or the hematopoietic progenitor cells that will be used may be one
of these cell types alone or may be both of the cell types. In
addition, the cells should include at least the hematopoietic stem
cells or the hematopoietic progenitor cells, and may include other
hematopoietic cells. Further, polypeptides of the invention can be
used for hematopoietic stem or progenitor expansion of purified
hematopoietic stem cell fraction or progenitor cell fractions from
the cell populations that contain the hematopoietic stem cells or
progenitor cells.
[0193] Examples of sources of hematopoietic stem cells and
hematopoietic progenitor cells in the methods in accordance with
the present invention are fetal liver, bone marrow, fetal bone
marrow, peripheral blood, peripheral blood from persons from whom
stem cells are mobilized by cytokines and/or dosing of antitumor
drugs, cord blood, and the like of mammals such as human and mouse
and the like. Any sources may be used as long as the tissue
includes the hematopoietic stem cells.
[0194] A culture method using petri dishes and flasks for culture
may be employed to culture the hematopoietic stem cells or the
hematopoietic progenitor cells. The cultivation of the
hematopoietic stem cells and/or progenitor cells may be improved by
mechanically controlling medium composition, pH, and the like, and
employing a bioreactor capable of high density cultivation
(Schwartz, Proc. Natl. Acad. Sci. U.S.A., 88: 6760, 1991; Koller,
M. R., Bio/Technology, 11: 358, 1993; Koller, M. R., Blood, 82:
378, 1993; Palsson, B. O., Bio/Technology, 11: 368, 1993).
[0195] Since SCR-1 can increase activities of stromal cells to
support the hematopoietic stem cells under the conditions of
co-culture of stromal cells and hematopoietic cells, the
hematopoietic stem cells and/or progenitor cells can be efficiently
expanded when whole bone marrow cells are cultured in the presence
of SCR-1. This type of co-culture of the stromal cells and the
hematopoietic cells can be performed simply after the collection of
the bone marrow cells without complicated cell separation.
Furthermore, one can perform co-culture with separate components
such as hematopoietic stem cells, progenitor cells and stromal
cells from collected bone marrow cells and combine the
hematopoietic cells and stromal cells from different individuals.
Furthermore, one can grow stromal cells and establish stromal cell
culture prior to co-culture with the hematopoietic stem cells for
the hematopoietic stem cells or progenitor cell expansion. At this
time, one can utilize cell stimulating factors to promote growth
and survival of stromal cells to establish stromal cell culture.
Examples of cell stimulating factors includes growth factors which
are typically a cytokine such as SCF (stem cell factor), IL-3
(interleukin-3), GM-CSF (granulocyte/macrophage colony-stimulating
factor), IL-6 (interleukin-6), TPO (thrombopoietin), G-CSF
(granulocyte colony-stimulating factor), TGF-b (transforming growth
factor-b), MIP-1a (Davatelis, G., J. Exp. Med. 167: 1939, 1988);
differentiation and proliferation control factors such as
hematopoietic hormones such as EPO (erythropoietin), chemokine, Wnt
gene product, and notch ligand; and development control
factors.
[0196] In addition, the proliferation and the survival of
hematopoietic stem cells or hematopoietic progenitor cells can be
retained by culturing the hematopoietic stem and/or progenitor
cells with recombinant SCR-1 alone or combination with the cell
stimulating factors without stromal cells. Examples of the cell
stimulating factors used in this case are above described cell
stimulating factors and the like.
[0197] Medium used for culture is not specially restricted as long
as the proliferation or the survival of the hematopoietic stem
cells or the hematopoietic progenitor cells is not perturbed.
Preferable media are, for example, MEM-.alpha. medium (GIBCO BRL),
SF-02 medium (Sanko Junyaku), Opti-MEM medium (GIBCO BRL), IMDM
medium (GIBCO BRL), and PRMI1640 medium (GIBCO BRL). A culture
temperature is usually ranging from 25 to 39.degree. C., and
preferably ranging from 33 to 39.degree. C. Examples of additives
to the medium are fetal bovine serum, human serum, horse serum,
insulin, transferrin, lactoferrin, ethanolamine, sodium selenite,
monothiolglycerol, 2-mercaptoethanol, bovine serum albumin, sodium
pyruvate, polyethylene glycol, a variety of vitamins, and a variety
of amino acids. A concentration of CO.sub.2 is usually ranging from
four to six percent, and preferably five percent.
[0198] Since hematopoietic stem cells can differentiate into all
hematopoietic cell lineages, hematopoietic stem cells can be
manipulated to be differentiated into a specific cell type in
vitro, and then the specific cells can be transplanted. For
example, when erythrocytes are necessary, after cultivation and
expansion of the patient's stem cells, hemopoietic cells whose main
component is the erythrocyte can be artificially produced using an
erythrocyte differentiation induction or promoting factors such as
EPO.
[0199] The hematopoietic stem cells or the hematopoietic progenitor
cells cultured using the polypeptides in accordance with the
present invention can replace as a graft for the conventional bone
marrow transplantation or cord blood transplantation.
Transplantation of the hematopoietic stem cells is superior to the
conventional hematopoietic cell transplantation therapy, since the
graft can take semipermanently.
[0200] The transplantation of the hematopoietic stem cells can be
employed as therapy for a variety of diseases in addition to as
combination therapy for total body X-ray irradiation therapy or
advanced chemotherapy for leukemia. For example, when therapy
accompanied with myelosuppression as an adverse reaction such as
chemotherapy, radiation therapy, and the like is performed for the
patient with solid cancer, hematological disorder, hematological
failure can be early improved as follows. The bone marrow is
collected before the therapy and the hematopoietic stem cells or
the hematopoietic progenitor cells are allowed to expand in vitro.
Then, the expanded cells are infused to the patient after the
therapy, so that the patient can get benefit of early recovery and
stronger chemotherapy than the conventional one can be performed to
improve the therapeutic effect of the chemotherapy. In addition,
the hematopoietic stem cells or the hematopoietic progenitor cells
obtained according to the present invention are differentiated into
a variety of hematopoietic cells. The transplantation of these
cells into a patient with hypoplasia of a given hematopoietic cells
can improve the patient's deficient status. In addition, this
therapy can improve dyshemopoietic anemia to develop anemia such as
aplastic anemia caused by bone marrow hypoplasia. Furthermore,
examples of diseases in which the transplantation of the
hematopoietic stem cells according to the present invention is
effective are immunodeficiency syndrome such as chronic
granulomatous disease, duplicated immunodeficiency syndrome,
agammaglobulinemia, Wiskott-Aldrich syndrome, acquired
immunodeficiency syndrome (AIDS), and the like, thalassemia,
hemolytic anemia due to enzyme defect, congenital anemia such as
sicklemia, Gaucher's disease, lysosomal storage disease such as
mucopolysaccharidosis, adrenal white matter degeneration, a variety
of cancers and tumors, and the like.
[0201] Transplantation of hematopoietic stem cells may be performed
in the same manner as conventional bone marrow transplantation or
cord blood transplantation other than the differences of the cells
used.
[0202] The hematopoietic stem cells which may be used for the above
described hematopoietic stem cell transplantation are derived from
not only bone marrow but also the above described fetal liver,
fetal bone marrow, peripheral blood, peripheral blood with stem
cells induced by cytokines and/or dosing of antitumor drugs, cord
blood, and the like.
[0203] The graft may be a composition including buffer solution and
the like in addition to the hematopoietic stem cells and the
hematopoietic progenitor cells produced by the method according to
the present invention.
[0204] The hematopoietic stem cells or the hematopoietic progenitor
cells produced according to the present invention may be used for
ex vivo gene therapy. Since the incidence of recombination of
target genes to the chromosome is low due to dormancy of the stem
cells, differentiation of stem cells during the culture period, and
the like, gene therapy to the hematopoietic stem cells has been
hard to established. However, the present invention can amplify
stem cells without differentiation, so that efficacy of gene
transfer is expected to be remarkably improved. In gene therapy, a
foreign gene (a gene for therapy) is transferred into the
hematopoietic stem cells or the hematopoietic progenitor cells, and
then the obtained gene-transferred cells are used. The foreign gene
to be transferred is appropriately selected according to disease.
Examples of diseases in which the target cells of gene therapy is
the hematopoietic cells include immunodeficiency syndrome such as
chronic granulomatous disease, duplicated immunodeficiency
syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, acquired
immunodeficiency syndrome (AIDS), and the like, thalassemia,
hemolytic anemia due to enzyme defect, congenital anemia such as
sicklemia, Gaucher's disease, lysosomal storage disease such as
mucopolysaccharidosis, adrenal white matter degeneration, a variety
of cancers and tumors, and the like.
[0205] Usual method used for transfer of a gene into animal cells
is employed for the transfer of the gene for the therapy into
hematopoietic stem cells or hematopoietic progenitor cells.
Examples are a method using a vector for animal cells derived from
virus utilized for gene therapy such as retrovirus vector such as
Moloney mouse leukemia virus, adenovirus vector, adeno-associated
virus (AAV) vector, herpes simplex virus vector, and HIV vector
(with respect to a vector for gene therapy, see Verma, I. M.,
Nature, 389: 239, 1997); calcium phosphate transfection,
DEAE-dextran transfection, electroporation, liposome method,
lipofection method, microinjection method, and the like. Among
them, methods using retrovirus vector, adeno-associated virus
vector, or HIV vector are preferable, since expression of a gene is
permanently expected due to insertion into the chromosome DNA of a
target cell.
[0206] For example, adeno-associated virus (AAV) vector can be
prepared as follows. First, a vector plasmid inserted a gene for
therapy into ITR (inverted terminal repeat) at both ends of
wild-type adeno-associated virus DNA and a helper plasmid for
supplementing virus protein are transfected into 293 cell strain.
Next, adenovirus as helper virus is infected, so that virus
particles including the AAV vector are produced. Alternatively,
instead of adenovirus, a plasmid which expresses adenovirus gene
coding helper function may be transfected. The obtained virus
particles are infected to the hematopoietic stem cells or the
hematopoietic progenitor cells. Preferably, appropriate promoter
and enhancer are inserted into upstream region of the target gene
in the vector DNA, so that the expression of the gene is regulated.
When marker gene such as a drug resistant gene is used in addition
to the gene for therapy, cells transferred with the gene for
therapy are easily selected. The gene for therapy may be sense gene
or antisense gene.
[0207] A composition for gene therapy may include buffer solution
and a novel active ingredient and the like in addition to the
hematopoietic stem cells or the hematopoietic progenitor cells by
the method according to the present invention.
[0208] A vector for gene therapy can be produced by transferring
SCR-1 in expression vector using a usual method. This vector for
gene therapy is useful to treat diseases which need survival and
proliferation of the human hematopoietic stem cells. That is, a
vector producing SCR-1 is transferred into the hematopoietic stem
cells and the cells are cultured in vitro, so that the
hematopoietic stem cells or the hematopoietic progenitor cells can
proliferate dominatingly. The hematopoietic stem cells can
proliferate in vivo caused by returning these hematopoietic stem
cells thus treated. The hematopoietic stem cells can significantly
proliferate in vivo by introducing this vector for gene therapy
into the body. Alternatively, the bone marrow cells derived from a
patient are cultured and transferred with this vector for gene
therapy, so that the hematopoietic stem cells or the hematopoietic
progenitor cells can be proliferated in culture system.
Alternatively, this vector for gene therapy is transferred into
stromal cell derived from bone marrow and cultivated and
mesenchaymal stem cell, so that the activity to support
hematopoietic stem cells can be added or increased.
[0209] As shown in Examples, since it is possible that the stromal
cells without the activity to support the hematopoietic stem cells
can be modified to include this activity using SCR-1, stromal cells
derived from human or mouse can have the activity to support the
hematopoietic stem cells by gene transferring SCR-1. The stromal
cells expressing SCR-1 and hematopoietic stem cells or
hematopoietic progenitor cells are co-cultured, so that the
hematopoietic stem cells or the hematopoietic progenitor cells can
exist and proliferate so as to be useful for a variety
treatment.
[0210] Since the hematopoietic stem cells or the hematopoietic
progenitor cells can survive and proliferate by expression of SCR-1
in the stromal cell, an activity to support the hematopoietic stem
cells of the stromal cells can be assessed using the expression of
SCR-1 as an index. The expression of SCR-1 in the stromal cells can
be confirmed using antibody to SCR-1. PCR primers can be prepared
from genes included in SEQ ID NO: 31, SEQ ID NO: 33 or other
polynucleotides of the invention and RNA is prepared from the
stromal cells of interest, and RT-PCR is performed, so that the
expression of SCR-1 can be confirmed. Antibody to SCR-1 will be
described below.
[0211] The pharmaceutical composition in accordance with the
present invention can be administered to human. The pharmaceutical
composition having an activity to proliferate or to support the
human hematopoietic stem cells or the hematopoietic progenitor
cells can be produced by mixing medically acceptable diluent,
stabilizer, carrier, and/or other additives with the polypeptides
in accordance with the present invention. At this time, in order to
increase the stability of the protein in vivo the polypeptides in
accordance with the present invention may be modified by a
modifying agent. Examples of the modifying agent are polyethylene
glycol (PEG), dextran, poly(N-vinyl-pyrrolidone), polypropylene
glycol homopolymer, polypropylene oxide/ethylene oxide copolymer,
polyoxyethylated polyol, polyvinyl alcohol, and the like. Examples
of modification of protein with PEG are a method in which activated
ester derivatives of PEG is reacted with the protein, a method in
which aldehyde derivatives at end portion of PEG is reacted with
protein under the presence of a reducing agent, and the like.
Japanese Unexamined Patent Application No. 10-510980 discloses
modification of such protein in detail.
[0212] When the pharmaceutical composition in accordance with the
present invention is administered to human, recovery from
hematological suppression due to an adverse drug reaction of
carcinostatics; early recovery of hematopoietic cells at bone
marrow transplantation, peripheral blood stem cell transplantation,
or cord blood transplantation; and recovery of hematopoietic
function at pancytopenia such as aplastic anemia (AA) and
myelodysplastic syndrome (MDS) are expected.
[0213] The antibody in accordance with the present invention reacts
specifically to the above described polypeptides in accordance with
the present invention. This antibody may be monoclonal antibodies
or polyclonal antibodies as long as they react specifically to the
above described polypeptides.
[0214] The antibody in accordance with the present invention can be
prepared according to usual methods. For example, the antibody can
be prepared either in vivo method in which animals are additionally
immunized by antigen together with adjuvant once or several times
at an interval of several weeks or in vitro method in which immune
cells are isolated and sensitized in an appropriate culture system.
Examples of immune cells which can produce the antibody in
accordance with the present invention are splenic cells, tonsillar
cells, lymph gland cells, and the like.
[0215] The whole polypeptide according to the present invention is
not necessarily used as an antigen. A part of a polypeptide of the
invention may be used as an antigen. When the antigen is a short
peptide, particularly, a peptide made of about 20 amino acid
residues, it may be used by binding it to a carrier protein having
high antigenicity such as keyhole lympet hemocyanin or bovine serum
albumin using chemical modification and the like. Alternatively,
the antigen may be used by covalently binding it to a peptide
having branching skeleton such as lysine core MAP peptide instead
of the carrier protein (Posnett et al., J. Biol. Chem., 263,
1719-1725, 1988; Lu et al., Mol. Immunol., 28, 623-630, 1991;
Briand et al., J. Immunol. Methods, 156, 255-265, 1992).
[0216] Examples of adjuvants are Freund's complete adjuvant,
Freund's incomplete adjuvant, aluminum hydroxide gel, and the like.
Animals given the antigen are, for example, mouse, rat, rabbit,
sheep, goat, chicken, bovine, horse, guinea pig, hamster, and the
like. The blood is collected from these animals and the serum is
separated. Then, immunoglobulin is purified from the serum using an
ammonium sulfate precipitation method, anion exchange
chromatography, protein A chromatography, or protein G
chromatography to obtain polyclonal antibodies.
[0217] With respect to chicken, antibodies can be purified from an
egg. Monoclonal antibodies can be purified and prepared from
supernatant of culture of hybridoma cells or ascites from animals
which received intrapertoneal administration of hybridoma cells.
Hybridomas are made by fusion of the immune cells sensitized in
vitro, or immune cells from the above described animals with parent
cells capable of cultivation. Examples of parent cells are X63,
NS-1, P3U1, X63.653, SP2/O, Y3, SKO-007, GM1500, UC729-6, HM2.0,
NP4-1 cell strains, and the like. Preparation may be performed by
cultivating the immortalized antibody-forming cells obtained by
sensitization in vitro, or infection of a proper virus such as EB
virus to the immune cells of the above described animals.
[0218] In addition to these cell engineering methods, antibodies
can be obtained using gene engineering methods. For example, the
antibody gene obtained from the in vitro sensitized cells or immune
cells derived from the above described animals is amplified by PCR
(polymerase chain reaction) and isolated, and the amplified genes
are transferred into microorganisms such as E. coli to produce the
antibodies. Alternatively, the antibodies may be expressed on
surfaces of phages as fused protein. Antibodies of the invention
are also addressed herein, infra.
[0219] SCR-1 can be measured in vivo using antibodies in accordance
with the present invention. Thus, the relationship between SCR-1
and pathological status of a variety of diseases can be clarified.
Moreover, the antibodies can be used for diagnosis and treatment of
diseases, and efficient affinity purification of SCR-1.
[0220] The present invention provides polypeptides having an
activity to support survival or proliferation of hematopoietic stem
cells or hematopoietic progenitor cells by effecting or acting
thereon, or an activity to give an activity to support the
hematopoietic stem cells to stromal cells by effecting thereon, and
also provides DNA coding thereof. The polypeptides in accordance
with the present invention can efficiently maintain the
proliferation or the survival of the hematopoietic stem cells or
the hematopoietic progenitor cells.
[0221] In addition, the polypeptides in accordance with the present
invention can be used as a medicine to proliferate or to support
human hematopoietic stem cells or human hematopoietic progenitor
cells.
[0222] Alternatively, the invention is described as set out
below.
5.2 NUCLEIC ACIDS AND POLYPEPTIDES OF THE INVENTION
[0223] Nucleotide and amino acid sequences of the invention are set
forth as SEQ ID NO: 1-24, and 31-34. Fragments of the proteins of
the present invention which are capable of exhibiting biological
activity are also encompassed by the present invention. Fragments
of the protein may be in linear form or they may be cyclized using
known methods, for example, as described in H. U. Saragovi, et al.,
Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J.
Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are
incorporated herein by reference. Such fragments may be fused to
carrier molecules such as immunoglobulins for many purposes,
including increasing the valency of protein binding sites. For
example, fragments of the protein may be fused through "linker"
sequences to the Fc portion of an immunoglobulin. For a bivalent
form of the protein, such a fusion could be to the Fc portion of an
IgG molecule. Other immunoglobulin isotypes may also be used to
generate such fusions. For example, a protein-IgM fusion would
generate a decavalent form of the protein of the invention.
[0224] The present invention also provides both full-length and
mature forms (for example, without a signal sequence or precursor
sequence) of the disclosed proteins. The protein coding sequence is
identified in the sequence listing by translation of the disclosed
nucleotide sequences. The mature form of such protein may be
obtained by expression of a full-length polynucleotide in a
suitable mammalian cell or other host cell. The sequence of the
mature form of the protein is also determinable from the amino acid
sequence of the full-length form. Where proteins of the present
invention are membrane bound, soluble forms of the proteins are
also provided. In such forms, part or all of the regions causing
the proteins to be membrane bound are deleted so that the proteins
are fully secreted from the cell in which it is expressed.
[0225] The polynucleotides of the invention include naturally
occurring or wholly or partially synthetic DNA, e.g., cDNA and
genomic DNA, and RNA, e.g., mRNA. SEQ ID NO: 1-9, 11, 12, 31 or 33
may include the entire coding region of the cDNA or may represent a
portion of the coding region of the cDNA. Further 5' and 3'
sequence can be obtained using methods known in the art. For
example, full length cDNA or genomic DNA that corresponds to any of
the polynucleotides of the SEQ ID NO: 1-9, 11, 12, 31 or 33 can be
obtained by screening appropriate cDNA or genomic DNA libraries
under suitable hybridization conditions using any of the
polynucleotides of the SEQ ID NO: 1-9, 11, 12, 31 or 33 or a
portion thereof as a probe. Alternatively, the polynucleotides of
the SEQ ID NO: 1-9, 11, 12, 31 or 33 may be used as the basis for
suitable primer(s) that allow identification and/or amplification
of genes in appropriate genomic DNA or cDNA libraries.
[0226] The nucleic acid sequences of the invention can be assembled
ESTs and sequences (including cDNA and genomic sequences) obtained
from one or more public databases, such as dbEST, gbpri, and
UniGene. The sequences falling within the scope of the present
invention are not limited to these specific sequences, but also
include allelic and species variations thereof. Allelic and species
variations can be routinely determined by comparing the sequence
provided in SEQ ID NO: 8-9, 11-12, 31 or 33 a representative
fragment thereof, or a nucleotide sequence at least 90% identical,
preferably 99.9% identical, to SEQ ID NO: 8-9, 11-12, 31 or 33 with
a sequence from another isolate of the same species. Furthermore,
to accommodate codon variability, the invention includes nucleic
acid molecules coding for the same amino acid sequences as do the
specific ORFs disclosed herein. In other words, in the coding
region of an ORF, substitution of one codon for another which
encodes the same amino acid is expressly contemplated.
[0227] The nucleic acids of the present invention, designated as
SEQ ID NO. 8 and 9 were assembled using an EST sequence as a seed.
The EST sequence can be extended into a full-length nucleic acid
sequence by programs or algorithms known in the art. Preferably, a
recursive algorithm is used to extend the seed EST into an extended
assemblage, by pulling additional sequences from different
databases (e.g., Hyseq's database containing EST sequences, dbEST
version 114, gb pri 114, and UniGene version 101) that belong to
this assemblage. The algorithm terminates when there was no
additional sequences from the databases that will extend the
assemblage. Further, the inclusion of component sequences into the
assemblage is preferably based on a BLASTN hit to the extending
assemblage with BLAST score greater than 300 and percent identity
greater than 95%. BLAST, which stands for Basic Local Alignment
Search Tool, is used to search for local sequence alignments
(Altschul, S. F., J. Mol. Evol. 36: 290-300 (1993) and Altschul S.
F. et al., J. Mol. Biol. 21: 403-10 (1990)). BLAST produces
alignments of both nucleotide and amino acid sequences to determine
sequence similarity. Because of the local nature of the alignments,
BLAST is especially useful in determining exact matches.
[0228] The EST sequences (SEQ ID NO. 1-7) can provide identifying
sequence information, representative fragment or segment
information, or novel segment information for the full-length
gene.
[0229] The nearest neighbor result for the nucleic acids of the
present invention, including SEQ ID NO. 9, can be obtained by
searching a database using an algorithm or a program. Preferably, a
FASTA version 3 search against Genpept, using Fastxy algorithm. The
nearest neighbor result shows the closest homologue for each
assemblage from Genpept (and contains the translated amino acid
sequences for which the assemblage encodes).
[0230] The present invention also provides genes corresponding to
the cDNA sequences disclosed herein. The corresponding genes can be
isolated in accordance with known methods using the sequence
information disclosed herein. Such methods include the preparation
of probes or primers from the disclosed sequence information for
identification and/or amplification of genes in appropriate genomic
libraries or other sources of genomic materials.
[0231] Species homologs (or orthologs) of the disclosed
polynucleotides and proteins are also provided by the present
invention. Species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source from the
desired species.
[0232] The invention also encompasses allelic variants of the
disclosed polynucleotides or proteins; that is, naturally-occurring
alternative forms of the isolated polynucleotide which also encode
proteins which are identical, homologous or related to that encoded
by the polynucleotides.
5.3 NUCLEIC ACIDS OF THE INVENTION
[0233] The isolated polynucleotides of the invention include, but
are not limited to, a polynucleotide encoding a polypeptide
comprising SEQ ID NO: 10, 13-24, 32 and 34, or the mature protein
portion thereof. Preferred nucleic acid sequences are set forth as
SEQ ID NO: 9, 11, 12, 31 or 33.
[0234] The isolated polynucleotides of the invention further
include, but are not limited to a polynucleotide comprising any of
the nucleotide sequence of the SEQ ID NO: 1-9, 11, 12, 31 or 33; a
polynucleotide comprising the full length protein coding sequence
of the polynucleotides of the SEQ ID NO: 1-9, 11, 12, 31 or 33; and
a polynucleotide comprising the nucleotide sequence encoding the
mature protein coding sequence of the polynucleotides of the SEQ ID
NO: 1-9, 11, 12, 31 or 33. The polynucleotides of the present
invention also include, but are not limited to, a polynucleotide
that preferably has stem cell growth factor activity and that
hybridizes under stringent conditions (a) to the complement of any
of the nucleotides sequences of the SEQ ID NO: 1-9, 11, 12, 31 or
33 (b) to a polynucleotide encoding the polypeptide of SEQ ID NO:
10, 13-24, 32 or 34, a polynucleotide which is an allelic variant
of any polynucleotide recited above; a polynucleotide which encodes
a species homolog of any of the proteins recited above; or a
polynucleotide that encodes a polypeptide comprising a specific
domain or truncation of the polypeptide of SEQ ID NO: 10, 13-24, 32
or 34. Domains of interest may depend on the nature of the encoded
polypeptide; e.g., domains in receptor-like polypeptides include
ligand-binding, extracellular, transmembrane, or cytoplasmic
domains, or combinations thereof; domains in immunoglobulin-like
proteins include the variable immunoglobulin-like domains; domains
in enzyme-like polypeptides include catalytic and substrate binding
domains; and domains in ligand polypeptides include
receptor-binding domains.
[0235] Polynucleotides encoding preferred polypeptide truncations
of the invention can be used to generate polynucleotides encoding
chimeric or fusion proteins comprising one or more domains of the
invention and heterologous protein sequences.
[0236] The polynucleotides of the invention additionally include
the complement of any of the polynucleotides recited above.
[0237] The polynucleotides of the invention also provide
polynucleotides including nucleotide sequences that are
substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least
about 65%, at least about 70%, at least about 75%, at least about
80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%,
87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%,
94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%
sequence identity to a polynucleotide recited above. The invention
also provides the complement of such polynucleotides. The
polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic)
or RNA. Methods and algorithms for obtaining such polynucleotides
are well known to those of skill in the art and can include, for
example, methods for determining hybridization conditions which can
routinely isolate polynucleotides of the desired sequence
identities.
[0238] A polynucleotide according to the invention can be joined to
any of a variety of other nucleotide sequences by well-established
recombinant DNA techniques (see Sambrook J et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include
an assortment of vectors, e.g., plasmids, cosmids, lambda phage
derivatives, phagernids, and the like, that are well known in the
art. Accordingly, the invention also provides a vector including a
polynucleotide of the invention and a host cell containing the
polynucleotide. In general, the vector contains an origin of
replication functional in at least one organism, convenient
restriction endonuclease sites, and a selectable marker for the
host cell. Vectors according to the invention include expression
vectors, replication vectors, probe generation vectors, and
sequencing vectors. A host cell according to the invention can be a
prokaryotic or eukaryotic cell and can be a unicellular organism or
part of a multicellular organism.
[0239] The sequences falling within the scope of the present
invention are not limited to the specific sequences herein
described, but also include allelic variations thereof. Allelic
variations can be routinely determined by comparing the nucleotide
sequences provided in the SEQ ID NO: 1-9, 11, 12, 31 or 33, a
representative fragment thereof, or a nucleotide sequence at least
99.9% identical to any of the nucleotide sequences of the SEQ ID
NO: 1-9, 11, 12, 31 or 33 with a sequence from another isolate of
the same species. To accommodate codon variability, the invention
includes nucleic acid molecules coding for the same amino acid
sequences as do the specific ORFs disclosed herein. In other words,
in the coding region of an ORF, substitution of one codon for
another which encodes the same amino acid is expressly
contemplated. Any specific sequence disclosed herein can be readily
screened for errors by resequencing a particular fragment, such as
an ORF, in both directions (i.e., sequence both strands).
[0240] The present invention further provides recombinant
constructs comprising a nucleic acid having any of the nucleotide
sequences of the SEQ ID NO: 1-9, 11, 12, 31 or 33 or a fragment
thereof or any other polynucleotides of the invention. In one
embodiment, the recombinant constructs of the present invention
comprise a vector, such as a plasmid or viral vector, into which a
nucleic acid having any of the nucleotide sequences of the SEQ ID
NO: 1-9, 11, 12, 31 or 33 or a fragment thereof is inserted, in a
forward or reverse orientation. In the case of a vector comprising
one of the ORFs of the present invention, the vector may further
comprise regulatory sequences, including for example, a promoter,
operably linked to the ORF. For vectors comprising the EMFs and
UMFs of the present invention, the vector may further comprise a
marker sequence or heterologous ORF operably linked to the EMF or
UMF. Large numbers of suitable vectors and promoters are known to
those of skill in the art and are commercially available for
generating the recombinant constructs of the present invention. The
following vectors are provided by way of example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
[0241] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the protein
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in R. Kaufman, Methods in Enzymology
185, 537-566 (1990). As defined herein "operably linked" means that
the isolated polynucleotide of the invention and an expression
control sequence are situated within a vector or cell in such a way
that the protein is expressed by a host cell which has been
transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0242] 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 lac, lacZ, T3, T7,
gpt, lambda PR, and trc. 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. 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), a-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
amino terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product. 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.
[0243] As a representative but non-limiting 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 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. Following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced or derepressed by appropriate means (e.g., temperature
shift or chemical induction) and cells are cultured for an
additional period. Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0244] Included within the scope of the nucleic acid sequences of
the invention are nucleic acid sequence fragments that hybridize
under stringent conditions to any of the nucleotide sequences of
the SEQ ID NO: 1-9, 11, 12, 31 or 33 or complements thereof, which
fragment is greater than about 10 bp, preferably 20 to 50 bp, and
even greater than 100 bp, greater than 300 bp, or greater than 500
bp. Fragments of, e.g. 15, 16, or 20 bp or more that are selective
for (i.e. specifically hybridize to any one of the polynucleotides
of the invention) are contemplated. Probes capable of specifically
hybridizing to a polynucleotide can differentiate polynucleotide
sequences of the invention from other polynucleotide sequences in
the same family of genes or can differentiate human genes from
genes of other species, and are preferably based on unique
nucleotide sequences.
[0245] In accordance with the invention, polynucleotide sequences
comprising the mature protein coding sequences corresponding to the
SEQ ID NO: 10, 13-24, 32 or 34 or functional equivalents thereof,
may be used to generate recombinant DNA molecules that direct the
expression of that nucleic acid, or a functional equivalent
thereof, in appropriate host cells. Also included are the cDNA
inserts of any of the clones identified herein.
[0246] The nucleic acid sequences of the invention are further
directed to sequences which encode variants of the described
nucleic acids. These amino acid sequence variants may be prepared
by methods known in the art by introducing appropriate nucleotide
changes into a native or variant polynucleotide. There are two
variables in the construction of amino acid sequence variants: the
location of the mutation and the nature of the mutation. Nucleic
acids encoding the amino acid sequence variants are preferably
constructed by mutating the polynucleotide to encode an amino acid
sequence that does not occur in nature. These nucleic acid
alterations can be made at sites that differ in the nucleic acids
from different species (variable positions) or in highly conserved
regions (constant regions). Sites at such locations will typically
be modified in series, e.g., by substituting first with
conservative choices (e.g., hydrophobic amino acid to a different
hydrophobic amino acid) and then with more distant choices (e.g.,
hydrophobic amino acid to a charged amino acid), and then deletions
or insertions may be made at the target site. Amino acid sequence
deletions generally range from about 1 to 30 residues, preferably
about 1 to 10 residues, and are typically contiguous. Amino acid
insertions include amino- and/or carboxyl-terminal fusions ranging
in length from one to one hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions may range generally from about 1 to 10
amino residues, preferably from 1 to 5 residues. Examples of
terminal insertions include the heterologous signal sequences
necessary for secretion or for intracellular targeting in different
host cells and sequences such as FLAG or poly-histidine sequences
useful for purifying the expressed protein.
[0247] In a preferred method, polynucleotides encoding the novel
amino acid sequences are changed via site-directed mutagenesis.
This method uses oligonucleotide sequences to alter a
polynucleotide to encode the desired amino acid variant, as well as
a sufficient adjacent nucleotides on both sides of the changed
amino acid to form a stable duplex on either side of the site of
being changed. In general, the techniques of site-directed
mutagenesis are well known to those of skill in the art and this
technique is exemplified by publications such as, Edelman et al.,
DNA 2:183 (1983). A versatile and efficient method for producing
site-specific changes in a polynucleotide sequence was published by
Zoller and Smith, Nucleic Acids Res. 10:64876500 (1982). PCR may
also be used to create amino acid sequence variants of the novel
nucleic acids. When small amounts of template DNA are used as
starting material, primer(s) that differs slightly in sequence from
the corresponding region in the template DNA can generate the
desired amino acid variant. PCR amplification results in a
population of product DNA fragments that differ from the
polynucleotide template encoding the polypeptide at the position
specified by the primer. The product DNA fragments replace the
corresponding region in the plasmid and this gives a polynucleotide
encoding the desired amino acid variant.
[0248] A further technique for generating amino acid variants is
the cassette mutagenesis technique described in Wells et al., Gene
34:315 (1985); and other mutagenesis techniques well known in the
art, such as, for example, the techniques in Sambrook et al.,
supra, and Current Protocols in Molecular Biology, Ausubel et al.
Due to the inherent degeneracy of the genetic code, other DNA
sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be used in the practice of the
invention for the cloning and expression of these novel nucleic
acids. Such DNA sequences include those which are capable of
hybridizing to the appropriate novel nucleic acid sequence under
stringent conditions.
[0249] Polynucleotides of the invention can also be used to induce
immune responses. For example, as described in Fan et al., Nat.
Biotech. 17:870-872 (1999), incorporated herein by reference,
nucleic acid sequences encoding a polypeptide may be used to
generate antibodies against the encoded polypeptide following
topical administration of naked plasmid DNA or following injection,
and preferably intramuscular injection of the DNA. The nucleic acid
sequences are preferably inserted in a recombinant expression
vector and may be in the form of naked DNA.
5.3.1 ANTISENSE NUCLEIC ACIDS
[0250] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that can hybridize to, or are
complementary to, the nucleic acid molecule comprising the stem
cell growth factor-like nucleotide sequence, or fragments, analogs
or derivatives thereof. An "antisense" nucleic acid comprises a
nucleotide sequence that is complementary to a "sense" nucleic acid
encoding a protein (e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA
sequence). In specific aspects, antisense nucleic acid molecules
are provided that comprise a sequence complementary to at least
about 10, 25, 50, 100, 250 or 500 nucleotides or an entire stem
cell growth factor-like coding strand, or to only a portion
thereof. Nucleic acid molecules encoding fragments, homologs,
derivatives, and analogs of a stem cell growth factor-like or
antisense nucleic acids complementary to a stem cell growth
factor-like nucleic acid sequence of are additionally provided.
[0251] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding a stem cell growth factor-like protein. The term
"coding region" refers to the region of the nucleotide sequence
comprising codons which are translated into amino acid residues. In
another embodiment, the antisense nucleic acid molecule is
antisense to a "conceding region" of the coding strand of a
nucleotide sequence encoding the stem cell growth factor-like
protein. The term "conceding region" refers to 5' and 3' sequences
which flank the coding region that are not translated into amino
acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0252] Given the coding strand sequences encoding the stem cell
growth factor-like protein disclosed herein, antisense nucleic
acids of the invention can be designed according to the rules of
Watson and Crick or Hoogsteen base pairing. The antisense nucleic
acid molecule can be complementary to the entire coding region of
stem cell growth factor-like mRNA, but more preferably is an
oligonucleotide that is antisense to only a portion of the coding
or noncoding region of stem cell growth factor-like mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of stem cell growth
factor-like mRNA. An antisense oligonucleotide can be, for example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in
length. An antisense nucleic acid of the invention can be
constructed using chemical synthesis or enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids
(e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used).
[0253] Examples of modified nucleotides that can be used to
generate the antisense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following section).
[0254] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a stem cell growth factor-like protein thereby inhibit
expression of the protein (e.g., by inhibiting transcription and
translation). The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule that binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention includes direct injection
at a tissue site. Alternatively, antisense nucleic acid molecules
can be modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface (e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell surface receptors or antigens). The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient nucleic
acid molecules, vector constructs in which the antisense nucleic
acid molecule is placed under the control of a strong pol II or pol
III promoter are preferred.
[0255] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an alpha-anomeric nucleic acid
molecule. An alpha-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual alpha-units, the strands run parallel to each other.
See, e.g., Gaultier, et al. 1987. Nucl. Acids Res. 15, 6625-6641.
The antisense nucleic: acid molecule can also comprise a
2'-o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl.
Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (see,
e.g., Inoue, et al., 1987. FEBS Lett. 215, 327-330).
5.3.2 RIBOZYMES AND PNA MOIETIES
[0256] Nucleic acid modifications include, by way of non-limiting
example, modified bases, and nucleic acids whose sugar phosphate
backbones are modified or derivatized. These modifications are
carried out at least in part to enhance the chemical stability of
the modified nucleic acid, such that they can be used, for example,
as antisense binding nucleic acids in therapeutic applications in a
subject.
[0257] In one embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
as described in Haselhoff and Gerlach 1988. Nature 334, 585-591)
can be used to catalytically cleave stem cell growth factor-like
mRNA transcripts to thereby inhibit translation of stem cell growth
factor-like mRNA. A ribozyme having specificity for a stem cell
growth factor-like-encoding nucleic acid can be designed based upon
the nucleotide sequence of a stem cell growth factor-like cDNA
disclosed herein. For example, a derivative of a Tetrahymena L-19
IVS RNA can be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence to be
cleaved in a stem cell growth factor-like-encoding mRNA. See, e.g.,
U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No. 5,116,742
to Cech, et al. Stem cell growth factor-like mRNA can also be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules. See, e.g., Bartel et al., (1993)
Science 261, 1411-1418.
[0258] Alternatively, stem cell growth factor-like gene expression
can be inhibited by targeting nucleotide sequences complementary to
the regulatory region of the stem cell growth factor-like nucleic
acid (e.g., the stem cell growth factor-like promoter and/or
enhancers) to form triple-helical structures that prevent
transcription of the stem cell growth factor-like gene in target
cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6, 569-84;
Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660, 27-36; Maher, 1992.
Bioassays 14, 807-15.
[0259] In various embodiments, the stem cell growth factor-like
nucleic acids can be modified at the base moiety, sugar moiety or
phosphate backbone to improve, e.g., the stability, hybridization,
or solubility of the molecule. For example, the deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med
Chem 4, 5-23. As used herein, the terms "peptide nucleic acids" or
"PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc.
Natl. Acad. Sci. USA 93, 14670-14675.
[0260] PNAs of stem cell growth factor-like can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, e.g., inducing transcription or
translation arrest or inhibiting replication. PNAs of stem cell
growth factor-like can also be used, for example, in the analysis
of single base pair mutations in a gene (e.g., PNA directed PCR
clamping; as artificial restriction enzymes when used in
combination with other enzymes, e.g., S1 nucleases (see, Hyrup, et
al., 1996.supra); or as probes or primers for DNA sequence and
hybridization (see, Hyrup, et al., 1996, supra; Perry-O'Keefe, et
al., 1996. supra).
[0261] In another embodiment, PNAs of stem cell growth factor-like
can be modified, e.g., to enhance their stability or cellular
uptake, by attaching lipophilic or other helper groups to PNA, by
the formation of PNA-DNA chimeras, or by the use of liposomes or
other techniques of drug delivery known in the art. For example,
PNA-DNA chimeras of stem cell growth factor-like can be generated
that may combine the advantageous properties of PNA and DNA. Such
chimeras allow DNA recognition enzymes (e.g., RNase H and DNA
polymerases) to interact with the DNA portion while the PNA portion
would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The
synthesis of PNA-DNA chimeras can be performed as described in
Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res
24, 3357-3363. For example, a DNA chain can be synthesized on a
solid support using standard phosphoramidite coupling chemistry,
and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used between the PNA and the 5' end of DNA. See, e.g., Mag, et
al., 1989. Nucl Acid Res 17, 5973-5988. PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra. Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al.,
1975. Bioorg. Med. Chem. Lett. 5, 1119-11124.
[0262] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86, 6553-6556; Lemaitre, et al., 1987. Proc.
Natl. Acad. Sci. 84, 648-652; PCT Publication No. WO88/09810) or
the blood-brain barrier (see, e.g., PCT Publication No.
WO89/10134). In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol, et al.,
1988. BioTechniques 6, 958-976) or intercalating agents (see, e.g.,
Zon, 1988. Pharm. Res. 5, 539-549). To this end, the
oligonucleotide can be conjugated to another molecule, e.g., a
peptide, a hybridization triggered cross-linking agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
5.4 HOSTS
[0263] The present invention further provides host cells
genetically engineered to contain the polynucleotides of the
invention. For example, such host cells may contain nucleic acids
of the invention introduced into the host cell using known
transformation, transfection or infection methods. The present
invention still further provides host cells genetically engineered
to express the polynucleotides of the invention, wherein such
polynucleotides are in operative association with a regulatory
sequence heterologous to the host cell which drives expression of
the polynucleotides in the cell.
[0264] Knowledge of stem cell growth factor-like DNA sequences
allows for modification of cells to permit, or increase, expression
of stem cell growth factor-like polypeptide. Cells can be modified
(e.g., by homologous recombination) to provide increased stem cell
growth factor-like polypeptide expression by replacing, in whole or
in part, the naturally occurring stem cell growth factor-like
promoter with all or part of a heterologous promoter so that the
cells stem cell growth factor-like polypeptide is expressed at
higher levels. The heterologous promoter is inserted in such a
manner that it is operatively linked to stem cell growth
factor-like encoding sequences. See, for example, PCT International
Publication No. WO94/12650, PCT International Publication No.
WO92/20808, and PCT International Publication No. WO91/09955. It is
also contemplated that, in addition to heterologous promoter DNA,
amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional
CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydroorotase) and/or intron DNA may be
inserted along with the heterologous promoter DNA. If linked to the
stem cell growth factor-like coding sequence, amplification of the
marker DNA by standard selection methods results in
co-amplification of the stem cell growth factor-like coding
sequences in the cells.
[0265] The host cell can be a higher eukaryotic host cell, such as
a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran mediated transfection, or electroporation (Davis, L. et
al., Basic Methods in Molecular Biology (1986)). The host cells
containing one of polynucleotides of the invention, can be used in
conventional manners to produce the gene product encoded by the
isolated fragment (in the case of an ORF) or can be used to produce
a heterologous protein under the control of the EMF.
[0266] Any host/vector system can be used to express one or more of
the ORFs of the present invention. These include, but are not
limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS
cells, and Sf9 cells, as well as prokaryotic host such as E. coli
and B. subtilis. The most preferred cells are those which do not
normally express the particular polypeptide or protein or which
expresses the polypeptide or protein at low natural level. 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.,
in Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y. (1989), the disclosure of which is hereby
incorporated by reference.
[0267] 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 tines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter, 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 viral genome, for example, SV40 origin, early
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture
are usually isolated by initial extraction from cell pellets,
followed by one or more salting-out, aqueous ion exchange or size
exclusion chromatography steps. 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. 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.
[0268] A number of types of cells may act as suitable host cells
for expression of the protein. Mammalian host cells include, for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human
kidney 293 cells, human epidermal A431 cells, human Colo205 cells,
3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary-explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells.
[0269] Alternatively, it may be possible to produce the protein in
lower eukaryotes such as yeast or in prokaryotes such as bacteria.
Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins. If
the protein is made in yeast or bacteria, it may be necessary to
modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the
functional protein. Such covalent attachments may be accomplished
using known chemical or enzymatic methods.
[0270] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites, and
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting, including polyadenylation signals,
mRNA stability elements, splice sites, leader sequences for
enhancing or modifying transport or secretion properties of the
protein, or other sequences which alter or improve the function or
stability of protein or RNA molecules.
[0271] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the host cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0272] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
5.5 POLYPEPTIDES OF THE INVENTION
[0273] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising: the amino acid sequence
set forth as SEQ ID NO: 10, 13-24, 32 or 34 or an amino acid
sequence encoded by any one of the nucleotide sequences SEQ ID NO:
1-9, 11, 12, 31 or 33 or the corresponding full length or mature
protein. Polypeptides of the invention also include polypeptides
preferably with biological or immunological activity that are
encoded by: (a) a polynucleotide having any one of the nucleotide
sequences set forth in the SEQ ID NO: 1-9, 11, 12, 31 or 33 or (b)
polynucleotides encoding the amino acid sequence set forth as SEQ
ID NO: 10, 13-24, 32 or 34 or (c) polynucleotides that hybridize to
the complement of the polynucleotides of either (a) or (b) under
stringent hybridization conditions. The invention also provides
biologically active or immunologically active variants of any of
the polypeptide amino acid sequences set forth as SEQ ID NO: 10,
13-24, 32 or 32 or the corresponding full length or mature protein;
and "substantial equivalents" thereof (e.g., with at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%,
93%, 94%, typically at least about 95%, 96%, 97%, more typically at
least about 98%, or most typically at least about 99% amino acid
identity) that retain biological activity. Polypeptides encoded by
allelic variants may have a similar, increased, or decreased
activity compared to polypeptides comprising SEQ ID NO: 10, 13-24,
32 or 34.
[0274] Protein compositions of the present invention may further
comprise an acceptable carrier, such as a hydrophilic, e.g.,
pharmaceutically acceptable, carrier.
[0275] The invention also relates to methods for producing a
polypeptide comprising growing a culture of host cells of the
invention in a suitable culture medium, and purifying the protein
from the cells or the culture in which the cells are grown. For
example, the methods of the invention include a process for
producing a polypeptide in which a host cell containing a suitable
expression vector that includes a polynucleotide of the invention
is cultured under conditions that allow expression of the encoded
polypeptide. The polypeptide can be recovered from the culture,
conveniently from the culture medium, or from a lysate prepared
from the host cells and further purified. Preferred embodiments
include those in which the protein produced by such process is a
full length or mature form of the protein.
[0276] The present invention further provides isolated polypeptides
encoded by the nucleic acid fragments of the present invention or
by degenerate variants of the nucleic acid fragments of the present
invention. By "degenerate variant" is intended nucleotide fragments
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the genetic code, encode an identical polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the
ORFs that encode proteins. A variety of methodologies known in the
art can be utilized to obtain any one of the isolated polypeptides
or proteins of the present invention. At the simplest level, the
amino acid sequence can be synthesized using commercially available
peptide synthesizers. This technique is particularly useful in
producing small peptides and fragments of larger polypeptides.
Fragments are useful, for example, in generating antibodies against
the native polypeptide. In an alternative method, the polypeptide
or protein is purified from bacterial cells which naturally produce
the polypeptide or protein. One skilled in the art can readily
follow known methods for isolating polypeptides and proteins in
order to obtain one of the isolated polypeptides or proteins of the
present invention. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography, and immuno-affinity chromatography.
See, e.g., Scopes, Protein Purification: Principles and Practice,
Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory Manual; Ausubel et al., Current Protocols in Molecular
Biology. Polypeptide fragments that retain biological/immunological
activity include fragments encoding greater than about 100 amino
acids, or greater than about 200 amino acids, and fragments that
encode specific protein domains.
[0277] The polypeptides and proteins of the present invention can
alternatively be purified from cells which have been altered to
express the desired polypeptide or protein. As used herein, a cell
is said to be altered to express a desired polypeptide or protein
when the cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. One skilled in the art
can readily adapt procedures for introducing and expressing either
recombinant or synthetic sequences into eukaryotic or prokaryotic
cells in order to generate a cell which produces one of the
polypeptides or proteins of the present invention.
[0278] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
protein.
[0279] The protein may also be produced by known conventional
chemical synthesis. Methods for constructing the proteins of the
present invention by synthetic means are known to those skilled in
the art. The synthetically-constructed protein sequences, by virtue
of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common therewith, including protein activity. Thus,
they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
[0280] The proteins provided herein also include proteins
characterized by amino acid sequences similar to those of purified
proteins but into which modification are naturally provided or
deliberately engineered. For example, modifications in the peptide
or DNA sequences can be made by those skilled in the art using
known techniques. Modifications of interest in the protein
sequences may include the alteration, substitution, replacement,
insertion or deletion of a selected amino acid residue in the
coding sequence. For example, one or more of the cysteine residues
may be deleted or replaced with another amino acid to alter the
conformation of the molecule. Techniques for such alteration,
substitution, replacement, insertion or deletion are well known to
those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).
Preferably, such alteration, substitution, replacement, insertion
or deletion retains the desired activity of the protein. Regions of
the protein that are important for the protein function can be
determined by various methods known in the art including the
alanine-scanning method which involved systematic substitution of
single or strings of amino acids with alanine, followed by testing
the resulting alanine-containing variant for biological activity.
This type of analysis determines the importance of the substituted
amino acid(s) in biological activity.
[0281] Other fragments and derivatives of the sequences of proteins
which would be expected to retain protein activity in whole or in
part and are useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are encompassed by
the present invention.
[0282] The protein may also be produced by operably linking the
isolated polynucleotide of the invention to suitable control
sequences in one or more insect expression vectors, and employing
an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, e.g., Invitrogen, San Diego, Calif.,
U.S.A. (the MaxBat.TM. kit), and such methods are well known in the
art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0283] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl.TM. or
Cibacrom blue 3GA Sepharose.TM. one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0284] Alternatively, the protein of the invention may also be
expressed in a form which will facilitate purification. For
example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin (TRX), or as a His tag. Kits for expression and
purification of such fusion proteins are commercially available
from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway,
N.J.) and Invitrogen, respectively. The protein can also be tagged
with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("FLAG.RTM.")
is commercially available from Kodak (New Haven, Conn.).
[0285] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. The protein thus purified is substantially
free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein. "
[0286] The polypeptides of the invention include analogs
(variants). Analogs embrace fragments, as well as antagonists which
comprise one or more amino acids deleted, inserted, or substituted.
Analogs of the invention also embrace fusions of the polypeptide of
the invention or modifications of the polypeptide of the invention
or analog is fused to another moiety or moieties, e.g., targeting
moiety, imaging moiety or another therapeutic agent. Such analogs
may exhibit improved properties such as activity and/or stability.
Examples of moieties which may be fused to polypeptides of the
invention or analogs thereof include, for example, targeting
moieties which provide for the delivery of polypeptide to desired
cell types. Other moieties which may be fused to the polypeptides
of the invention include therapeutic agents which are used for
treatment of disorders described herein.
5.5.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY AND
SIMILARITY
[0287] Preferred identity and/or similarity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in computer programs
including, but are not limited to, the GCG program package,
including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):
387 (1984); Genetics Computer Group, University of Wisconsin,
Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et
al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F.
et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein
incorporated by reference), the eMatrix software (Wu et al., J.
Comp. Biol., vol. 6, pp. 219-235 (1999), herein incorporated by
reference), eMotif software (Nevill-Manning et al, ISMB-97, vol 4,
pp. 202-209, herein incorporated by reference), the GeneAtlas
software (Molecular Simulations Inc. (MSI), San Diego, Calif.)
(Sanchez and Sali (1998) Proc. Natl. Acad. Sci., 95, 13597-13602;
Kitson D H et al, (2000) "Remote homology detection using
structural modeling--an evaluation" Submitted; Fischer and
Eisenberg (1996) Protein Sci. 5, 947-955), Neural Network SignalP
V1.1 program (from Center for Biological Sequence Analysis, The
Technical University of Denmark) and the Kyte-Doolittle
hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31
(1982), incorporated herein by reference). The BLAST programs are
publicly available from the National Center for Biotechnology
Information (NCBI) and other sources (BLAST Manual, Altschul, S.,
et al. NCB NLM NIH Bethesda, MD 20894; Altschul, S., et al., J.
Mol. Biol. 215:403-410 (1990).
5.6 CHIMERIC AND FUSION PROTEINS
[0288] The invention also provides stem cell growth factor-like
chimeric or fusion proteins. As used herein, a stem cell growth
factor-like "chimeric protein" or "fusion protein" comprises a stem
cell growth factor-like polypeptide operatively linked to either a
different stem cell growth factor-like polypeptide or a non-stem
cell growth factor-like polypeptide. An "stem cell growth
factor-like polypeptide" refers to a polypeptide having an amino
acid sequence corresponding to a stem cell growth factor-like
protein, whereas a "non-stem cell growth factor-like polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to a protein that is not substantially homologous to the stem cell
growth factor-like protein, e.g., a protein that is different from
the stem cell growth factor-like protein and that is derived from
the same or a different organism. Within a stem cell growth
factor-like fusion protein the stem cell growth factor-like
polypeptide can correspond to all or a portion of a stem cell
growth factor-like protein. In one embodiment, a stem cell growth
factor-like fusion protein comprises at least one biologically
active portion of a stem cell growth factor-like protein. In
another embodiment, a stem cell growth factor-like fusion protein
comprises at least two biologically active portions of a stem cell
growth factor-like protein. In yet another embodiment, a stem cell
growth factor-like fusion protein comprises at least three
biologically active portions of a stem cell growth factor-like
protein. Within the fusion protein, the term "operatively-linked"
is intended to indicate that the stem cell growth factor-like
polypeptide(s) and/or the non-stem cell growth factor-like
polypeptide are fused in-frame with one another. The non-stem cell
growth factor-like polypeptide can be fused to the N-terminus or
C-terminus of the stem cell growth factor-like polypeptide.
[0289] In one embodiment, the fusion protein is a GST-stem cell
growth factor-like fusion protein in which-the stem cell growth
factor-like sequences are fused to the C-terminus of the GST
(glutathione S-transferase) sequences. Such fusion proteins can
facilitate the purification of recombinant stem cell growth
factor-like polypeptides.
[0290] In another embodiment, the fusion protein is a stem cell
growth factor-like protein containing a heterologous signal
sequence at its N-terminus. In certain host cells (e.g., mammalian
host cells), expression and/or secretion of stem cell growth
factor-like can be increased through use of a heterologous signal
sequence.
[0291] In yet another embodiment, the fusion protein is a stem cell
growth factor-like-immunoglobulin fusion protein in which the stem
cell growth factor-like sequences are fused to sequences derived
from a member of the immunoglobulin protein family. The stem cell
growth factor-like-immunoglobulin fusion proteins of the invention
can be incorporated into pharmaceutical compositions and
administered to a subject to inhibit an interaction between a stem
cell growth factor-like ligand and a stem cell growth factor-like
protein on the surface of a cell, to thereby suppress stem cell
growth factor-like-mediated signal transduction in vivo. The stem
cell growth factor-like-immunoglobulin fusion proteins can be used
to affect the bioavailability of a stem cell growth factor-like
cognate ligand. Inhibition of the stem cell growth factor-like
ligand/stem cell growth factor-like interaction can be useful
therapeutically for both the treatment of proliferative and
differentiative disorders, as well as modulating (e.g. promoting or
inhibiting) cell survival. Moreover, the stem cell growth
factor-like-immunoglobulin fusion proteins of the invention can be
used as immunogens to produce anti-stem cell growth factor-like
antibodies in a subject, to purify stem cell growth factor-like
ligands, and in screening assays to identify molecules that inhibit
the interaction of stem cell growth factor-like with a stem cell
growth factor-like ligand.
[0292] Stem cell growth factor-like chimeric or fusion protein of
the invention can be produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance
with conventional techniques, e.g., by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers that give
rise to complementary overhangs between two consecutive gene
fragments that can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, e.g., Ausubel, et al.
(eds.) Current Protocols in Molecular Biology, John Wiley &
Sons, 1992). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST
polypeptide). Stem cell growth factor-like-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the stem cell growth factor-like
protein.
5.7 GENE THERAPY
[0293] Mutations in the polynucleotides of the invention gene may
result in loss of normal function of the encoded protein. The
invention thus provides gene therapy to restore normal activity of
the polypeptides of the invention; or to treat disease states
involving polypeptides of the invention. Delivery of a functional
genes encoding polypeptides of the invention to appropriate cells
is effected ex vivo, in situ, or in vivo by use of vectors, and
more particularly viral vectors (e.g., adenovirus, adeno-associated
virus, or a retrovirus), or ex vivo by use of physical DNA transfer
methods (e.g., liposomes or chemical treatments). See, for example,
Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20
(1998). For additional reviews of gene therapy technology see
Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific
American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Introduction of any one of the nucleotides of the present invention
or a gene encoding the polypeptides of the present invention can
also be accomplished with extrachromosomal substrates (transient
expression) or artificial chromosomes (stable expression). Cells
may also be cultured ex vivo in the presence of proteins of the
present invention in order to proliferate or to produce a desired
effect on or activity in such cells. Treated cells can then be
introduced in vivo for therapeutic purposes. Alternatively, it is
contemplated that in other human disease states, preventing the
expression of or inhibiting the activity of polypeptides of the
invention will be useful in treating the, disease states. It is
contemplated that antisense therapy or gene therapy could be
applied to negatively regulate the expression of polypeptides of
the invention.
[0294] Other methods inhibiting expression of a protein include the
introduction of antisense molecules to the nucleic acids of the
present invention, their complements, or their translated RNA
sequences, by methods known in the art, the removal of the nucleic
acids of the present invention such as using targeted deletion
methods, or the insertion of a negative regulatory element such as
a silencer, which is tissue specific. Further, the polypeptides of
the present invention can be inhibited by the introduction of
antisense molecules that hybridize to nucleic acids that encode for
the polypeptides of the present invention and by the removal of a
gene that encode for the polypeptides of the present invention.
[0295] The present invention still further provides cells
genetically engineered in vivo to express the polynucleotides of
the invention, wherein such polynucleotides are in operative
association with a regulatory sequence heterologous to the host
cell which drives expression of the polynucleotides in the cell.
These methods can be used to increase or decrease the expression of
the polynucleotides of the present invention.
[0296] Knowledge of DNA sequences provided by the invention allows
for modification of cells to permit, increase, or decrease,
expression of endogenous polypeptide. Cells can be modified (e.g.,
by homologous recombination) to provide increased polypeptide
expression by replacing, in whole or in part, the naturally
occurring promoter with all or part of a heterologous promoter so
that the cells express the protein at higher levels. The
heterologous promoter is inserted in such a manner that it is
operatively linked to the desired protein encoding sequences. See,
for example, PCT International Publication No. WO 94/12650, PCT
International Publication No. WO 92/20808, and PCT International
Publication No. WO 91/09955. It is also contemplated that, in
addition to heterologous promoter DNA, amplifiable marker DNA
(e.g., ada, dhfr, and the multifunctional CAD gene which encodes
carbamyl phosphate synthase, aspartate transcarbamylase, and
dihydroorotase) and/or intron DNA may be inserted along with the
heterologous promoter DNA. If linked to the desired protein coding
sequence, amplification of the marker DNA by standard selection
methods results in co-amplification of the desired protein coding
sequences in the cells.
[0297] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequence include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0298] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0299] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
5.8 TRANSGENIC ANIMALS
[0300] In preferred methods to determine biological functions of
the polypeptides of the invention in vivo, one or more genes
provided by the invention are either over expressed or inactivated
in the germ line of animals using homologous recombination
[Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene
is over expressed, under the regulatory control of exogenous or
endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by
homologous recombination are referred to as "knockout" animals.
Knockout animals, preferably non-human mammals, can be prepared as
described in U.S. Pat. No. 5,557,032, incorporated herein by
reference. Transgenic animals are useful to determine the roles
polypeptides of the invention play in biological processes, and
preferably in disease states. Transgenic animals are useful as
model systems to identify compounds that modulate lipid metabolism.
Transgenic animals, preferably non-human mammals, are produced
using methods as described in U.S. Pat. No. 5,489,743 and PCT
Publication No. WO94/28122, incorporated herein by reference.
[0301] Transgenic animals can be prepared wherein all or part of a
polynucleotides of the invention promoter is either activated or
inactivated to alter the level of expression of the polypeptides of
the invention. Inactivation can be carried out using homologous
recombination methods described above. Activation can be achieved
by supplementing or even replacing the homologous promoter to
provide for increased protein expression. The homologous promoter
can be supplemented by insertion of one or more heterologous
enhancer elements known to confer promoter activation in a
particular tissue.
5.9 USES AND BIOLOGICAL ACTIVITY OF STEM CELL GROWTH FACTOR-LIKE
POLYPEPTIDE
[0302] Stem cell growth factor-like polypeptide is based on
polynucleotides isolated from cDNA libraries prepared from human
testis cells (Hyseq clone identification numbers 2880984 and
2881695), from human fetal skin (Hyseq clone identification number
15375176), adult spleen (Hyseq clone identification number
14856094), and human endothelial cells (Hyseq clone identification
numbers 13804756, 13687487, 13804756).
[0303] FIG. 1 shows the alignment of polynucleotide SEQ ID NO: 9
and EST sequences SEQ ID NO: 1-7. The nucleic acid sequences of the
present invention (SEQ ID NO: 1-9) are expected encode polypeptides
having stem cell growth factor activity, including hematopoietic
stem cell growth factor activity, as described herein. The
polypeptide of SEQ ID NO: 10, fragments thereof, sequences having
at least 90% homology, are also expected to have stem cell growth
factor activity, including hematopoietic stem cell growth factor
activity, as described herein.
[0304] The stem cell growth factor-like polypeptide of SEQ ID NO:
10 is an approximately 272-amino acid protein with a predicted
molecular mass of approximately 30 kDa unglycosylated. Protein
database searches with the BLASTX algorithm (Altschul S. F. et al.,
J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.
Biol. 21:403-10 (1990), herein incorporated by reference) indicate
that SEQ ID NO: 10 is homologous to thrombospondin type I domain
and a human secreted protein clone da 228.sub.--6. Protein database
search with eMATRIX software (Stanford University, Stanford Calif.)
further show that a portion of SEQ ID NO: 10 has a laminin-type
EGF-like (LE) domain, a vertebrate metallothioneins domain, an
endogenous opioids neuropeptides precursors proteins domain, a
membrane attack complex components/perforin proteins domain, an
HMG-1 and HMG-Y DNA-binding domain proteins (Ahook), an HMG1/2
protein domain, a vertebrate metallothionein signature domain, and
a neurohypophysial hormone signature domain.
[0305] A predicted approximately twenty-one residue signal peptide
is encoded from approximately residue 1 to residue 21 of SEQ ID NO:
10 (SEQ ID NO: 15). The extracellular portion is useful on its own.
This can be confirmed by expression in mammalian cells and
sequencing of the cleaved product. The signal peptide region was
predicted using Neural Network SignalP V1.1 program (Nielsen et al,
(1997) Int. J. Neural Syst. 8, 581-599). One of skill in the art
will recognize that the actual cleavage site may be different than
that predicted by the computer program. SEQ ID NO: 16 is the
peptide resulting when the predicted signal peptide is removed from
SEQ ID NO: 10.
[0306] Using eMATRIX software package (Stanford University,
Stanford, Calif.) (Wu et al., J. Comp. Biol., vol. 6, pp. 219-235
(1999), herein incorporated by reference), Siglec-like polypeptide
of SEQ ID NO: 10 is expected to have following domains, wherein
A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,
F=Phenylalanine, G=Glycine, H=Histidine,. I=Isoleucine, K=Lysine,
L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,
R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,
Y=Tyrosine:
TABLE-US-00002 Laminin-type EGF-like (LE) domain proteins at 100
ADCDTCFNKNFCTKCKSGFYLIIL 122 (SEQ ID NO: 17) Vertebrate
metallothioneins proteins at 92
INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANN 137 (SEQ ID NO: 18)
Endogenous opioids neuropeptides precursors proteins at 33
MHPNVSQGCQGGCATCSDYN 52 (SEQ ID NO: 19) Membrane attack complex
components/perform proteins at 145
IVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQ 181 (SEQ ID NO: 20) HMG-I and
HMG-Y DNA-binding domain proteins (Ahook) at 213 KKGRERKRKK 222
(SEQ ID NO: 21) HMG1/2 proteins at 198
KCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLE 239 (SEQ ID NO: 22)
VERTEBRATE METALLOTHIONEIN SIGNATURE at 104 TCFNKNFCTKCKSG 117 (SEQ
ID NO: 23) NEUROHYPOPHYSIAL HORMONE SIGNATURE at 148
CEVSEWNPWSPCTKKGKTCG 167 (SEQ ID NO: 24)
[0307] Motif 100-122, a laminin-type EGF-like domain, is a
component of extracellular matrix which promotes cell growth. The
membrane attack complex component/perforin domain (145-185) is
postulated to mediate cell-cell interaction and thus cell growth
and differentiation. Neurohypophysial hormone is itself regulated
by many other factors including Interleukin-1 beta and
Interleukin-6. The presence of these motifs are expected in stem
cell growth factor activity.
[0308] Stem cell growth factor-like protein and/or fragments or
derivatives would have similar activity to stem cell growth factors
and anabolic growth factors and receptors.
[0309] Polypeptides of the invention having stem cell growth
factor-like activity are useful for but not limited to cell growth
and morphogenesis, including hematopoietic stem cell growth and/or
growth of a particular hematopoietic cell type (such as B or T
cells), tissue specific stem cell growth, epithelial cell growth
and regulation, ovarian follicle development, promoting nerve cell
growth, sustaining neuronal populations, cartilage remodeling,
wound repair, bone growth, imnmunosuppression, immune response
modulation, modulating antibody and cell mediated immunity and
vascular remodeling. The polypeptides of the invention can
therefore be employed in but not limited to the prophylaxis or
treatment of disorders and diseases caused by or involving wound
healing, growth and development, regulation of cartilage growth and
development, vascular remodeling (angiogenesis),
imnmunosuppression, follicle growth and development and neurite
growth and development. Polypeptides of the invention can also be
used in the production of and maintenance of transplants or
epidermal grafts.
[0310] The polynucleotides and proteins of the present invention
are expected to exhibit one or more of the uses or biological
activities (including those associated with assays cited herein)
identified herein. Uses or activities described for proteins of the
present invention may be provided by administration or use of such
proteins or of polynucleotides encoding such proteins (such as, for
example, in gene therapies or vectors suitable for introduction of
DNA). The mechanism underlying the particular condition or
pathology will dictate whether the polypeptides of the invention,
the polynucleotides of the invention or modulators (activators or
inhibitors) thereof would be beneficial to the subject in need of
treatment. Thus, "therapeutic compositions of the invention"
include compositions comprising isolated polynucleotides (including
recombinant DNA molecules, cloned genes and degenerate variants
thereof) or polypeptides of the invention (including full length
protein, mature protein and truncations or domains thereof), or
compounds and other substances that modulate the overall activity
of the target gene products, either at the level of target
gene/protein expression or target protein activity. Such modulators
include polypeptides, analogs, (variants), including fragments and
fusion proteins, antibodies and other binding proteins; chemical
compounds that directly or indirectly activate or inhibit the
polypeptides of the invention (identified, e.g., via drug screening
assays as described herein); antisense polynucleotides and
polynucleotides suitable for triple helix formation; and in
particular antibodies or other binding partners that specifically
recognize one or more epitopes of the polypeptides of the
invention.
[0311] The protein of the present invention may likewise be
involved in cellular activation or in one of the other
physiological pathways described herein.
5.9.1 RESEARCH USES AND UTILITIES
[0312] The polynucleotides provided by the present invention can be
used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; as markers for
tissues in which the corresponding protein is preferentially
expressed (either constitutively or at a particular stage of tissue
differentiation or development or in disease states); as molecular
weight markers on gels; as chromosome markers or tags (when
labeled) to identify chromosomes or to map related gene positions;
to compare with endogenous DNA sequences in patients to identify
potential genetic disorders; as probes to hybridize and thus
discover novel, related DNA sequences; as a source of information
to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out" known sequences in the process of discovering other
novel polynucleotides; for selecting and making oligomers for
attachment to a "gene chip" or other support, including for
examination of expression patterns; to raise anti-protein
antibodies using DNA immunization techniques; and as an antigen to
raise anti-DNA antibodies or elicit another immune response. Where
the polynucleotide encodes a protein which binds or potentially
binds to another protein (such as, for example, in a
receptor-ligand interaction), the polynucleotide can also be used
in interaction trap assays (such as, for example, that described in
Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides
encoding the other protein with which binding occurs or to identify
inhibitors of the binding interaction.
[0313] The proteins provided by the present invention can similarly
be used in assays to determine biological activity, including in a
panel of multiple proteins for high-throughput screening; to raise
antibodies or to elicit another immune response; as a reagent
(including the labeled reagent) in assays designed to
quantitatively determine levels of the protein (or its receptor) in
biological fluids; as markers for tissues in which the
corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate
correlative receptors or ligands. Where the protein binds or
potentially binds to another protein (such as, for example, in a
receptor-ligand interaction), the protein can be used to identify
the other protein with which binding occurs or to identify
inhibitors of the binding interaction. Proteins involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0314] The polypeptides of the invention are also useful for making
antibody substances that are specifically immunoreactive with stem
cell growth factor-like proteins. Antibodies and portions thereof
(e.g., Fab fragments) which bind to the polypeptides of the
invention can be used to identify the presence of such polypeptides
in a sample. For example, the level of the native protein
corresponding to SEQ ID NO: 10 in a tissue sample can be determined
as an indication of chrondrocyte differentiation or embryonic
status. Such determinations are carried out using any suitable
immunoassay format, and any polypeptide of the invention that is
specifically bound by the antibody can be employed as a positive
control.
[0315] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0316] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
5.9.2 NUTRITIONAL USES
[0317] Polynucleotides and proteins of the present invention can
also be used as nutritional sources or supplements. Such uses
include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and
use as a source of carbohydrate. In such cases the protein or
polynucleotide of the invention can be added to the feed of a
particular organism or can be administered as a separate solid or
liquid preparation, such as in the form of powder, pills,
solutions, suspensions or capsules. In the case of microorganisms,
the protein or polynucleotide of the invention can be added to the
medium in or on which the microorganism is cultured.
[0318] Additionally, the polypeptides of the invention can be used
as molecular weight markers, and as a food supplement. A
polypeptide consisting of SEQ ID NO: 10, for example, has a
molecular mass of approximately 30 kDa in its unprocessed and
unglycosylated state. Protein food supplements are well known and
the formulation of suitable food supplements including polypeptides
of the invention is within the level of skill in the food
preparation art.
5.9.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION ACTIVITY
[0319] A protein of the present invention may exhibit activity
relating to cytokine, cell proliferation (either inducing or
inhibiting) or cell differentiation (either inducing or inhibiting)
activity or may induce production of other cytokines in certain
cell populations. A polynucleotide of the invention can encode a
polypeptide exhibiting such attributes. Many protein factors
discovered to date, including all known cytokines, have exhibited
activity in one or more factor-dependent cell proliferation assays,
and hence the assays serve as a convenient confirmation of cytokine
activity. The activity of therapeutic compositions of the present
invention is evidenced by any one of a number of routine factor
dependent cell proliferation assays for cell lines including,
without limitation, 32D, DA2, DAIG, T10, B9, B9/11, BaF3, MC9/G,
M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e,
CMK, HUVEC, and Caco. Therapeutic compositions of the invention can
be used in the following:
[0320] Assays for T-cell or thymocyte proliferation include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai
et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J.
Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular
Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.
149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,
1994.
[0321] Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
interleukin-, Schreiber, R. D. In Current Protocols in Inmmunology.
J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
[0322] Assays for proliferation and differentiation of
hematopoietic and lymphopoietic cells include, without limitation,
those described in: Measurement of Human and Murine Interleukin 2
and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In
Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.
6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al.,
J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:690-692, 1988; Greenberger et al., Proc. Nad. Acad. Sci. U.S.A.
80:2931-2938, 1983; Measurement of mouse and human interleukin
6-Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds.
Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et
al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement
of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C.
and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan
eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;
Measurement of mouse and human Interleukin 9-Ciarletta, A.,
Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols
in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and
Sons, Toronto. 1991.
[0323] Assays for T-cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W
Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter
6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA
77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405411,
1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988.
5.9.4 STEM CELL GROWTH FACTOR ACTIVITY
[0324] Polypeptides of the present invention have been shown to
exhibit stem cell growth factor activity and to be involved in the
proliferation, differentiation and survival of pluripotent and
totipotent stem cells including primordial germ cells, embryonic
stem cells, neural stem cells, skeletal muscle stem cells,
mesencymal stem cells, hematopoietic stem cells and/or germ line
stem cells. Administration of the polypeptide of the invention to
stem cells in vivo or ex vivo maintains and expands cell
populations in a totipotential or pluripotential state which would
be useful for re-engineering damaged or diseased tissues,
transplantation including solid organs and bone marrow transplants,
manufacture of bio-pharmaceuticals and the development of
bio-sensors. The ability to produce large quantities of human cells
has important working applications for the production of human
proteins which currently must be obtained from non-human sources or
donors, implantation of cells to treat diseases such as
Parkinson's, Alzheimer's and other neurodegenerative diseases;
tissues for grafting such as bone marrow, skin, cartilage, tendons,
bone, muscle (including cardiac muscle), blood vessels, cornea,
neural cells, gastrointestinal cells and others; and organs for
transplantation such as kidney, liver, pancreas (including islet
cells), heart and lung.
[0325] It is contemplated that multiple different exogenous growth
factors and/or cytokines may be administered in combination with
the polypeptide of the invention to achieve the desired effect,
including any of the growth factors listed herein, other stem cell
maintenance factors, and specifically including stem cell factor
(SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any
of the interleukins, recombinant soluble IL-6 receptor fused to
IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF,
GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4),
platelet-derived growth factor (PDGF), neural growth factors and
basic fibroblast growth factor (bFGF).
[0326] Since totipotent stem cells can give rise to virtually any
mature cell type, expansion of these cells in culture will
facilitate the production of large quantities of mature cells.
Techniques for culturing stem cells are known in the art and
administration of polypeptides of the invention, optionally with
other growth factors and/or cytokines, is expected to enhance the
survival and proliferation of the stem cell populations. This can
be accomplished by direct administration of the polypeptide of the
invention to the culture medium. Alternatively, stroma cells
transfected with a polynucleotide that encodes for the polypeptide
of the invention can be used as a feeder layer for the stem cell
populations in culture or in vivo. Stromal support cells for feeder
layers may include embryonic bone marrow fibroblasts, bone marrow
stromal cells, fetal liver cells, or cultured embryonic fibroblasts
(see U.S. Pat. No. 5,690,926).
[0327] Stem cells themselves can be transfected with a
polynucleotide of the invention to induce autocrine expression of
the polypeptide of the invention. This will allow for generation of
undifferentiated totipotential/pluripotential stem cell lines that
are useful as is or that can then be differentiated into the
desired mature cell types. These stable cell lines can also serve
as a source of undifferentiated totipotential/pluripotential mRNA
to create cDNA libraries and templates for polymerase chain
reaction experiments. These studies would allow for the isolation
and identification of differentially expressed genes in stem cell
populations that regulate stem cell proliferation and/or
maintenance.
[0328] Expansion and maintenance of totipotent stem cell
populations is useful in the treatment of many pathological
conditions. For example, polypeptides of the present invention may
be used to manipulate stem cells in culture to give rise to
neuroepithelial cells that can be used to augment or replace cells
damaged by illness, autoimmune disease, accidental damage or
genetic disorders, inflammatory disease, immunodeficiency, leukemia
and neoplastic myeloid disorders. The polypeptide of the invention
can be useful for inducing the proliferation of neural cells and
for the regeneration of nerve and brain tissue, i.e. for the
treatment of central and peripheral nervous system diseases and
neuropathies, as well as mechanical and traumatic disorders which
involve degeneration, death or trauma to neural cells or nerve
tissue. In addition, the expanded stem cell populations can also be
genetically altered for gene therapy purposes and to decrease host
rejection of replacement tissues after grafting or implantation.
The polypeptide of the invention can also be useful for inducing
the proliferation of cardiac stem cells and for regenerating
functional heart tissue following cardiac damage induced by cardiac
disorders such as myocardial infarctions and artery blockage. In
addition, the polypeptides of the invention may strengthen cardiac
muscle cells and prevent and/or repair the heart tissue damage due
to heart failure. See Weismann, Science, 287: 1442-1446, 2001;
Vogel, Science, 290: 1672-1674, 2000 Kajstura et al., Nature, 410:
701-705, 2001.
[0329] Expression of the polypeptide of the invention and its
effect on stem cells can also be manipulated to achieve controlled
differentiation of the stem cells into more differentiated cell
types. A broadly applicable method of obtaining pure populations of
a specific differentiated cell type from undifferentiated stem cell
populations involves the use of a cell-type specific promoter
driving a selectable marker. The selectable marker allows only
cells of the desired type to survive. For example, stem cells can
be induced to differentiate into cardiomyocytes (Wobus et al.,
Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder,
L. W. In: Principles of Tissue Engineering eds. Lanza et al.,
Academic Press (1997)). Alternatively, directed differentiation of
stem cells can be accomplished by culturing the stem cells in the
presence of a differentiation factor such as retinoic acid and an
antagonist of the polypeptide of the invention which would inhibit
the effects of endogenous stem cell factor activity and allow
differentiation to proceed.
[0330] In vitro cultures of stem cells can be used to determine if
the polypeptide of the invention exhibits stem cell growth factor
activity. Stem cells are isolated from any one of various cell
sources (including hematopoietic stem cells and embryonic stem
cells) and cultured on a feeder layer, as described by Thompson et
al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the
presence of the polypeptide of the invention alone or in
combination with other growth factors or cytokines. The ability of
the polypeptide of the invention to induce stem cells proliferation
is determined by colony formation on semi-solid support e.g. as
described by Bernstein et al., Blood, 77: 2316-2321 (1991).
5.9.5 HEMATOPOIESIS REGULATING ACTIVITY
[0331] A protein of the present invention may be involved in
regulation of hematopoiesis and, consequently, in the treatment of
myeloid or lymphoid cell disorders. Even marginal biological
activity in support of colony forming cells or of factor-dependent
cell lines indicates involvement in regulating hematopoiesis, e.g.
in supporting the growth and proliferation of erythroid progenitor
cells alone or in combination with other cytokines, thereby
indicating utility, for example, in treating various anemias or for
use in conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors and/or erythroid cells; in
supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional
circulating soluble factor activity) useful, for example, in
conjunction with chemotherapy to prevent or treat consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and consequently of platelets thereby allowing
prevention or treatment of various platelet disorders such as
thrombocytopenia, and generally for use in place of or
complimentary to platelet transfusions; and/or in supporting the
growth and proliferation of hematopoietic stem cells which are
capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post irradiation/chemotherapy, either
in-vivo or ex-vivo (i.e., in conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically
manipulated for gene therapy.
[0332] Therapeutic compositions of the invention can be used in the
following:
[0333] Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
[0334] Assays for embryonic stem cell differentiation (which will
identify, among others, proteins that influence embryonic
differentiation hematopoiesis) include, without limitation, those
described in: Johansson et al. Cellular Biology 15:141-151, 1995;
Keller et al., Molecular and Cellular Biology 13:473-486, 1993;
McClanahan et al., Blood 81:2903-2915, 1993.
[0335] Assays for stem cell survival and differentiation (which
will identify, among others, proteins that regulate
lympho-hematopoiesis) include, without limitation, those described
in: Methylcellulose colony forming assays, Freshney, M. G. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.
265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al.,
Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative
potential, McNiece, I. K. and Briddell, R. A. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay,
Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York,
N.Y. 1994; Long term bone marrow cultures in the presence of
stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179,
Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating
cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R.
I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New
York, N.Y. 1994.
5.9.6 TISSUE GROWTH ACTIVITY
[0336] A protein of the present invention also may be involved in
bone, cartilage, tendon, ligament and/or nerve tissue growth or
regeneration, as well as in wound healing and tissue repair and
replacement, and in healing of burns, incisions and ulcers.
[0337] For example, induction of cartilage and/or bone growth in
circumstances where bone is not normally formed, has application in
the healing of bone fractures and cartilage damage or defects in
humans and other animals. Compositions of a protein, antibody,
binding partner, or other modulator of the invention may have
prophylactic use in closed as well as open fracture reduction and
also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair
of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and also is useful in cosmetic plastic
surgery.
[0338] A protein of this invention may also be involved in
attracting bone-forming cells, stimulating growth of bone-forming
cells, or inducing differentiation of progenitors of bone-forming
cells. Treatment of osteoporosis, osteoarthritis, bone degenerative
disorders, or periodontal disease, such as through stimulation of
bone and/or cartilage repair or by blocking inflammation or
processes of tissue destruction (collagenase activity, osteoclast
activity, etc.) mediated by inflammatory processes may also be
possible using the composition of the invention.
[0339] Another category of tissue regeneration activity that may
involve the protein of the present invention is tendon/ligament
formation. Induction of tendon/ligament-like tissue or other tissue
formation in circumstances where such tissue is not normally
formed, has application in the healing of tendon or ligament tears,
deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a tendon/ligament-like
tissue inducing protein may have prophylactic use in preventing
damage to tendon or ligament tissue, as well as use in the improved
fixation of tendon or ligament to bone or other tissues, and in
repairing defects to tendon or ligament tissue. De novo
tendon/ligament-like tissue formation induced by a composition of
the present invention contributes to the repair of congenital,
trauma induced, or other tendon or ligament defects of other
origin, and is also useful in cosmetic plastic surgery for
attachment or repair of tendons or ligaments. The compositions of
the present invention may provide environment to attract tendon- or
ligament-forming cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of
tendon- or ligament-forming cells, or induce growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to
effect tissue repair. The compositions of the invention may also be
useful in the treatment of tendinitis, carpal tunnel syndrome and
other tendon or ligament defects. The compositions may also include
an appropriate matrix and/or sequestering agent as a carrier as is
well known in the art.
[0340] The compositions of the present invention may also be useful
for proliferation of neural cells and for regeneration of nerve and
brain tissue, i.e. for the treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to
neural cells or nerve tissue. More specifically, a composition may
be used in the treatment of diseases of the peripheral nervous
system, such as peripheral nerve injuries, peripheral neuropathy
and localized neuropathies, and central nervous system diseases,
such as Alzheimer's, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further
conditions which may be treated in accordance with the present
invention include mechanical and traumatic disorders, such as
spinal cord disorders, head trauma and cerebrovascular diseases
such as stroke. Peripheral neuropathies resulting from chemotherapy
or other medical therapies may also be treatable using a
composition of the invention.
[0341] Compositions of the invention may also be useful to promote
better or faster closure of non-healing wounds, including without
limitation pressure ulcers, ulcers associated with vascular
insufficiency, surgical and traumatic wounds, and the like.
[0342] Compositions of the present invention may also be involved
in the generation or regeneration of other tissues, such as organs
(including, for example, pancreas, liver, intestine, kidney, skin,
endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the
growth of cells comprising such tissues. Inhibition or modulation
of fibrotic scarring may allow normal tissue to regenerate.
[0343] A composition of the present invention may also be useful
for gut protection or regeneration and treatment of lung or liver
fibrosis, reperfusion injury in various tissues, and conditions
resulting from systemic cytokine damage.
[0344] A composition of the present invention may also be useful
for promoting or inhibiting differentiation of tissues described
above from precursor tissues or cells; or for inhibiting the growth
of tissues described above.
[0345] Therapeutic compositions of the invention can be used in the
following:
[0346] Assays for tissue generation activity include, without
limitation, those described in: International Patent Publication
No. WO95/16035 (bone, cartilage, tendon); International Patent
Publication No. WO95/05846 (nerve, neuronal); International Patent
Publication No. WO91/07491 (skin, endothelium).
[0347] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest. Dermatol 71:382-84 (1978).
5.9.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
[0348] Compositions of the present invention may also exhibit
immune stimulating or immune suppressing activity, including
without limitation the activities for which assays are described
herein. A polynucleotide of the invention can encode a polypeptide
involved in such activities. A protein or antibody, other binding
partner, or other modulator of the invention may be useful in the
treatment of various immune deficiencies and disorders (including
severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down) growth and proliferation of T and/or B lymphocytes, as
well as effecting the cytolytic activity of NK cells and other cell
populations. These immune deficiencies may be genetic or be caused
by viral (e.g., HIV) as well as bacterial or fungal infections, or
may result from autoimmune disorders. More specifically, infectious
diseases caused by viral, bacterial, fungal or other infection may
be treatable using a protein, antibody, binding partner, or other
modulator of the invention, including infections by HIV, hepatitis
viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp.
and various fungal infections such as candidiasis, as well as other
conditions where a boost to the immune system generally may be
desirable, e.g., in the treatment of cancer.
[0349] Autoimmune disorders which may involve a protein of the
present invention include, for example, connective tissue disease,
multiple sclerosis, systemic lupus erythematosus, rheumatoid
arthritis, autoimmune pulmonary inflammation, Guillain-Barre
syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis, myasthenia gravis, graft-versus-host disease and
autoimmune inflammatory eye disease. Such a protein of the present
invention may also to be involved in allergic reactions and
conditions, such as asthma (particularly allergic asthma) or other
respiratory problems.
[0350] Using the proteins, antibody, binding partners, or other
modulators of the invention it may also be possible to modulate
immune responses, in a number of ways. The immune response may be
enhanced or suppressed. Down regulation may be in the form of
inhibiting or blocking an immune response already in progress or
may involve preventing the induction of an immune response. The
functions of activated T cells may be inhibited by suppressing T
cell responses or by inducing specific tolerance in T cells, or
both. Immunosuppression of T cell responses is generally an active,
non-antigen-specific, process which requires continuous exposure of
the T cells to the suppressive agent. Tolerance, which involves
inducing non-responsiveness or anergy in T cells, is
distinguishable from immunosuppression in that it is generally
antigen-specific and persists after exposure to the tolerizing
agent has ceased. Operationally, tolerance can be demonstrated by
the lack of a T cell response upon reexposure to specific antigen
in the absence of the tolerizing agent.
[0351] Down regulating or preventing the immune response, e.g.,
preventing high level lymphokine synthesis by activated T cells,
will be useful in situations of tissue, skin and organ
transplantation and in graft-versus-host disease (GVHD). For
example, blockage of T cell function should result in reduced
tissue destruction in tissue transplantation. Typically, in tissue
transplants, rejection of the transplant is initiated through its
recognition as foreign by T cells, followed by an immune reaction
that destroys the transplant. The administration of a molecule
which inhibits or blocks the immune response (e.g. a receptor
fragment, binding partner, or other modulator such as antisense
polynucleotides) may act as an immunosuppressant.
[0352] The efficacy of particular immune response modulators in
preventing organ transplant rejection or GVHD can be assessed using
animal models that are predictive of efficacy in humans. Examples
of appropriate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of
GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York,
1989, pp. 846-847) can be used to determine the effect of blocking
B lymphocyte antigen function in vivo on the development of that
disease.
[0353] Blocking the inflammatory response may also be
therapeutically useful for treating autoimmune diseases. Many
autoimmune disorders are the result of inappropriate activation of
T cells that are reactive against self tissue and which promote the
production of cytokines and autoantibodies involved in the
pathology of the diseases. Preventing the activation of
autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells can
be used to inhibit T cell activation and prevent production of
autoantibodies or T cell-derived cytokines which may be involved in
the disease process. Additionally, blocking reagents may induce
antigen-specific tolerance of autoreactive T cells which could lead
to long-term relief from the disease. The efficacy of blocking
reagents in preventing or alleviating autoimmune disorders can be
determined using a number of well-characterized animal models of
human autoimmune diseases. Examples include murine experimental
autoimmune encephalitis, systemic lupus erythematosus in
MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen
arthritis, diabetes mellitus in NOD mice and BB rats, and murine
experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856).
[0354] Upregulation of immune responses, may also be useful in
therapy. Upregulation of immune responses may be in the form of
enhancing an existing immune response or eliciting an initial
immune response. For example, enhancing an immune response may be
useful in cases of viral infection such as influenza, the common
cold, and encephalitis.
[0355] Alternatively, anti-viral immune responses may be enhanced
in an infected patient by removing T cells from the patient,
costimulating the T cells in vitro and reintroducing the in vitro
activated T cells into the patient.
[0356] The activity of therapeutic compositions of the invention
may, among other means, be measured by the following methods:
[0357] Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current Protocols
in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology
61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et
al., J. Immunol. 153:3079-3092, 1994.
[0358] Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins that
modulate T-cell dependent antibody responses and that affect
Th1/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro antibody production, Mond, J. J. and Brunswick,
M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0359] Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Th1 and CTL
responses) include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et
al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
[0360] Dendritic cell-dependent assays (which will identify, among
others, proteins expressed by dendritic cells that activate naive
T-cells) include, without limitation, those described in: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:40624069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0361] Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Datzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
[0362] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
88:7548-7551, 1991.
5.9.8 ACTIVIN/INHIBIN ACTIVITY
[0363] A protein of the present invention may also exhibit activin-
or inhibin-related activities. A polynucleotide of the invention
may encode a polypeptide exhibiting such characteristics. Inhibins
are characterized by their ability to inhibit the release of
follicle stimulating hormone (FSH), while activins and are
characterized by their ability to stimulate the release of follicle
stimulating hormone (FSH). Thus, a protein of the present
invention, alone or in heterodimers with a member of the inhibin
family, may be useful as a contraceptive based on the ability of
inhibins to decrease fertility in female mammals and decrease
spermatogenesis in male mammals. Administration of sufficient
amounts of other inhibins can induce infertility in these mammals.
Alternatively, the protein of the invention, as a homodimer or as a
heterodimer with other protein subunits of the inhibin group, may
be useful as a fertility inducing-therapeutic, based upon the
ability of activin molecules in stimulating FSH release from cells
of the anterior pituitary. See, for example, U.S. Pat. No.
4,798,885. A protein of the invention may also be useful for
advancement of the onset of fertility in sexually immature mammals,
so as to increase the lifetime reproductive performance of domestic
animals such as, but not limited to, cows, sheep and pigs.
[0364] The activity of a protein of the invention may, among other
means, be measured by the following methods.
[0365] Assays for activin/inhibin activity include, without
limitation, those described in: Vale et al., Endocrinology
91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095,
1986.
5.9.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
[0366] A protein of the present invention may be involved in
chemotactic or chemokinetic activity for mammalian cells,
including, for example, monocytes, fibroblasts, neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells. A polynucleotide of the invention can encode a polypeptide
exhibiting such attributes. Chemotactic and chemokinetic receptor
activation can be used to mobilize or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic
compositions (e.g. proteins, antibodies, binding partners, or
modulators of the invention) provide particular advantages in
treatment of wounds and other trauma to tissues, as well as in
treatment of localized infections. For example, attraction of
lymphocytes, monocytes or neutrophils to tumors or sites of
infection may result in improved immune responses against the tumor
or infecting agent.
[0367] A protein or peptide has chemotactic activity for a
particular cell population if it can stimulate, directly or
indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability to
directly stimulate directed movement of cells. Whether a particular
protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any
known assay for cell chemotaxis.
[0368] Therapeutic compositions of the invention can be used in the
following:
[0369] Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et
al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768,
1994.
5.9.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
[0370] A protein of the invention may also be involved in
hemostatis or thrombolysis or thrombosis. A polynucleotide of the
invention can encode a polypeptide exhibiting such attributes.
Compositions may be useful in treatment of various coagulation
disorders (including hereditary disorders, such as hemophilias) or
to enhance coagulation and other hemostatic events in treating
wounds resulting from trauma, surgery or other causes. A
composition of the invention may also be useful for dissolving or
inhibiting formation of thromboses and for treatment and prevention
of conditions resulting therefrom (such as, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
[0371] Therapeutic compositions of the invention can be used in the
following:
[0372] Assay for hemostatic and thrombolytic activity include,
without limitation, those described in: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
5.9.11 CANCER DIAGNOSIS AND THERAPY
[0373] Polypeptides of the invention may be involved in cancer cell
generation, proliferation or metastasis. Detection of the presence
or amount of polynucleotides or polypeptides of the invention may
be useful for the diagnosis and/or prognosis of one or more types
of cancer. For example, the presence or increased expression of a
polynucleotide/polypeptide of the invention may indicate a
hereditary risk of cancer, a precancerous condition, or an ongoing
malignancy. Conversely, a defect in the gene or absence of the
polypeptide may be associated with a cancer condition.
Identification of single nucleotide polymorphisms associated with
cancer or a predisposition to cancer may also be useful for
diagnosis or prognosis.
[0374] Cancer treatments promote tumor regression by inhibiting
tumor cell proliferation, inhibiting angiogenesis (growth of new
blood vessels that is necessary to support tumor growth) and/or
prohibiting metastasis by reducing tumor cell motility or
invasiveness. Therapeutic compositions of the invention may be
effective in adult and pediatric oncology including in solid phase
tumors/malignancies, locally advanced tumors, human soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood
cell malignancies including multiple myeloma, acute and chronic
leukemias, and lymphomas, head and neck cancers including mouth
cancer, larynx cancer and thyroid cancer, lung cancers including
small cell carcinoma and non-small cell cancers, breast cancers
including small cell carcinoma and ductal carcinoma,
gastrointestinal cancers including esophageal cancer; stomach
cancer, colon cancer, colorectal cancer and polyps associated with
colorectal neoplasia, pancreatic cancers, liver cancer, urologic
cancers including bladder cancer and prostate cancer, malignancies
of the female genital tract including ovarian carcinoma, uterine
(including endometrial) cancers, and solid tumor in the ovarian
follicle, kidney cancers including renal cell carcinoma, brain
cancers including intrinsic brain tumors, neuroblastoma, astrocytic
brain tumors, gliomas, metastatic tumor cell invasion in the
central nervous system, bone cancers including osteomas, skin
cancers including malignant melanoma, tumor progression of human
skin keratinocytes, squamous cell carcinoma, basal cell carcinoma,
hemangiopericytoma and Karposi's sarcoma.
[0375] Polypeptides, polynucleotides, or modulators of polypeptides
of the invention (including inhibitors and stimulators of the
biological activity of the polypeptide of the invention) may be
administered to treat cancer. Therapeutic compositions can be
administered in therapeutically effective dosages alone or in
combination with adjuvant cancer therapy such as surgery,
chemotherapy, radiotherapy, thermotherapy, and laser therapy, and
may provide a beneficial effect, e.g. reducing tumor size, slowing
rate of tumor growth, inhibiting metastasis, or otherwise improving
overall clinical condition, without necessarily eradicating the
cancer.
[0376] The composition can also be administered in therapeutically
effective amounts as a portion of an anti-cancer cocktail. An
anti-cancer cocktail is a mixture of the polypeptide or modulator
of the invention with one or more anti-cancer drugs in addition to
a pharmaceutically acceptable carrier for delivery. The use of
anti-cancer cocktails as a cancer treatment is routine. Anti-cancer
drugs that are well known in the art and can be used as a treatment
in combination with the polypeptide or modulator of the invention
include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin,
Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin
(cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside),
Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl,
Estramustine phosphate sodium, Etoposide (V16-213),. Floxuridine,
5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide),
Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide
acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine
HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna,
Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,
Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,
Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,
Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2,
Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine
sulfate.
[0377] In addition, therapeutic compositions of the invention may
be used for prophylactic treatment of cancer. There are hereditary
conditions and/or environmental situations (e.g. exposure to
carcinogens) known in the art that predispose an individual to
developing cancers. Under these circumstances, it may be beneficial
to treat these individuals with therapeutically effective doses of
the polypeptide of the invention to reduce the risk of developing
cancers.
[0378] In vitro models can be used to determine the effective doses
of the polypeptide of the invention as a potential cancer
treatment. These in vitro models include proliferation assays of
cultured tumor cells, growth of cultured tumor cells in soft agar
(see Freshney, (1987) Culture of Animal Cells: A Manual of Basic
Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor
systems in nude mice as described in Giovanella et al., J. Natl.
Can. Inst., 52: 921-30 (1974), mobility and invasive potential of
tumor cells in Boyden Chamber assays as described in Pilkington et
al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays
such as induction of vascularization of the chick chorioallantoic
membrane or induction of vascular endothelial cell migration as
described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97
(1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999)
respectively. Suitable tumor cells lines are available, e.g. from
American Type Tissue Culture Collection catalogs.
5.9.12 RECEPTOR/LIGAND ACTIVITY
[0379] A protein of the present invention may also demonstrate
activity as receptor, receptor ligand or inhibitor or agonist of
receptor/ligand interactions. A polynucleotide of the invention can
encode a polypeptide exhibiting such characteristics. Examples of
such receptors and ligands include, without limitation, cytokine
receptors and their ligands, receptor kinases and their ligands,
receptor phosphatases and their ligands, receptors involved in
cell-cell interactions and their ligands (including without
limitation, cellular adhesion molecules (such as selectins,
integrins and their ligands) and receptor/ligand pairs involved in
antigen presentation, antigen recognition and development of
cellular and humoral immune responses. Receptors and ligands are
also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein
of the present invention (including, without limitation, fragments
of receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand interactions.
[0380] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0381] Suitable assays for receptor-ligand activity include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion
under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl.
Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994;
Stitt et al., Cell 80:661-670, 1995.
[0382] By way of example, the polypeptides of the invention may be
used as a receptor for a ligand(s) thereby transmitting the
biological activity of that ligand(s). Ligands may be identified
through binding assays, affinity chromatography, dihybrid screening
assays, BIAcore assays, gel overlay assays, or other methods known
in the art.
[0383] Studies characterizing drugs or proteins as agonist or
antagonist or partial agonists or a partial antagonist require the
use of other proteins as competing ligands. The polypeptides of the
present invention or ligand(s) thereof may be labeled by being
coupled to radioisotopes, colorimetric molecules or a toxin
molecules by conventional methods. ("Guide to Protein Purification"
Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990)
Academic Press, Inc. San Diego). Examples of radioisotopes include,
but are not limited to, tritium and carbon-14. Examples of
colorimetric molecules include, but are not limited to, fluorescent
molecules such as fluorescamine, or rhodamine or other colorimetric
molecules. Examples of toxins include, but are not limited, to
ricin.
5.9.13 DRUG SCREENING
[0384] This invention is particularly useful for screening chemical
compounds by using the novel polypeptides or binding fragments
thereof in any of a variety of drug screening techniques. The
polypeptides or fragments employed in such a test may either be
free in solution, affixed to a solid support, borne on a cell
surface or located intracellularly. One method of drug screening
utilizes eukaryotic or prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the
polypeptide or fragment. Drugs are screened against such
transformed cells in competitive binding assays. Such cells, either
in viable or fixed form, can be used for standard binding assays.
One may measure, for example, the formation of complexes between
polypeptides of the invention or fragments and the agent being
tested or examine the diminution in complex formation between the
novel polypeptides and an appropriate cell line, which are well
known in the art.
[0385] Sources for test compounds that may be screened for ability
to bind to or modulate (i.e., increase or decrease) the activity of
polypeptides of the invention include (1) inorganic and organic
chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries comprised of either random or mimetic
peptides, oligonucleotides or organic molecules.
[0386] Chemical libraries may be readily synthesized or purchased
from a number of commercial sources, and may include structural
analogs of known compounds or compounds that are identified as
"hits" or "leads" via natural product screening.
[0387] The sources of natural product libraries are microorganisms
(including bacteria and fungi), animals, plants or other
vegetation, or marine organisms, and libraries of mixtures for
screening may be created by: (1) fermentation and extraction of
broths from soil, plant or marine microorganisms or (2) extraction
of the organisms themselves. Natural product libraries include
polyketides, non-ribosomal peptides, and (non-naturally occurring)
variants thereof. For a review, see Science 282:63-68 (1998).
[0388] Combinatorial libraries are composed of large numbers of
peptides, oligonucleotides or organic compounds and can be readily
prepared by traditional automated synthesis methods, PCR, cloning
or proprietary synthetic methods. Of particular interest are
peptide and oligonucleotide combinatorial libraries. Still other
libraries of interest include peptide, protein, peptidomimetic,
multiparallel synthetic collection, recombinatorial, and
polypeptide libraries. For a review of combinatorial chemistry and
libraries created therefrom, see Myers, Curr. Opin. Biotechnol.
8:701-707 (1997). For reviews and examples of peptidomimetic
libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23
(1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997);
Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated
dipeptides).
[0389] Identification of modulators through use of the various
libraries described herein permits modification of the candidate
"hit" (or "lead") to optimize the capacity of the "hit" to bind a
polypeptide of the invention. The molecules identified in the
binding assay are then tested for antagonist or agonist activity in
in vivo tissue culture or animal models that are well known in the
art. In brief, the molecules are titrated into a plurality of cell
cultures or animals and then tested for either cell/animal death or
prolonged survival of the animal/cells.
[0390] The binding molecules thus identified may be complexed with
toxins, e.g., ricin or cholera, or with other compounds that are
toxic to cells such as radioisotopes. The toxin-binding molecule
complex is then targeted to a tumor or other cell by the
specificity of the binding molecule for a polypeptide of the
invention. Alternatively, the binding molecules may be complexed
with imaging agents for targeting and imaging purposes.
5.9.14 ASSAY FOR RECEPTOR ACTIVITY
[0391] The invention also provides methods to detect specific
binding of a polypeptide e.g. a ligand or a receptor. The art
provides numerous assays particularly useful for identifying
previously unknown binding partners for receptor polypeptides of
the invention. For example, expression cloning using mammalian or
bacterial cells, or dihybrid screening assays can be used to
identify polynucleotides encoding binding partners. As another
example, affinity chromatography with the appropriate immobilized
polypeptide of the invention can be used to isolate polypeptides
that recognize and bind polypeptides of the invention. There are a
number of different libraries used for the identification of
compounds, and in particular small molecule, that modulate (i.e.,
increase or decrease) biological activity of a polypeptide of the
invention. Ligands for receptor polypeptides of the invention can
also be identified by adding exogenous ligands, or cocktails of
ligands to two cells populations that are genetically identical
except for the expression of the receptor of the invention: one
cell population expresses the receptor of the invention whereas the
other does not. The response of the two cell populations to the
addition of ligands(s) are then compared. Alternatively, an
expression library can be co-expressed with the polypeptide of the
invention in cells and assayed for an autocrine response to
identify potential ligand(s). As stiff another example, BIAcore
assays, gel overlay assays, or other methods known in the art can
be used to identify binding partner polypeptides, including, (1)
organic and inorganic chemical libraries, (2) natural product
libraries, and (3) combinatorial libraries comprised of random
peptides, oligonucleotides or organic molecules.
[0392] The role of downstream intracellular signaling molecules in
the signaling cascade of the polypeptide of the invention can be
determined. For example, a chimeric protein in which the
cytoplasmic domain of the polypeptide of the invention is fused to
the extracellular portion of a protein, whose ligand has been
identified, is produced in a host cell. The cell is then incubated
with the ligand specific for the extracellular portion of the
chimeric protein, thereby activating the chimeric receptor. Known
downstream proteins involved in intracellular signaling can then be
assayed for expected modifications i.e. phosphorylation. Other
methods known to those in the art can also be used to identify
signaling molecules involved in receptor activity.
5.9.15 ANTI-INFLAMMATORY ACTIVTY
[0393] Compositions of the present invention may also exhibit
anti-inflammatory activity. The anti-inflammatory activity may be
achieved by providing a stimulus to cells involved in the
inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
directly inhibit or promote an inflammatory response. Compositions
with such activities can be used to treat inflammatory conditions
including chronic or acute conditions), including without
limitation intimation associated with infection (such as septic
shock, sepsis or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's
disease or resulting from over production of cytokines such as TNF
or IL-1. Compositions of the invention may also be useful to treat
anaphylaxis and hypersensitivity to an antigenic substance or
material. Compositions of this invention may be utilized to prevent
or treat condition such as, but not limited to, utilized, for
example, as part of methods for the prevention and/or treatment of
disorders involving sepsis, acute pancreatitis, endotoxin shock,
cytokine induced shock, rheumatoid arthritis, chronic inflammatory
arthritis, pancreatic cell damage from diabetes mellitus type 1,
graft versus host disease, inflammatory bowel disease, inflamation
associated with pulmonary disease, other autoimmune disease or
inflammatory disease, an antiproliferative agent such as for acute
or chronic mylegenous leukemia or in the prevention of premature
labor secondary to intrauterine infections.
5.9.16 LEUKEMIAS
[0394] Leukemias and related disorders may be treated or prevented
by administration of a therapeutic that promotes or inhibits
function of the polynucleotides and/or polypeptides of the
invention. Such leukemias and related disorders include but are not
limited to acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic,
monocytic, erythroleukemia, chronic leukemia, chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia).
5.9.17 NERVOUS SYSTEM DISORDERS
[0395] Nervous system disorders, involving cell types which can be
tested for efficacy of intervention with compounds that modulate
the activity of the polynucleotides and/or polypeptides of the
invention, and which can be treated upon thus observing an
indication of therapeutic utility, include but are not limited to
nervous system injuries, and diseases or disorders which result in
either a disconnection of axons, a diminution or degeneration of
neurons, or demyelination. Nervous system lesions which may be
treated in a patient (including human and non-human mammalian
patients) according to the invention include but are not limited to
the following lesions of either the central (including spinal cord,
brain) or peripheral nervous systems:
[0396] (i) traumatic lesions, including lesions caused by physical
injury or associated with surgery, for example, lesions which sever
a portion of the nervous system, or compression injuries;
[0397] (ii) ischemic lesions, in which a lack of oxygen in a
portion of the nervous system results in neuronal injury or death,
including cerebral infarction or ischemia, or spinal cord
infarction or ischemia;
[0398] (iii) infectious lesions, in which a portion of the nervous
system is destroyed or injured as a result of infection, for
example, by an abscess or associated with infection by human
immunodeficiency virus, herpes zoster, or herpes simplex virus or
with Lyme disease, tuberculosis, syphilis;
[0399] (iv) degenerative lesions, in which a portion of the nervous
system is destroyed or injured as a result of a degenerative
process including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis;
[0400] (v) lesions associated with nutritional diseases or
disorders, in which a portion of the nervous system is destroyed or
injured by a nutritional disorder or disorder of metabolism
including but not limited to, vitamin B12 deficiency, folic acid
deficiency, Wernicke disease, tobacco-alcohol amblyopia,
Marchiafava-Bignami disease (primary degeneration of the corpus
callosum), and alcoholic cerebellar degeneration;
[0401] (vi) neurological lesions associated with systemic diseases
including but not limited to diabetes (diabetic neuropathy, Bell's
palsy), systemic lupus erythematosus, carcinoma, or
sarcoidosis;
[0402] (vii) lesions caused by toxic substances including alcohol,
lead, or particular neurotoxins; and
[0403] (viii) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including but not limited to multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[0404] Therapeutics which are useful according to the invention for
treatment of a nervous system disorder may be selected by testing
for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, therapeutics which elicit any of the following effects
may be useful according to the invention:
[0405] (i) increased survival time of neurons in culture;
[0406] (ii) increased sprouting of neurons in culture or in
vivo;
[0407] (iii) increased production of a neuron-associated molecule
in culture or in vivo, e.g., choline acetyltransferase or
acetylcholinesterase with respect to motor neurons; or (iv)
decreased symptoms of neuron dysfunction in vivo.
[0408] Such effects may be measured by any method known in the art.
In preferred, non-limiting embodiments, increased survival of
neurons may be measured by the method set forth in Arakawa et al.
(1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons
may be detected by methods set forth in Pestronk et al. (1980, Exp.
Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci.
4:17-42); increased production of neuron-associated molecules may
be measured by bioassay, enzymatic assay, antibody binding,
Northern blot assay, etc., depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0409] In specific embodiments, motor neuron disorders that may be
treated according to the invention include but are not limited to
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including but not limited to
progressive spinal muscular atrophy, progressive bulbar palsy;
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
5.9.18 ARTHRITIS AND INFLAMMATION
[0410] The immunosuppressive effects of the compositions of the
invention against rheumatoid arthritis is determined in an
experimental animal model system. The experimental model system is
adjuvant induced arthritis in rats, and the protocol is described
by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et
al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of
the disease can be caused by a single injection, generally
intradermally, of a suspension of killed Mycobacterium tuberculosis
in complete Freund's adjuvant (CFA). The route of injection can
vary, but rats may be injected at the base of the tail with an
adjuvant mixture. The inhibitor is administered in phosphate
buffered solution (PBS) at a dose of about 1-5 mg/kg. The control
consists of administering PBS only.
[0411] The procedure for testing the effects of the test compound
would consist of intradermally injecting killed Mycobacterium
tuberculosis in CFA followed by immediately administering the
inhibitor and subsequent treatment every other day until day 24. At
14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium
CFA, an overall arthritis score may be obtained as described by J.
Holoskitz above. An analysis of the data would reveal that the test
compound would have a dramatic affect on the swelling of the joints
as measured by a decrease of the arthritis score.
5.9.19 OTHER ACTIVITIES
[0412] A protein of the invention may also exhibit one or more of
the following additional activities or effects: inhibiting the
growth, infection or function of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other
parasites; effecting (suppressing or enhancing) bodily
characteristics, including, without limitation, height, weight,
hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ or body part size or shape (such as, for
example, breast augmentation or diminution, change in bone form or
shape); effecting biorhythms or circadian cycles or rhythms;
effecting the fertility of male or female subjects; effecting the
metabolism, catabolism, anabolism, processing, utilization, storage
or elimination of dietary fat, lipid, protein, carbohydrate,
vitamins, minerals, co-factors or other nutritional factors or
component(s); effecting behavioral characteristics, including,
without limitation, appetite, libido, stress, cognition (including
cognitive disorders), depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain
reducing effects; promoting differentiation and growth of embryonic
stem cells in lineages other than hematopoietic lineages; hormonal
or endocrine activity; in the case of enzymes, correcting
deficiencies of the enzyme and treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for
example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the
ability to act as an antigen in a vaccine composition to raise an
immune response against such protein or another material or entity
which is cross-reactive with such protein.
5.9.20 IDENTIFICATION OF POLYMORPHISMS
[0413] The demonstration of polymorphisms makes possible the
identification of such polymorphisms in human subjects and the
pharmacogenetic use of this information for diagnosis and
treatment. Such polymorphisms may be associated with, e.g.,
differential predisposition or susceptibility to various disease
states (such as disorders involving inflammation or immune
response) or a differential response to drug administration, and
this genetic information can be used to tailor preventive or
therapeutic treatment appropriately. For example, the existence of
a polymorphism associated with a predisposition to inflammation or
autoimmune disease makes possible the diagnosis of this condition
in humans by identifying the presence of the polymorphism.
[0414] Polymorphisms can be identified in a variety of ways known
in the art which all generally involve obtaining a sample from a
patient, analyzing DNA from the sample, optionally involving
isolation or amplification of the DNA, and identifying the presence
of the polymorphism in the DNA. For example, PCR may be used to
amplify an appropriate fragment of genomic DNA which may then be
sequenced. Alternatively, the DNA may be subjected to
allele-specific oligonucleotide hybridization (in which appropriate
oligonucleotides are hybridized to the DNA under conditions
permitting detection of a single base mismatch) or to a single
nucleotide extension assay (in which an oligonucleotide that
hybridizes immediately adjacent to the position of the polymorphism
is extended with one or more labeled nucleotides). In addition,
traditional restriction fragment length polymorphism analysis
(using restriction enzymes that provide differential digestion of
the genomic DNA depending on the presence or absence of the
polymorphism) may be performed. Arrays with nucleotide sequences of
the present invention can be used to detect polymorphisms. The
array can comprise modified nucleotide sequences of the present
invention in order to detect the nucleotide sequences of the
present invention. In the alternative, any one of the nucleotide
sequences of the present invention can be placed on the array to
detect changes from those sequences.
[0415] Alternatively a polymorphism resulting in a change in, the
amino acid sequence could also be detected by detecting a
corresponding change in amino acid sequence of the protein, e.g.,
by an antibody specific to the variant sequence.
5.10 THERAPEUTIC METHODS
[0416] The compositions (including polypeptide fragments, analogs,
variants and antibodies or other binding partners or modulators
including antisense polynucleotides) of the invention have numerous
applications in a variety of therapeutic methods. Examples of
therapeutic applications include, but are not limited to, those
exemplified herein.
5.10.1 EXAMPLES
[0417] Another embodiment of the invention is the administration of
an effective amount of the polypeptide or other composition of the
invention to individuals affected by a disease or disorder which
can be modulated by regulating the IgSF member of the invention.
While the mode of administration is not particularly important,
parenteral administration is preferred. An exemplary mode of
administration is to deliver an intravenous bolus. The dosage of
the polypeptide or composition of the invention will normally be
determined by the prescribing physician. It is to be expected that
the dosage will vary according to the age, weight, condition and
response of the individual patient. Typically, the amount of
protein or other active ingredient administered per dose will be in
the range of about 0.1 to 25 mg/kg of body weight, with the
preferred dose being about 0.1 to 10 mg/kg of patient body weight.
For parenteral administration, the polypeptides or other active
ingredient of the invention will be formulated in an injectable
form that includes a pharmaceutically acceptable parenteral
vehicle. Such vehicles are well known in the art and examples
include water, saline, Ringer's solution, dextrose solution, and
solutions consisting of small amounts of the human serum albumin.
The vehicle may contain minor amounts of additives that maintain
the isotonicity and stability of the polypeptide or other active
ingredient. The preparation of such solutions is within the skill
of the art. Typically, the cytokine inhibitor will be formulated in
such vehicles at a concentration of about 1-8 mg/ml to about 10
mg/ml.
5.11 PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION
[0418] A protein or other composition of the present invention
(from whatever source derived, including without limitation from
recombinant and non-recombinant sources and including antibodies
and other binding partners of the polypeptides of the invention)
may be administered to a patient in need, by itself, or in
pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s) at doses to treat or ameliorate a variety
of disorders. Such a composition may optionally contain (in
addition to protein or other active ingredient and a carrier)
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration. The pharmaceutical composition of the
invention may also contain cytokines, lymphokines, or other
hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8; IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IFN, TNF-, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem cell factor, and erythropoietin. In further
compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage
defect, wound, or tissue in questions. These agents include various
growth factors such as epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), transforming growth factors
(TGF- and TGF-), insulin-like growth factor (IGF), as well as
cytokines described herein.
[0419] The pharmaceutical composition may further contain other
agents which either enhance the activity of the protein or other
active ingredient or compliment its activity or use in treatment.
Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with
protein or other active ingredient of the invention, or to minimize
side effects. Conversely, protein or other active ingredient of the
present invention may be included in formulations of the particular
cytokine, lymphokine, other hematopoietic factor, thrombolytic or
anti-thrombotic factor, or anti-inflammatory agent to minimize side
effects of the cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers
(e.g., heterodimers or homodimers) or complexes with itself or
other proteins. As a result, pharmaceutical compositions of the
invention may comprise a protein of the invention in such
multimeric or complexed form.
[0420] As an alternative to being included in a pharmaceutical
composition of the invention including a first protein, a second
protein or a therapeutic agent may be concurrently administered
with the first protein (e.g., at the same time, or at differing
times provided that therapeutic concentrations of the combination
of agents is achieved at the treatment site). Techniques for
formulation and administration of the compounds of the instant
application may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co.; Easton, Pa., latest edition. A therapeutically
effective dose further refers to that amount of the compound
sufficient to result in amelioration of symptoms, e.g., treatment,
healing, prevention or amelioration of the relevant medical
condition, or an increase in rate of treatment, healing, prevention
or amelioration of such conditions. When applied to an individual
active ingredient, administered alone, a therapeutically effective
dose refers to that ingredient alone. When applied to a
combination, a therapeutically effective dose refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0421] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of protein or other
active ingredient of the present invention is administered to a
mammal having a condition to be treated. Protein or other active
ingredient of the present invention may be administered in
accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing
cytokines, lymphokines or other hematopoietic factors. When
co-administered with one or more cytokines, lymphokines or other
hematopoietic factors, protein or other active ingredient of the
present invention may be administered either simultaneously with
the cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially, the attending physician will decide on
the appropriate sequence of administering protein or other active
ingredient of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors.
5.11.1 ROUTES OF ADMINISTRATION
[0422] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Administration of protein or other active
ingredient of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can
be carried out in a variety of conventional ways, such as oral
ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
[0423] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a arthritic joints or in fibrotic tissue,
often in a depot or sustained release formulation. In order to
prevent the scarring process frequently occurring as complication
of glaucoma surgery, the compounds may be administered topically,
for example, as eye drops. Furthermore, one may administer the drug
in a targeted drug delivery system, for example, in a liposome
coated with a specific antibody, targeting, for example, arthritic
or fibrotic tissue. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0424] The polypeptides of the invention are administered by any
route that delivers an effective dosage to the desired site of
action. The determination of a suitable route of administration and
an effective dosage for a particular indication is within the level
of skill in the art. Preferably for wound treatment, one
administers the therapeutic compound directly to the site. Suitable
dosage ranges for the polypeptides of the invention can be
extrapolated from these dosages or from similar studies in
appropriate animal models. Dosages can then be adjusted as
necessary by the clinician to provide maximal therapeutic
benefit.
5.11.2 COMPOSITIONS/FORMULATIONS
[0425] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in a conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. These pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Proper formulation is dependent upon the route of
administration chosen. When a therapeutically effective amount of
protein or other active ingredient of the present invention is
administered orally, protein or other active ingredient of the
present invention will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein or
other active ingredient of the present invention, and preferably
from about 25 to 90% protein or other active ingredient of the
present invention. When administered in liquid form, a liquid
carrier such as water, petroleum, oils of animal or plant origin
such as peanut oil, mineral oil, soybean oil, or sesame oil, or
synthetic oils may be added. The liquid form of the pharmaceutical
composition may further contain physiological saline solution,
dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When administered
in liquid form, the pharmaceutical composition contains from about
0.5 to 90% by weight of protein or other active ingredient of the
present invention, and preferably from about 1 to 50% protein or
other active ingredient of the present invention.
[0426] When a therapeutically effective amount of protein or other
active ingredient of the present invention is administered by
intravenous, cutaneous or subcutaneous injection, protein or other
active ingredient of the present invention will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein or other active
ingredient solutions, having due regard to pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to protein or
other active ingredient of the present invention, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art. For injection, the
agents of the invention may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks'
solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0427] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-inked polyvinyl pyrrolidone,
agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses.
[0428] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
[0429] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. The compounds may
be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
[0430] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0431] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas. e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides. In addition to the formulations described previously,
the compounds may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0432] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The cosolvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:5 W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose. Alternatively, other
delivery systems for hydrophobic pharmaceutical compounds may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Various types of sustained-release materials
have been established and are well known by those skilled in the
art. Sustained-release capsules may, depending on their chemical
nature, release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein or other
active ingredient stabilization may be employed.
[0433] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the active ingredients of the invention may be provided as
salts with pharmaceutically compatible counterions. Such
pharmaceutically acceptable base addition salts are those salts
which retain the biological effectiveness and properties of the
free acids and which are obtained by reaction with inorganic or
organic bases such as sodium hydroxide, magnesium hydroxide,
ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino
acids, sodium acetate, potassium benzoate, triethanol amine and the
like.
[0434] The pharmaceutical composition of the invention may be in
the form of a complex of the protein(s) or other active ingredient
of present invention along with protein or peptide antigens. The
protein and/or peptide antigen will deliver a stimulatory signal to
both B and T lymphocytes. B lymphocytes will respond to antigen
through their surface immunoglobulin receptor. T lymphocytes will
respond to antigen through the T cell receptor (TCR) following
presentation of the antigen by MHC proteins. MHC and structurally
related proteins including those encoded by class I and class II
MHC genes on host cells will serve to present the peptide
antigen(s) to T lymphocytes. The antigen components could also be
supplied as purified MHC-peptide complexes alone or with
co-stimulatory molecules that can directly signal T cells.
Alternatively antibodies able to bind surface immunoglobulin and
other molecules on B cells as well as antibodies able to bind the
TCR and other molecules on T cells can be combined with the
pharmaceutical composition of the invention. The pharmaceutical
composition of the invention may be in the form of a liposome in
which protein of the present invention is combined, in addition to
other pharmaceutically acceptable carriers, with amphipathic agents
such as lipids which exist in aggregated form as micelles,
insoluble monolayers, liquid crystals, or lamellar layers in
aqueous solution. Suitable lipids for liposomal formulation
include, without limitation, monoglycerides, diglycerides,
sulfatides, lysolecithins, phospholipids, saponin, bile acids, and
the like. Preparation of such liposomal formulations is within the
level of skill in the art, as disclosed, for example, in U.S. Pat.
Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which
are incorporated herein by reference.
[0435] The amount of protein or other active ingredient of the
present invention in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments which the
patient has undergone. Ultimately, the attending physician will
decide the amount of protein or other active ingredient of the
present invention with which to treat each individual patient.
Initially, the attending physician will administer low doses of
protein or other active ingredient of the present invention and
observe the patient's response. Larger doses of protein or other
active ingredient of the present invention may be administered
until the optimal therapeutic effect is obtained for the patient,
and at that point the dosage is not increased further. It is
contemplated that the various pharmaceutical compositions used to
practice the method of the present invention should contain about
0.01 .mu.g to about 100 mg (preferably about 0.1 .mu.g to about 10
mg, more preferably about 0.1 .mu.g to about 1 mg) of protein or
other active ingredient of the present invention per kg body
weight. For compositions of the present invention which are useful
for bone, cartilage, tendon or ligament regeneration, the
therapeutic method includes administering the composition
topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention
is, of course, in a pyrogen-free, physiologically acceptable form.
Further, the composition may desirably be encapsulated or injected
in a viscous form for delivery to the site of bone, cartilage or
tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Therapeutically useful agents other than
a protein or other active ingredient of the invention which may
also optionally be included in the composition as described above,
may alternatively or additionally, be administered simultaneously
or sequentially with the composition in the methods of the
invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the
protein-containing or other active ingredient-containing
composition to the site of bone and/or cartilage damage, providing
a structure for the developing bone and cartilage and optimally
capable of being resorbed into the body. Such matrices may be
formed of materials presently in use for other implanted medical
applications.
[0436] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalcium phosphate, hydroxyapatite,
polylactic acid, polyglycolic acid and polyanhydrides. Other
potential materials are biodegradable and biologically
well-defined, such as bone or dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are nonbiodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be comprised of combinations of any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The
bioceramics may be altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size,
particle size, particle shape, and biodegradability. Presently
preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic acid in the form of porous particles having diameters
ranging from 150 to 800 microns. In some applications, it will be
useful to utilize a sequestering agent, such as carboxymethyl
cellulose or autologous blood clot, to prevent the protein
compositions from disassociating from the matrix.
[0437] A preferred family of sequestering agents is cellulosic
materials such as alkylcelluloses (including
hydroxyallylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorption of the protein from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the protein the
opportunity to assist the osteogenic activity of the progenitor
cells. In further compositions, proteins or other active ingredient
of the invention may be combined with other agents beneficial to
the treatment of the bone and/or cartilage defect, wound, or tissue
in question. These agents include various growth factors such as
epidermal growth factor (EGF), platelet derived growth
factor-(PDGF), transforming growth factors (TGF- and TGF-), and
insulin-like growth factor (IGF).
[0438] The therapeutic compositions are also presently valuable for
veterinary applications. Particularly domestic animals and
thoroughbred horses, in addition to humans, are desired patients
for such treatment with proteins or other active ingredient of the
present invention. The dosage regimen of a protein-containing
pharmaceutical composition to be used in tissue regeneration will
be determined by the attending physician considering various
factors which modify the action of the proteins, e.g., amount of
tissue weight desired to be formed, the site of damage, the
condition of the damaged tissue, the size of a wound, type of
damaged tissue (e.g., bone), the patient's age, sex, and diet, the
severity of any infection, time of administration and other
clinical factors. The dosage may vary with the type of matrix used
in the reconstitution and with inclusion of other proteins in the
pharmaceutical composition. For example, the addition of other
known growth factors, such as IGF I (insulin like growth factor I),
to the final composition, may also effect the dosage. Progress can
be monitored by periodic assessment of tissue/bone growth and/or
repair, for example, X-rays, histomorphometric determinations and
tetracycline labeling.
5.11.3. EFFECTIVE DOSAGE
[0439] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amount is well within the capability of those skilled
in the art, especially in light of the detailed disclosure provided
herein. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from
appropriate in vitro assays. For example, a dose can be formulated
in animal models to achieve a circulating concentration range that
can be used to more accurately determine useful doses in humans.
For example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC.sub.50 as
determined in cell culture (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of the IgSF
protein's biological activity). Such information can be used to
more accurately determine useful doses in humans.
[0440] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1. Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
which are sufficient to maintain the desired effects, or minimal
effective concentration (MEC). The MEC will vary for each compound
but can be estimated from in vitro data. Dosages necessary to
achieve the MEC will depend on individual characteristics and route
of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
[0441] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which-maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0442] An exemplary dosage regimen for polypeptides or other
compositions of the invention will be in the range of about 0.01 to
100 mg/kg of body weight daily, with the preferred dose being about
0.1 to 25 mg/kg of patient body weight daily, varying in adults and
children. Dosing may be once daily, or equivalent doses may be
delivered at longer or shorter intervals.
[0443] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's age and
weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician.
5.11.4. PACKAGING
[0444] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
5.12 ANTIBODIES
[0445] Also included in the invention are antibodies to proteins,
or fragments of proteins of the invention. The term "antibody" as
used herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin (Ig) molecules, i.e., molecules
that contain an antigen-binding site that specifically binds
(immunoreacts with) an antigen. Such antibodies include, but are
not limited to, polyclonal, monoclonal, chimeric, single chain,
F.sub.ab, F.sub.ab' and F.sub.(ab')2 fragments, and an F.sub.ab
expression library. In general, an antibody molecule obtained from
humans relates to any of the classes IgG, IgM, IgA, IgE, and IgD,
which differ from one another by the nature of the heavy chain
present in the molecule. Certain classes have subclasses as well,
such as IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans,
the light chain may be a kappa chain or a lambda chain. Reference
herein to antibodies includes a reference to all such classes,
subclasses, and types of human antibody species.
[0446] An isolated related protein of the invention may be intended
to serve as an antigen, or a portion or fragment thereof, and
additionally can be used as an immunogen to generate antibodies
that immunospecifically bind the antigen, using standard techniques
for polyclonal and monoclonal antibody preparation. The full-length
protein can be used or, alternatively, the invention provides
antigenic peptide fragments of the antigen for use as immunogens.
An antigenic peptide fragment comprises at least 6 amino acid
residues of the amino acid sequence of the full length protein,
such as an amino acid sequence shown in SEQ ID NO: 10, 13-24, 32 or
34 and encompasses an epitope thereof such that an antibody raised
against the peptide forms a specific immune complex with the full
length protein or with any fragment that contains the epitope.
Preferably, the antigenic peptide comprises at least 10 amino acid
residues, or at least 15 amino acid residues, or at least 20 amino
acid residues, or at least 30 amino acid residues. Preferred
epitopes encompassed by the antigenic peptide are regions of the
protein that are located on its surface; commonly these are
hydrophilic regions.
[0447] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of TGF
alpha-like protein that is located on the surface of the protein,
e.g., a hydrophilic region. A hydrophobicity analysis of the human
related protein sequence will indicate which regions of a related
protein are particularly hydrophilic and, therefore, are likely to
encode surface residues useful for targeting antibody production.
As a means for targeting antibody production, hydropathy plots
showing regions of hydrophilicity and hydrophobicity may be
generated by any method well known in the art, including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with
or without Fourier transformation. See, e.g., Hopp and Woods, 1981,
Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982,
J. Mol. Biol. 157: 105-142, each of which is incorporated herein by
reference in its entirety. Antibodies that are specific for one or
more domains within an antigenic protein, or derivatives,
fragments, analogs or homologs thereof, are also provided
herein.
[0448] A protein of the invention, or a derivative, fragment,
analog, homolog or ortholog thereof, may be utilized as an
immunogen in the generation of antibodies that immunospecifically
bind these protein components.
[0449] The term "specific for" indicates that the variable regions
of the antibodies of the invention recognize and bind polypeptides
of the invention exclusively (i.e., able to distinguish the
polypeptide of the invention from other similar polypeptides
despite sequence identity, homology, or similarity found in the
family of polypeptides), but may also interact with other proteins
(for example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the
variable region of the antibodies, and in particular, in the
constant region of the molecule. Screening assays to determine
binding specificity of an antibody of the invention are well known
and routinely practiced in the art. For a comprehensive discussion
of such assays, see Harlow et al. (Eds), Antibodies A Laboratory
Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.
(1988), Chapter 6. Antibodies that recognize and bind fragments of
the polypeptides of the invention are also contemplated, provided
that the antibodies are first and foremost specific for, as defined
above, full-length polypeptides of the invention. As with
antibodies that are specific for full length polypeptides of the
invention, antibodies of the invention that recognize fragments are
those which can distinguish polypeptides from the same family of
polypeptides despite inherent sequence identity, homology, or
similarity found in the family of proteins.
[0450] Antibodies of the invention are useful for, for example,
therapeutic purposes (by modulating activity of a polypeptide-of
the invention), diagnostic purposes to detect or quantitate a
polypeptide of the invention, as well as purification of a
polypeptide of the invention. Kits comprising an antibody of the
invention for any of the purposes described herein are also
comprehended. In general, a kit of the invention also includes a
control antigen for which the antibody is immunospecific. The
invention further provides a hybridoma that produces an antibody
according to the invention. Antibodies of the invention are useful
for detection and/or purification of the polypeptides of the
invention.
[0451] Monoclonal antibodies binding to the protein of the
invention may be useful diagnostic agents for the immunodetection
of the protein. Neutralizing monoclonal antibodies binding to the
protein may also be useful therapeutics for both conditions
associated with the protein and also in the treatment of some forms
of cancer where abnormal expression of the protein is involved. In
the case of cancerous cells or leukemic cells, neutralizing
monoclonal antibodies against the protein may be useful in
detecting and preventing the metastatic spread of the cancerous
cells, which may be mediated by the protein.
[0452] The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues in which a fragment of the polypeptide of interest is
expressed. The antibodies may also be used directly in therapies or
other diagnostics. The present invention further provides the
above-described antibodies immobilized on a solid support. Examples
of such solid supports include plastics such as polycarbonate,
complex carbohydrates such as agarose and Sepharose.RTM., acrylic
resins and such as polyacrylamide and latex beads. Techniques for
coupling antibodies to such solid supports are well known in the
art (Weir, D. M. et al., "Handbook of Experimental Immunology" 4th
Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10
(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N. Y.
(1974)). The immobilized antibodies of the present invention can be
used for in vitro, in vivo, and in situ assays as well as for
immuno-affinity purification of the proteins of the present
invention.
[0453] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs, or orthologs thereof (see, for example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Some of these antibodies are
discussed below.
5.12.1 POLYCLONAL ANTIBODIES
[0454] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by one or more injections with the native protein,
a synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example, the
naturally occurring immunogenic protein, a chemically synthesized
polypeptide representing the immunogenic protein, or a
recombinantly expressed immunogenic protein. Furthermore, the
protein may be conjugated to a second protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. The preparation can further include an adjuvant.
Various adjuvants used to increase the immunological response
include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide),
surface-active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and
Corynebacterium parvum, or similar immunostimulatory agents.
Additional examples of adjuvants that can be employed include
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
[0455] The polyclonal antibody molecules directed against the
immunogenic protein can be isolated from the mammal (e.g., from the
blood) and further purified by well known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
5.12.2 MONOCLONAL ANTIBODIES
[0456] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs thus contain an
antigen-binding site capable of immunoreacting with a particular
epitope of the antigen characterized by a unique binding affinity
for it.
[0457] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes can be immunized in
vitro.
[0458] The immunizing agent will typically include the protein
antigen, a fragment thereof, or a fusion protein thereof.
Generally, either peripheral blood lymphocytes are used if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine, and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells can be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
[0459] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0460] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980). Preferably, antibodies having a high
degree of specificity and a high binding affinity for the target
antigen are isolated.
[0461] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods. Suitable culture media for this purpose include,
for example, Dulbecco's Modified Eagle's Medium and RPMI-1640
medium. Alternatively, the hybridoma cells can be grown in vivo as
ascites in a mammal.
[0462] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0463] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
5.12.3 HUMANIZED ANTIBODIES
[0464] The antibodies directed against the protein antigens of the
invention can further comprise humanized antibodies or human
antibodies. These antibodies are suitable for administration to
humans without engendering an immune response by the human against
the administered immunoglobulin. Humanized forms of antibodies are
chimeric immunoglobulins, immunoglobulin chains, or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that are principally
comprised of the sequence of a human immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. (See also U.S.
Pat. No. 5,225,539). In some instances, Fv framework residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
5.12.4 HUMAN ANTIBODIES
[0465] Fully human antibodies relate to antibody molecules in which
essentially the entire sequences of both the light chain and the
heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0466] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by introducing human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks
et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature
368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild
et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
[0467] Human antibodies may additionally be produced using
transgenic nonhuman animals that are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells that secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0468] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method including deleting the J segment
genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0469] A method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. It
includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0470] In a further improvement on this procedure, a method for
identifying a clinically relevant epitope on an immunogen, and a
correlative method for selecting an antibody that binds
immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT publication WO 99/53049.
5.12.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES
[0471] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to an antigenic
protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of F.sub.ab
expression libraries (see e.g., Huse, et al., 1989 Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal F.sub.ab fragments with the desired specificity for a
protein or derivatives, fragments, analogs or homologs thereof.
Antibody fragments that contain the idiotypes to a protein antigen
may be produced by techniques known in the art including, but not
limited to: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv)
F.sub.v fragments.
5.12.6 BISPECIFIC ANTIBODIES
[0472] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for an antigenic protein of the invention. The
second binding target is any other antigen, and advantageously is a
cell-surface protein or receptor or receptor subunit.
[0473] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
[0474] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0475] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers that are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0476] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0477] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab').sub.2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human
cytotoxic-lymphocytes against human breast tumor targets.
[0478] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1541-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0479] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0480] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in the protein antigen
of the invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc
R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD16) so as to
focus cellular defense mechanisms to the cell expressing the
particular antigen. Bispecific antibodies can also be used to
direct cytotoxic agents to cells which express a particular
antigen. These antibodies possess an antigen-binding arm and an arm
which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of
interest binds the protein antigen described herein and further
binds tissue factor (TF).
5.12.7 HETEROCONJUGATE ANTIBODIES
[0481] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target inmmune system cells to unwanted cells
(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
5.12.8 EFFECTOR FUNCTION ENGINEERING
[0482] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
5.12.9 IMMUNOCONJUGATES
[0483] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0484] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0485] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0486] In another embodiment, the antibody can be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is in turn
conjugated to a cytotoxic agent.
5.13 COMPUTER READABLE SEQUENCES
[0487] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
medium which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention. As used herein, "recorded" refers to a process for
storing information on computer readable medium. A skilled artisan
can readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0488] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0489] By providing any of the nucleotide sequences SEQ ID NO: 1-9,
11, 12, 31 or 33 or a representative fragment thereof; or a
nucleotide sequence at least 99.9% identical to any of the
nucleotide sequences of the SEQ ID NO: 1-9, 11, 12, 31 or 33 in
computer readable form, a skilled artisan can routinely access the
sequence information for a variety of purposes. Computer software
is publicly available which allows a skilled artisan to access
sequence information provided in a computer readable medium. The
examples which follow demonstrate how software which implements the
BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms
on a Sybase system is used to identify open reading frames (ORFs)
within a nucleic acid sequence. Such ORFs may be protein encoding
fragments and may be useful in producing commercially important
proteins such as enzymes used in fermentation reactions and in the
production of commercially useful metabolites.
[0490] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based systems are suitable for use in the
present invention. As stated above, the computer-based systems of
the present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, "data storage means"
refers to memory which can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention.
[0491] As used herein, "search means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of a known sequence
which match a particular target sequence or target motif. A variety
of known algorithms are disclosed publicly and a variety of
commercially available software for conducting search means are and
can be used in the computer-based systems of the present invention.
Examples of such software includes, but is not limited to,
Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA
(NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any
one of the available algorithms or implementing software packages
for conducting homology searches can be adapted for use in the
present computer-based systems. As used herein, a "target sequence"
can be any nucleic acid or amino acid sequence of six or more
nucleotides or two or more amino acids. A skilled artisan can
readily recognize that the longer a target sequence is, the less
likely a target sequence will be present as a random occurrence in
the database. The most preferred sequence length of a target
sequence is from about 10 to 100 amino acids or from about 30 to
300 nucleotide residues. However, it is well recognized that
searches for commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0492] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
5.14 EXPRESSION MODULATING SEQUENCES
[0493] EMF sequences can be identified within a genome by their
proximity to the ORFs. An intergenic segment, or a fragment of the
intergenic segment, from about 10 to 200 nucleotides in length,
taken 5' from any ORF will modulate the expression of an operably
linked 3' ORF in a fashion similar to that found with the naturally
linked ORF sequence. As used herein, an "intergenic segment" refers
to the fragments of a genome which are between two ORF(S) herein
described. Alternatively, EMFs can be identified using known EMFs
as a target sequence or target motif in the computer-based systems
of the present invention.
[0494] The presence and activity of an EMF can be confirmed using
an EMF trap vector. An EMF trap vector contains a cloning site 5'
to a marker sequence. A marker sequence encodes an identifiable
phenotype, such as antibiotic resistance or a complementing
nutrition auxotrophic factor, which can be identified or assayed
when the EMF trap vector is placed within an appropriate host under
appropriate conditions. As described above, an EMF will modulate
the expression of an operably linked marker sequence. A more
detailed discussion of various marker sequences is provided below.
A sequence which is suspected of being an EMF is cloned in all
three reading frames in one or more restriction sites upstream from
the marker sequence in the EMF trap vector. The vector is then
transformed into an appropriate host using known procedures and the
phenotype of the transformed host is examined under appropriate
conditions. As described above, an EMF will modulate the expression
of an operably linked marker sequence.
5.15 TRIPLE HELIX FORMATION
[0495] In addition, the fragments of the present invention, as
broadly described, can be used to control gene expression through
triple helix formation or antisense DNA or RNA, both of which
methods are based on the binding of a polynucleotide sequence to
DNA or RNA. Polynucleotides suitable for use in these methods are
usually 20 to 40 bases in length and are 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 15241:456 (1988); and Dervan et al., Science
251:1360 (1991)) or to the mRNA itself (antisense--Olmno, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Triple helix-formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks
translation of an mRNA molecule into polypeptide. Both techniques
have been demonstrated to be effective in model systems.
Information contained in the sequences of the present invention is
necessary for the design of an antisense or triple helix
oligonucleotide.
5.16 DIAGNOSTIC ASSAYS AND KITS
[0496] The present invention further provides methods to identify
the presence or expression of one of the ORFs of the present
invention, or homolog thereof, in a test sample, using a nucleic
acid probe or antibodies of the present invention, optionally
conjugated or otherwise associated with a suitable label.
[0497] In general, methods for detecting a polynucleotide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polynucleotide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polynucleotide of the invention is
detected in the sample. Such methods can also comprise contacting a
sample under stringent hybridization conditions with nucleic acid
primers that anneal to a polynucleotide of the invention under such
conditions, and amplifying annealed polynucleotides, so that if a
polynucleotide is amplified, a polynucleotide of the invention is
detected in the sample.
[0498] In general, methods for detecting a polypeptide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polypeptide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polypeptide of the invention is
detected in the sample.
[0499] In detail, such methods comprise incubating a test sample
with one or more of the antibodies or one or more of the nucleic
acid probes of the present invention and assaying for binding of
the nucleic acid probes or antibodies to components within the test
sample.
[0500] Conditions for incubating a nucleic acid probe or antibody
with a test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid probe or antibody used in the assay.
One skilled in the art will recognize that any one of the commonly
available hybridization, amplification or immunological assay
formats can readily be adapted to employ the nucleic acid probes or
antibodies of the present invention. Examples of such assays can be
found in Chard, T., An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The
test samples of the present invention include cells, protein or
membrane extracts of cells, or biological fluids such as sputum,
blood, serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are well known in the art and can be
readily be adapted in order to obtain a sample which is compatible
with the system utilized.
[0501] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention. Specifically, the invention
provides a compartment kit to receive, in close confinement, one or
more containers which comprises: (a) a first container comprising
one of the probes or antibodies of the present invention; and (b)
one or more other containers comprising one or more of the
following: wash reagents, reagents capable of detecting presence of
a bound probe or antibody.
[0502] In detail, a compartment kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allows one to efficiently
transfer reagents from one compartment to another compartment such
that the samples and reagents are not cross-contaminated, and the
agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the antibodies used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, etc.), and containers which contain
the reagents used to detect the bound antibody or probe. Types of
detection reagents include labeled nucleic acid probes, labeled
secondary antibodies, or in the alternative, if the primary
antibody is labeled, the enzymatic, or antibody binding reagents
which are capable of reacting with the labeled antibody. One
skilled in the art will readily recognize that the disclosed probes
and antibodies of the present invention can be readily incorporated
into one of the established kit formats which are well known in the
art.
5.17 MEDICAL IMAGING
[0503] The novel polypeptides and binding partners of the invention
are useful in medical imaging of sites expressing the molecules of
the invention (e.g., where the polypeptide of the invention is
involved in the immune response, for imaging sites of inflammation
or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778.
Such methods involve chemical attachment of a labeling or imaging
agent, administration of the labeled polypeptide to a subject in a
pharmaceutically acceptable carrier, and imaging the labeled
polypeptide in vivo at the target site.
5.18 SCREENING ASSAYS
[0504] Using the isolated proteins and polynucleotides of the
invention, the present invention further provides methods of
obtaining and identifying agents which bind to a polypeptide
encoded by an ORF corresponding to any of the nucleotide sequences
set forth in the SEQ ID NO: 1-9, 11, 12, 31 or 33 or bind to a
specific domain of the polypeptide encoded by the nucleic acid. In
detail, said method comprises the steps of:
[0505] (a) contacting an agent with an isolated protein encoded by
an ORF of the present invention, or nucleic acid of the invention;
and
[0506] (b) determining whether the agent binds to said protein or
said nucleic acid.
[0507] In general, therefore, such methods for identifying
compounds that bind to a polynucleotide of the invention can
comprise contacting a compound with a polynucleotide of the
invention for a time sufficient to form a polynucleotide/compound
complex, and detecting the complex, so that if a
polynucleotide/compound complex is detected, a compound that binds
to a polynucleotide of the invention is identified.
[0508] Likewise, in general, therefore, such methods for
identifying compounds that bind to a polypeptide of the invention
can comprise contacting a compound with a polypeptide of the
invention for a time sufficient to form a polypeptide/compound
complex, and detecting the complex, so that if a
polypeptide/compound complex is detected, a compound that binds to
a polynucleotide of the invention is identified.
[0509] Methods for identifying compounds that bind to a polypeptide
of the invention can also comprise contacting a compound with a
polypeptide of the invention in a cell for a time sufficient to
form a polypeptide/compound complex, wherein the complex drives
expression of a receptor gene sequence in the cell, and detecting
the complex by detecting reporter gene sequence expression, so that
if a polypeptide/compound complex is detected, a compound that
binds a polypeptide of the invention is identified.
[0510] Compounds identified via such methods can include compounds
which modulate the activity of a polypeptide of the invention (that
is, increase or decrease its activity, relative to activity
observed in the absence of the compound). Alternatively, compounds
identified via such methods can include compounds which modulate
the expression of a polynucleotide of the invention (that is,
increase or decrease expression relative to expression levels
observed in the absence of the compound). Compounds, such as
compounds identified via the methods of the invention, can be
tested using standard assays well known to those of skill in the
art for their ability to modulate activity/expression.
[0511] The agents screened in the above assay can be, but are not
limited to, peptides, carbohydrates, vitamin derivatives, or other
pharmaceutical agents. The agents can be selected and screened at
random or rationally selected or designed using protein modeling
techniques.
[0512] For random screening, agents such as peptides,
carbohydrates, pharmaceutical agents and the like are selected at
random and are assayed for their ability to bind to the protein
encoded by the ORF of the present invention. Alternatively, agents
may be rationally selected or designed. As used herein, an agent is
said to be "rationally selected or designed" when the agent is
chosen based on the configuration of the particular protein. For
example, one skilled in the art can readily adapt currently
available procedures to generate peptides, pharmaceutical agents
and the like capable of binding to a specific peptide sequence in
order to generate rationally designed antipeptide peptides, for
example see Hurby et al., Application of Synthetic Peptides:
Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H.
Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry
28:9230-8 (1989), or pharmaceutical agents, or the like.
[0513] In addition to the foregoing, one class of agents of the
present invention, as broadly described, can be used to control
gene expression through binding to one of the ORFs or EMFs of the
present invention. As described above, such agents can be randomly
screened or rationally designed/selected. Targeting the ORF or EMF
allows a skilled artisan to design sequence specific or element
specific agents, modulating the expression of either a single ORF
or multiple ORFs which rely on the same EMF for expression control.
One class of DNA binding agents are agents which contain base
residues which hybridize or form a triple helix formation by
binding to DNA or RNA. Such agents can be based on the classic
phosphodiester, ribonucleic acid backbone, or can be a variety of
sulfhydryl or polymeric derivatives which have base attachment
capacity.
[0514] Agents suitable for use in these methods usually contain 20
to 40 bases and are 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)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques have been demonstrated
to be effective in model systems. Information contained in the
sequences of the present invention is necessary for the design of
an antisense or triple helix oligonucleotide and other DNA binding
agents. Agents which bind to a protein encoded by one of the ORFs
of the present invention can be used as a diagnostic agent, in the
control of bacterial infection by modulating the activity of the
protein encoded by the ORF. Agents which bind to a protein encoded
by one of the ORFs of the present invention can be formulated using
known techniques to generate a pharmaceutical composition.
5.19 USE OF NUCLEIC ACIDS AS PROBES
[0515] Another aspect of the subject invention is to provide for
polypeptide-specific nucleic acid hybridization probes capable of
hybridizing with naturally occurring nucleotide sequences. The
hybridization probes of the subject invention may be derived from
any of the nucleotide sequences SEQ ID NO: 1-9, 11, 12, 31 or 33.
Because the corresponding gene is only expressed in a limited
number of tissues, a hybridization probe derived from of any of the
nucleotide sequences SEQ ID NO: 1-9, 11, 12, 31 or 33 can be used
as an indicator of the presence of RNA of cell type of such a
tissue in a sample.
[0516] Any suitable hybridization technique can be employed, such
as, for example, in situ hybridization. PCR as described in U.S.
Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for
oligonucleotides based upon the nucleotide sequences. Such probes
used in PCR may be of recombinant origin, may be chemically
synthesized, or a mixture of both. The probe will comprise a
discrete nucleotide sequence for the detection of identical
sequences or a degenerate pool of possible sequences for
identification of closely related genomic sequences.
[0517] Other means for producing specific hybridization probes for
nucleic acids include the cloning of nucleic acid sequences into
vectors for the production of mRNA probes. Such vectors are known
in the art and are commercially available and may be used to
synthesize RNA probes in vitro by means of the addition of the
appropriate RNA polymerase as T7 or SP6 RNA polymerase and the
appropriate radioactively labeled nucleotides. The nucleotide
sequences may be used to construct hybridization probes for mapping
their respective genomic sequences. The nucleotide sequence
provided herein may be mapped to a chromosome or specific regions
of a chromosome using well known genetic and/or chromosomal mapping
techniques. These techniques include in situ hybridization, linkage
analysis against known chromosomal markers, hybridization screening
with libraries or flow-sorted chromosomal preparations specific to
known chromosomes, and the like. The technique of fluorescent in
situ hybridization of chromosome spreads has been described, among
other places, in Verma et al (1988) Human Chromosomes: A Manual of
Basic Techniques, Pergamon Press, New York N.Y.
[0518] Fluorescent in situ hybridization of chromosomal
preparations and other physical chromosome mapping techniques may
be correlated with additional genetic map data. Examples of genetic
map data can be found in the 1994 Genome Issue of Science
(265:1981f). Correlation between the location of a nucleic acid on
a physical chromosomal map and a specific disease (or
predisposition to a specific disease) may help delimit the region
of DNA associated with that genetic disease. The nucleotide
sequences of the subject invention may be used to detect
differences in gene sequences between normal, carrier or affected
individuals. The nucleotide sequence may be used to produce
purified polypeptides using well known methods of recombinant DNA
technology. Among the many publications that teach methods for the
expression of genes after they have been isolated is Goeddel (1990)
Gene Expression Technology, Methods and Enzymology, Vol 185,
Academic Press, San Diego. Polypeptides may be expressed in a
variety of host cells, either prokaryotic or eukaryotic. Host cells
may be from the same species from which a particular polypeptide
nucleotide sequence was isolated or from a different species.
Advantages of producing polypeptides by recombinant DNA technology
include obtaining adequate amounts of the protein for purification
and the availability of simplified purification procedures.
5.20 PREPARATION OF SEQUENCING CHIPS AND ARRAYS
[0519] A basic example is using 6-mers attached to 50 micron
surfaces to give a chip with dimensions of 3.times.3 mm which can
be combined to give an array of 20.times.20 cm. Another example is
using 9-mer oligonucleotides attached to 10.times.10 microns
surface to create a 9-mer chip, with dimensions of 5.times.5 mm.
4000 units of such chips may be used to create a 30.times.30 cm
array. In an array in which 4,000 to 16,000 oligochips are arranged
into a square array. A plate, or collection of tubes, as also
depicted, may be packaged with the array as part of the sequencing
kit.
[0520] The arrays may be separated physically from each other or by
hydrophobic surfaces. One possible way to utilize the hydrophobic
strip separation is to use technology such as the Iso-Grid
Microbiology System produced by QA Laboratories, Toronto,
Canada.
[0521] Hydrophobic grid membrane filters (HGMF) have been in use in
analytical food microbiology for about a decade where they exhibit
unique attractions of extended numerical range and automated
counting of colonies. One commercially-available grid is
ISO-GRID.TM. from QA Laboratories Ltd. (Toronto, Canada) which
consists of a square (60.times.60 cm) of polysulfone polymer
(Gelman Tuffryn HT450, 0.45 u pore size) on which is printed a
black hydrophobic ink grid consisting of 1600 (40.times.40) square
cells. HGMF have previously been inoculated with bacterial
suspensions by vacuum filtration and incubated on the differential
or selective media of choice.
[0522] Because the microbial growth is confined to grid cells of
known position and size on the membrane, the HGMF functions more
like an MPN apparatus than a conventional plate or membrane filter.
Peterkin et al. (1987) reported that these HGMFs can be used to
propagate and store genomic libraries when used with a HGMF
replicator. One such instrument replicates growth from each of the
1600 cells of the ISO-GRID and enables many copies of the master
HGMF to be made (Peterkin et al., 1987).
[0523] Sharpe et al. (1989) also used ISO-GRID HGMF form QA
Laboratories and an automated HGMF counter (MI-100 Interpreter) and
RP-100 Replicator. They reported a technique for maintaining and
screening many microbial cultures.
[0524] Peterkin and colleagues later described a method for
screening DNA probes using the hydrophobic grid-membrane filter
(Peterkin et al., 1989). These authors reported methods for
effective colony hybridization directly on HGMFs. Previously, poor
results had been obtained due to the low DNA binding capacity of
the epoxysulfone polymer on which the HGMFs are printed. However,
Peterkin et al. (1989) reported that the binding of DNA to the
surface of the membrane was improved by treating the replicated and
incubated HGMF with polyethyleneimine, a polycation, prior to
contact with DNA. Although this early work uses cellular DNA
attachment, and has a different objective to the present invention,
the methodology described may be readily adapted for Format 3
SBH.
[0525] In order to identify useful sequences rapidly, Peterkin et
al. (1989) used radiolabeled plasmid DNA from various clones and
tested its specificity against the DNA on the prepared HGMFs. In
this way, DNA from recombinant plasmids was rapidly screened by
colony hybridization against 100 organisms on HGMF replicates which
can be easily and reproducibly prepared.
[0526] Manipulation with small (2-3 mm) chips, and parallel
execution of thousands of the reactions. The solution of the
invention is to keep the chips and the probes in the corresponding
arrays. In one example, chips containing 250,000 9-mers are
synthesized on a silicon wafer in the form of 8.times.8 mM plates
(15 M/oligonucleotide, Pease et al., 1994) arrayed in 8.times.12
format (96 chips) with a 1 mM groove in between. Probes are added
either by multichannel pipette or pin array, one probe on one chip.
To score all 4000 6-mers, 42 chip arrays have to be used, either
using different ones, or by reusing one set of chip arrays several
times.
[0527] In the above case, using the earlier nomenclature of the
application, F=9; P=6; and F+P=15. Chips may have probes of formula
BxNn, where x is a number of specified bases B; and n is a number
of non-specified bases, so that x=4 to 10 and n=1 to 4. To achieve
more efficient hybridization, and to avoid potential influence of
any support oligonucleotides, the specified bases can be surrounded
by unspecified bases, thus represented by a formula such as
(N)nBx(N)m.
5.21 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
[0528] Oligonucleotides, i.e., small nucleic acid segments, may be
readily prepared by, for example, directly synthesizing the
oligonucleotide by chemical means, as is commonly practiced using
an automated oligonucleotide synthesizer.
[0529] Support bound oligonucleotides may be prepared by any of the
methods known to those of skill in the art using any suitable
support such as glass, polystyrene or Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard
synthesizers. Immobilization can be achieved using passive
adsorption (Inouye & Hondo, 1990); using UV light (Nagata et
al., 1985; Dahlen et al., 1987; Morriey & Collins, 1989) or by
covalent binding of base modified DNA (Keller et al., 1988; 1989);
all references being specifically incorporated herein.
[0530] Another strategy that may be employed is the use of the
strong biotin-streptavidin interaction as a linker. For example,
Broude et al. (1994) describe the use of Biotinylated probes,
although these are duplex probes, that are immobilized on
streptavidin-coated magnetic beads. Streptavidin-coated beads may
be purchased from Dynal, Oslo. Of course, this same linking
chemistry is applicable to coating any surface with streptavidin.
Biotinylated probes may be purchased from various sources, such as,
e.g., Operon Technologies (Alameda, Calif.).
[0531] Nunc Laboratories (Naperville, Ill.) is also selling
suitable material that could be used. Nunc Laboratories have
developed a method by which DNA can be covalently bound to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene
surface grafted with secondary amino groups (>NH) that serve as
bridge-heads for further covalent coupling. CovaLink Modules may be
purchased from Nunc Laboratories. DNA molecules may be bound to
CovaLink exclusively at the 5'-end by a phosphoramidate bond,
allowing immobilization of more than 1 pmol of DNA (Rasmussen et
al., 1991).
[0532] The use of CovaLink NH strips for covalent binding of DNA
molecules at the 5'-end has been described (Rasmussen et al.,
1991). In this technology, a phosphoramidate bond is employed (Chu
et al., 1983). This is beneficial as immobilization using only a
single covalent bond is preferred. The phosphoramidate bond joins
the DNA to the CovaLink NH secondary amino groups that are
positioned at the end of spacer arms covalently grafted onto the
polystyrene surface through a 2 nm long spacer arm. To link an
oligonucleotide to CovaLink NH via an phosphoramidate bond, the
oligonucleotide terminus must have a 5'-end phosphate group. It is,
perhaps, even possible for biotin to be covalently bound to
CovaLink and then streptavidin used to bind the probes.
[0533] More specifically, the linkage method includes dissolving
DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95.degree.
C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole,
pH 7.0 (1-Melm.sub.7); is then added to a final concentration of 10
mM 1-Melm.sub.7. A ss DNA solution is then dispensed into CovaLink
NH strips (75 ul/well) standing on ice.
[0534] Carbodiimide 0.2 M
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in
10 mM 1-Melm.sub.7, is made fresh and 25 ul added per well. The
strips are incubated for 5 hours at 50.degree. C. After incubation
the strips are washed using, e.g., Nunc-Immuno Wash; first the
wells are washed 3 times, then they are soaked with washing
solution for 5 min., and finally they are washed 3 times (where in
the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50.degree.
C.).
[0535] It is contemplated that a further suitable method for use
with the present invention is that described in PCT Patent
Application WO 90/03382 (Southern & Maskos), incorporated
herein by reference. This method of preparing an oligonucleotide
bound to a support involves attaching a nucleoside 3'-reagent
through the phosphate group by a covalent phosphodiester link to
aliphatic hydroxyl groups carried by the support. The
oligonucleotide is then synthesized on the supported. nucleoside
and protecting groups removed from the synthetic oligonucleotide
chain under standard conditions that do not cleave the
oligonucleotide from the support. Suitable reagents include
nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
[0536] An on-chip strategy for the preparation of DNA probe for the
preparation of DNA probe arrays may be employed. For example,
addressable laser-activated photodeprotection may be employed in
the chemical synthesis of oligonucleotides directly on a glass
surface, as described by Fodor et al. (1991), incorporated herein
by reference. Probes may also be immobilized on nylon supports as
described by Van Ness et al. (1991); or linked to Teflon using the
method of Duncan & Cavalier (1988); all references being
specifically incorporated herein.
[0537] To link an oligonucleotide to a nylon support, as described
by Van Ness et al. (1991), requires activation of the nylon surface
via alkylation and selective activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
[0538] One particular way to prepare support bound oligonucleotides
is to utilize the light-generated synthesis described by Pease et
al., (1994, incorporated herein by reference). These authors used
current photolithographic techniques to generate arrays of
immobilized oligonucleotide probes (DNA chips). These methods, in
which light is used to direct the synthesis of oligonucleotide
probes in high-density, miniaturized arrays, utilize photolabile
5'-protected N-acyl-deoxynucleoside phosphoramidites, surface
linker chemistry and versatile combinatorial synthesis strategies.
A matrix of 256 spatially defined oligonucleotide probes may be
generated in this manner and then used in the advantageous Format 3
sequencing, as described herein.
5.22 PREPARATION OF NUCLEIC ACID FRAGMENTS
[0539] The nucleic acids to be sequenced may be obtained from any
appropriate source, such as cDNAs, genomic DNA, chromosomal DNA,
microdissected chromosome bands, cosmid or YAC inserts, and RNA,
including mRNA without any amplification steps. For example,
Sambrook et al. (1989) describes three protocols for the isolation
of high molecular weight DNA from mammalian cells (p.
9.14-9.23).
[0540] DNA fragments may be prepared as clones in M13, plasmid or
lambda vectors and/or prepared directly from genomic DNA or cDNA by
PCR or other amplification methods. Samples may be prepared or
dispensed in multiwell plates. About 100-1000 ng of DNA samples may
be prepared in 2-500 ml of final volume.
[0541] The nucleic acids would then be fragmented by any of the
methods known to those of skill in the art including, for example,
using restriction enzymes as described at 9.24-9.28 of Sambrook et
al. (1989), shearing by ultrasound and NaOH treatment.
[0542] Low pressure shearing is also appropriate, as described by
Schriefer et al. (1990, incorporated herein by reference). In this
method, DNA samples are passed through a small French pressure cell
at a variety of low to intermediate pressures. A lever device
allows controlled application of low to intermediate pressures to
the cell. The results of these studies indicate that low-pressure
shearing is a useful alternative to sonic and enzymatic DNA
fragmentation methods.
[0543] One particularly suitable way for fragmenting DNA is
contemplated to be that using the two base recognition
endonuclease, CviJI, described by Fitzgerald et al. (1992). These
authors described an approach for the rapid fragmentation and
fractionation of DNA into particular sizes that they contemplated
to be suitable for shotgun cloning and sequencing. The present
inventor envisions that this will also be particularly useful for
generating random, but relatively small, fragments of DNA for use
in the present sequencing technology.
[0544] The restriction endonuclease CviJI normally cleaves the
recognition sequence PuGCPy between the G and C to leave blunt
ends. Atypical reaction conditions, which alter the specificity of
this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUC19 (2688 base pairs).
Fitzgerald et al. (1992) quantitatively evaluated the randomness of
this fragmentation strategy, using a CviJI** digest of pUC19 that
was size fractionated by a rapid gel filtration method and directly
ligated, without end repair, to a lac Z minus M13 cloning vector.
Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy
and PuGCPu, in addition to PuGCPy sites, and that new sequence data
is accumulated at a rate consistent with random fragmentation.
[0545] As reported in the literature, advantages of this approach
compared to sonication and agarose gel fractionation include:
smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug);
and fewer steps are involved (no preligation, end repair, chemical
extraction, or agarose gel electrophoresis and elution are needed).
These advantages are also proposed to be of use when preparing DNA
for sequencing by Format 3.
[0546] Irrespective of the manner in which the nucleic acid
fragments are obtained or prepared, it is important to denature the
DNA to give single stranded pieces available for hybridization.
This is achieved by incubating the DNA solution for 2-5 minutes at
80-90.degree. C. The solution is then cooled quickly to 2.degree.
C. to prevent renaturation of the DNA fragments before they are
contacted with the chip. Phosphate groups must also be removed from
genomic DNA by methods known in the art.
5.23 PREPARATION OF DNA ARRAYS
[0547] Arrays may be prepared by spotting DNA samples on a support
such as a nylon membrane. Spotting may be performed by using arrays
of metal pins (the positions of which correspond to an array of
wells in a microtiter plate) to repeated by transfer of about 20 nl
of a DNA solution to a nylon membrane. By offset printing, a
density of dots higher than the density of the wells is achieved.
One to 25 dots may be accommodated in 1 mm.sup.2, depending on the
type of label used. By avoiding spotting in some preselected number
of rows and columns, separate subsets (subarrays) may be formed.
Samples in one subarray may be the same genomic segment of DNA (or
the same gene) from different individuals, or may be different,
overlapped genomic clones. Each of the subarrays may represent
replica spotting of the same samples. In one example, a selected
gene segment may be amplified from 64 patients. For each patient,
the amplified gene segment may be in one 96-well plate (all 96
wells containing the same sample). A plate for each of the 64
patients is prepared. By using a 96-pin device, all samples may be
spotted on one: 8.times.12 cm membrane. Subarrays may contain 64
samples, one from each patient. Where the 96 subarrays are
identical, the dot span may be 1 mm.sup.2 and there may be a 1 mm
space between subarrays.
[0548] Another approach is to use membranes or plates (available
from NUNC, Naperville, Ill.) which may be partitioned by physical
spacers e.g. a plastic grid molded over the membrane, the grid
being similar to the sort of membrane applied to the bottom of
multiwell plates, or hydrophobic strips. A fixed physical spacer is
not preferred for imaging by exposure to flat phosphor-storage
screens or x-ray films.
[0549] The present invention is illustrated in the following
examples. Upon consideration of the present disclosure, one of
skill in the art will appreciate that many other embodiments and
variations may be made in the scope of the present invention.
Accordingly, it is intended that the broader aspects of the present
invention not be limited to the disclosure of the following
examples. The present invention is not to be limited in scope by
the exemplified embodiments which are intended as illustrations of
single aspects of the invention, and compositions and methods which
are functionally equivalent are within the scope of the invention.
Indeed, numerous modifications and variations in the practice of
the invention are expected to occur to those skilled in the art
upon consideration of the present preferred embodiments.
Consequently, the only limitations which should be placed upon the
scope of the invention are those which appear in the appended
claims.
[0550] All references cited within the body of the instant
specification are hereby incorporated by reference in their
entirety.
6.0 EXAMPLES
Example 1
Isolation of SEQ ID NO: 1-7 from a cDNA Libraries of Human
Cells
[0551] A plurality of novel nucleic acids were obtained from a cDNA
library prepared from human testis cells (Hyseq clone
identification numbers 2880984 and 2881695), from human fetal skin
(Hyseq clone identification number 15375176), adult spleen (Hyseq
clone identification number 14856094), and human endothelial cells
(Hyseq clone identification numbers 13804756, 13687487, 13804756)
using standard PCR, sequencing by hybridization sequence signature
analysis, and Sanger sequencing techniques. The inserts of the
library were amplified with PCR using primers specific for vector
sequences flanking the inserts. These samples were spotted onto
nylon membranes and interrogated with oligonucleotide probes to
give sequence signatures. The clones were clustered into groups; of
similar or identical sequences, and single representative clones
were selected from each group for gel sequencing. The 5' sequence
of the amplified inserts was then deduced using the reverse M13
sequencing primer in a typical Sanger sequencing protocol. PCR
products were purified and subjected to fluorescent dye terminator
cycle sequencing. Single-pass gel sequencing was done using a 377
Applied Biosystems (ABI) sequencer. These inserts was identified as
a novel sequence not previously obtained from this library and not
previously reported in public databases. These sequences are
designated as SEQ ID NO: 1-7 in the attached sequence listing.
Example 2
Assemblage of SEQ ID NO: 8 and 9
[0552] The novel nucleic acids (SEQ ID NO: 8 and 9) of the
invention were assembled from sequences that were obtained from a
cDNA library by methods described in Example 1 above, and in some
cases sequences obtained from one or more public databases. The
sequence was assembled using an EST sequence (SEQ ID NO: 2) as a
seed. Then a recursive algorithm was used to extend the seed EST
into an extended assemblage, by pulling additional sequences from
different databases (i.e., Hyseq's database containing EST
sequences, dbEST version 114, gb pri 114, and UniGene version 101)
that belong to this assemblage. The algorithm terminated when there
was no additional sequences from the above databases that would
extend the assemblage. Inclusion of component sequences into the
assemblage was based on a BLASTN hit to the extending assemblage
with BLAST score greater than 300 and percent identity greater than
95%. SEQ ID NO: 8 was further manually edited to obtain SEQ ID NO:
9. FIG. 1 shows the alignment of SEQ ID NO. 9 with SEQ ID NO.
1-7.
[0553] The nearest neighbor result for the assembled sequence (SEQ
ID NO. 8) was obtained by a FASTA version 3 search against Genpept
release 114, using Fastxy algorithm. Fastxy is an improved version
of FASTA alignment which allows in-codon frame shifts. The nearest
neighbor result showed the closest homologue for each assemblage
from Genpept (and contains the translated amino acid sequences for
which the assemblage encodes). The nearest neighbor results is set
forth below:
TABLE-US-00003 Accession Smith- % No. Description Waterman Score
Identity AB016768 Mus musculus thrombospondin 56 43.750 type 1
domain
[0554] The predicted amino acid sequence for SEQ ID NO: 8 was
obtained by using a software program called FASTY (available from
http://fasta:bioch.virginia.edu) which selects a polypeptide based
on a comparison of translated novel polynucleotide to known
polynucleotides (W. R. Pearson, Methods in Enzymology, 183:63-98
(1990), incorporated herein by reference).
TABLE-US-00004 For SEQ ID NO: 8: Amino acid segment containing
signal peptide (A = Alanine, C = Cysteine, D = Aspartic Acid,
Predicted E = Glutamic Acid, F = Phenylalanine, beginning Predicted
end G = Glycine, H = Histidine, I = Isoleucine, nucleotide
nucleotide K = Lysine, L = Leucine, M = Methionine, location
location N = Asparagine, P = Proline, Q = Glutamine, corresponding
corresponding R = Arginine, S = Serine, T = Threonine, to first
amino to first amino V = Valine, W = Tryptophan, Y = Tyrosine, acid
residue acid residue X = Unknown, * = Stop Codon, / = possible of
amino acid of amino acid nucleotide deletion, \ = possible
nucleotide sequence sequence insertion) 575 1054 C T K C K A D C D
T C F N K N F C T K C K S G F Y L H L G K C L D N C P E G L E A N N
H T M E C V S I V H C E V S E W N P W S P C T K K G K T C G F K R G
T E T R V R E I I Q H P S A K G N L C P P T N E T R K C T V Q R K K
C Q K G E R G K K G R E R K R K K P N K G E S K E A I P D S K S L E
S S K E I P E Q R E N K Q Q Q (SEQ ID NO: 14)
Example 3
Assemblage of SEQ ID NO: 10
[0555] A polypeptide (SEQ ID NO: 10) was predicted to be encoded by
SEQ ID NO: 9 as set forth below. The polypeptide was predicted
using a software program called BLASTX which selects a polypeptide
based on a comparison of translated novel polynucleotide to known
polynucleotides. The initial methionine starts at position 291 of
SEQ ID NO: 9 and the putative stop codon, TAG, begins at position
1107 of the nucleotide sequence.
Example 4
Cloning of Stem Cell Growth Factor-Like Gene; and Expression and
Purification of Stem Cell Growth Factor-Like Protein
[0556] Stem cell growth factor-like polynucleotide (SEQ ID NO: 11
or 12) was cloned by PCR into pIB/V5-His TOPO TA cloning vector
(Invitrogen) from Hyseq's full-length stem cell growth factor-like
clone. Stem cell growth factor-like gene was further subcloned into
pCDNA3.1-Myc-His vectors (Invitrogen) and expressed with or without
V5-His tag. Insect cells (high Five, Invitrogen) were transfected
with stem cell growth factor-like gene with the His-5 tag by using
the InsectSelect system (Invitrogen) using manufacturer's suggested
protocols. Stem cell growth factor-like protein was purified from
the cell media by a combination of pH adjustment, cation exchange
chromatography, and affinity chromatography as described below.
Briefly, the pH of the medium was adjusted to 7.0 and the protease
inhibitors PMSF and EDTA were added. Column chromatography
purification was performed on Pharmacia Akta instrument system at
room temperature using sequential removal of contaminants on
appropriately sized columns of SP-Sepharose Fast Flow, Hitrap
heparin Sepharose, and Ni-NTA resins. Column elution fractions were
analyzed by separation on 16% SDS-PAGE gels, transfer to
Imrnmobilin membranes (Millipore), and detection of the tagged
protein by anti-V5 antibody using manufacturer's protocols.
Fractions containing stem cell growth factor-like activity eluted
from Ni-NTA column were pooled and equilibrated with PBS and stored
at -80.degree. C. until analyzed for stem cell growth factor-like
activity.
Example 5
Expression of Stem Cell Growth Factor-Like Protein in Primary Human
Cells
[0557] The product of the secondary nested PCR from Marathon spleen
library (SEQ ID NO: 11 or 12) or any other polynucleotide encoding
stem cell growth factor-like polypeptide were cloned into MSCV
retroviral vector (Clontech) into suitable cloning sites using
appropriate forward and reverse PCR primers. This retroviral
vectorwas then transfected using FUGENE-6 transfection reagent into
packaging cell lines to produce suitably large quantities of
retrovirus that will have the stem cell growth factor-like DNA
cloned in it. Retrovirus containing supernatants were prepared from
packaged cell lines and mixed with stromal or stem cells. Upon
retrovirus transduction these transduced cells may express the stem
cell growth factor-like protein.
Example 6
Assay for Growth and Differentiation of Stem Cells Using Coculture
Assay
[0558] 1.times.10.sup.4 mouse stem cells were co-cultured with
1.times.10.sup.4 stem cell growth factor-like
polynucleotide-transduced stromal cells or vector-transduced
stromal cells (produced by Example 5) in the serum-free medium. On
day seven, IL-3 (10 ng/ml) and IL-6 (10 ng/ml) were added as
additional growth factors. Cultures were monitored microscopically
every day. After appropriate further incubation, cells were
harvested, and counted using hemacytometer. Results from one
experiment are presented in the table below:
TABLE-US-00005 Day 15 (approximate Day 18 (approximate Conditions
number of cell/ml) number of cell/ml) Vector-transduced stroma
30000 105000 cells + stem cells Stem cell growth factor 405000
825000 polynucleotide-transduced stroma cells + stem cells
Example 7
Assay For Proliferation and Differentiation of Stem Cells
[0559] CD34.sup.+ hematopoietic stem cells (HSC) were purified from
mobilized peripheral blood (purchased from ALLCells). CD34.sup.+
cells were purified by positively selecting cells using Miltenyi
breads (Miltenyi). Stem cells were plated in 96-well plates at
10.sup.3/well. Purified stem cell growth factor-like protein and
other hematopoietic cytokines (purchased from R & D systems),
and the combinations thereof were added to the cultures for
assessing the stem cell growth factor activity. The growth and
differentiation of stem cells were examined 5 days after culture by
light microscope. The results of six experiments are shown in the
table below, wherein positive effect of stem cell growth factor
protein was observed in three out of six experiments.
[0560] Abbreviations: Stem cell growth factor-like protein=SCGF;
Interleukin-3=IL-3; thrombopoietin=TPO; Fms-like tyrosine kinase-3
ligand=flt-3 ligand (+) indicates growth and/or differentiation of
stem cells; (-) indicates no growth or differentiation and loss of
viability of stem cells.
TABLE-US-00006 Growth and Growth and morphological morphological
changes changes Growth factor(s) added Experiment 1 Experiment 2
None (-) (-) Stem cell growth factor (50 ng/ml) (-) (-) IL-3 (10
ng/ml) (+/-) (+) SCGF (50 ng/ml)+ IL-3 (10 ng/ml) (+) (+)
TABLE-US-00007 Growth and Growth and Growth and morphological
morphological morphological changes changes changes Growth
factor(s) added Experiment 3 Experiment 4 Experiment 5 None (-) (-)
(-) Stem cell growth (-) (-) (-) factor (50 ng/ml) TPO (100 ng/ml)
(-) (-) (-) kit ligand (50 ng/ml) + (-) (-) (-) flt-3 ligand (50
ng/ml) kit ligand (50 ng/ml) + (+) (-) (-) flt-3 ligand (50 ng/ml)
+ SCGF (50 ng/ml)
TABLE-US-00008 Growth and morphological changes Growth factor(s)
added Experiment 6 None (-) Stem cell growth factor (50 ng/ml) (-)
kit ligand (50 ng/ml) (-) flt-3 ligand (50 ng/ml) (-)
TABLE-US-00009 Growth and morphological Growth factor(s) added
changes kit ligand (50 ng/ml) + flt-3 ligand (50 ng/ml) (-) kit
ligand (50 ng/ml) + flt-3 ligand (50 ng/ml) + (+) SCGF (50
ng/ml)
Example 8
Establishment of Stromal Cell Strain Derived from Mouse AGM
[0561] (1) Isolation of AGM Region from Fetal Mouse
[0562] C3H/HeNSLc mouse of both genders (purchased from Japan SLC
INC.) was bred under a SPF (specific pathogen-free) circumstance.
One or two female mice and one male mouse were reared in the same
cage over a night. In the next morning, the female mice in which
the existence of a vaginal plug was confirmed were transferred to
other cages and breeded. The day when the existence of the vaginal
plug was confirmed was defined to be the 0.5th day of pregnancy. On
the 10.5th day of the pregnancy, after mouse was sacrificed by
cervical dislocation, fetuses Were extirpated. Isolation of AGM
regions was performed according to the method by Godin et al.
(Godin, I., Proc. Natl. Acad. Sci. U.S.A., 92: 773-777, 1995) and
the method by Medvinsky et-al. (Medvinsky, A. L., Blood 87:
557-565, 1996). The fetuses were placed in a culture dishes to
which PBS(-) (phosphate buffered saline) (produced by Nissui
Seiyaku) was added in a volume just sufficient to cover them. After
the AGM regions were carefully excised so as not to include other
regions under a stereoscopic microscope, they were put in another
24-well culture dish (Nunc).
(2) Establishment of Cell Lines Derived from AGM
[0563] One drop of MEM medium (Sigma) containing 10% FCS (Hyclone)
was added to the AGM regions in the 24-well culture dish (Nunc),
and AGM regions were cultured in incubator overnight. The cultures
were performed in the MEM medium (Sigma) including 10% FCS
(Hyclone) at 37.degree. C., in an atmosphere of 5% CO.sub.2, and at
a humidity of 100%. When the cells corresponding to the AGM regions
adhered to the culture dish due to overnight cultivation, two
milliliters of MEM medium containing 10% FCS was further added.
Stromal cells began to appear around the AGM region tissue fragment
after the continuous cultivation. After one-week cultivation,
adhesive cells were trypsinized (0.05% trypsin in PBS containing
0.53 mM EDTA (Gibco BRL) at 37.degree. C. for three to five
minutes) and dispersed. The stromal cells were then washed twice
with the medium, and seeded on 6-well culture dish (Nunc). On the
next day, the cells which did not adhered to the culture dish and
the medium were removed, and then, fresh medium were added. Two
weeks after transfer, the cells in the 6-well culture dish were
.gamma.-ray irradiated at 900 Rad to eliminate endogenous
hematopoietic cells. Although attempts of the direct cell cloning
by limiting dilution from this culture system was failed, so that
no cell proliferation was observed. Then, attempts were made
according to as follows: after adaptation of cells so as to be able
to proliferate from a small number of cells by increasing the
number of seeded cells in one well, the cells were cloned by
limiting dilution.
[0564] That is, the AGM was extirpated and cultured in the same
manner as described above. The culture system two weeks after the
.gamma.-ray radiation was trypsinized (0.05% trypsin in PBS
containing 0.53 mM EDTA at 37.degree. C. for three to five minutes)
and the cells were suspended, so that the cells-were-seeded in a
24-well culture dish ranging from 50 to 100 cells/well. After the
culture was continued for three weeks, the cells were seeded in a
96-well culture dish (Nunc) by means of limiting dilution so as to
be 0.3 cells/ well. The cells which were derived from the well
seeded only one cell and proliferated were allowed to enlarge
culture. As a result, the cells were successfully cloned to obtain
fibroblast like cells and cobble stone like cells.
[0565] CD34 positive cell fraction derived from the human cord
blood was co-cultured with the fibroblast like cells for two weeks.
Colony forming cells could not be found in the co-culture system
with the fibroblast like cells. Then, the similar examination was
performed for seven cell clones showing cobble stone like
morphotype. Three clones having activity to proliferate and support
the human hematopoietic stem cells were obtained and were named
AGM-s1, AGM-s2, and AGM-s3.
Example 9
Preparation of Hematopoietic Stem Cells from Mouse Bone Marrow
[0566] The bone marrow was collected from the femur of
C57BL/6-Ly5.1 pep (week ages ranging from eight to ten, and male)
(the gift from Professor K. Nakauchi, University of Tsukuba), and
suspended in PBS. After the mouse bone marrow mononuclear cells
were concentrated by specific gravity centrifugation according to
the usual method (S. Kouzu, Fundamental techniques for immunology,
YODOSHA, 1995), the cells were suspended with staining buffer (PBS
containing 5% FCS and 0.05% NaN.sub.3).
[0567] The most immature hematopoietic stem cell fraction was
obtained as follows (Osawa, M. et al., Science 273: 242-245,
1996).
[0568] The mononuclear cells were incubated with biotylated
anti-lineage monoclonal antibodies (CD45R, CD4, CD8, Gr-1, Ter119,
and CD11c, purchased from Pharmingen), fluorescein isothiocyanate
(FITC)-anti-CD34, phycoerythrin (PE)-anti-Sca-1, and
allophycocyanin (APC)-anti-c-Kit for 20 min on ice. After the
stained cells were washed twice with staining buffer, CD34
negative, Sca-1 positive, c-Kit positive, and Lin negative cells
were isolated on a FACS Vantage (Becton Dickinson).
Example 10
Subcloning of Mouse Stromal Cell Strain and Assessment of an
Activity to Support the Hematopoietic Stem Cells of a Variety of
Cell Strains
(1) Subcloning of Mouse Stromal Cell Strain
1) Isolation of AGM-s3 Subclone
[0569] Stromal cell strain AGM-s3 derived form AGM which was
subcultured in MEM.alpha. medium (GIBCO BRL) including non-active
10% FCS (bovine fetal serum, Hyclone) was suspended in PBS
containing 5% FCS (PBS-FCS). Clone sorting was performed in a
96-well culture dish (Falcon) at one cell/ well using a cell sorter
(FACS Vantage; Becton Dickinson). Among cells in the 96 wells,
cultures of the cells which proliferated were expanded, so that
thirteen kinds of AGM-s3 subclones were obtained. The activity to
support the hematopoietic cells of these AGM-s3 subclones were
assessed.
2) Isolation of Human Cord Blood CD34 Positive Stem Cell
[0570] The human cord blood was collected at normal delivery
according to the criteria approved by Drug Discovery Institute,
Ethics committee, Kirin Brewery Co., LTD. The cord blood was
collected using a syringe added with heparin so as not to
coagulate. The heparin treated cord blood was overlaid on
Lymphoprep (NYCOMED PHARMA), and mononuclear cells were separated
by centrifugation (at 400G, at room temperature, and for 30
minutes). Erythrocytes contaminated in the mononuclear cell
fraction were lyzed by treatment with ammonium chloride buffer
solution (0.83% NH.sub.4Cl-Tris HCl, 20 mM, pH 6.8) at room
temperature for two minutes. After the mononuclear cells were
washed with PBS-FCS, ten milligrams of human IgG was added and
allowed to stand on ice for ten minutes. Then, the cells were
further washed with PBS-FCS, added with biotinylated antibodies
against the antigens specific to the human differentiated blood
cells that is, CD2, CD11c (purified from ATCC. hybridoma), CD19
(Pharmingen), CD15, and CD41 (Leinco Technologies Inc.), and the
antibody against Glycophorin A (Cosmo. Bio), and allowed to stand
on ice for 20 min. After washing with PBS-FCS, the cells were
suspended in one milliliter of PBS containing 5% FCS, 10 mM EDTA,
and 0.05% NaN.sub.3 (PBS-FCS-EDTA-NaN.sub.3), added with magnetic
beads bound with streptavidin (BioMag. Per Septive Diagnostics),
and allowed to stand on ice for 40 min. The differentiated blood
cells which expressed differentiation antigens were removed using a
magnetic separator (Dynal MPC-1 Dynal). FITC labeled CD34 antibody
(Immunotech S. A., Marseilles, France) were added to the remaining
differentiated blood cell antigen negative cell fraction. After
incubation on ice for 20 min., CD34 positive fraction was recovered
using a cell sorter. This cell fraction was defined as a
hematopoietic stem cell fraction derived from the human cord
blood.
3) Co-Culture of the Human Hematopoietic Stem Cells and AGM-s3
Subclone
[0571] After 13 kinds of AGM-s3 subclones or stromal cell strain
MS-5 derived from the mouse bone marrow were seeded in a 24-well
culture dish (Falcon) at 1.times.10.sup.4 cells/well, and cells
were cultured in one milliliter of MEM.alpha. medium containing 10%
FCS until the cells covered all over the bottom surfaces of the
wells. CD34 positive hematopoietic stem cells derived from the
human cord blood were sorted on the above described stromal cells
at 500 cells/ well, and co-cultured in one milliliter of MEM.alpha.
medium containing 10% FCS. One week after the initiation of the
co-culture, one milliliter of the same medium was further added.
Two weeks after the initiation of the co-culture, the stromal cells
and the human blood cells were trypsinized (0.05% trypsin in PBS
containing 0.5 mM EDTA (GIBCO BRL) at 37.degree. C. and standing
for two to five min.) and dispersed from the culture dish.
Activities to support the hematopoietic stem cells were assessed
with a colony assay.
4) Assessment of Proliferation Statuses of the Hematopoietic Stem
Cells and Hemopoietic Precursor Cells by Clonogenic Assay
[0572] The cells which proliferated in the above described
co-culture system were appropriately diluted, added to one
milliliter of methylcellulose culture system, and analyzed in
triplicate. The analysis using the methylcellulose culture system
were performed using a 6-well culture dish (Falcon) in the presence
of 10 ng/ml of human SCF, human IL-3, human IL-6, human G-CSF,
human TPO, and EPO at 2 IU/ml to MethoCult H4230 (Stem Cell
Technologies Inc.). All of a variety of the above described
hematopoietic factors were recombinants and pure. Two weeks after
the culture, developed colonies were observed under a microscope
and counted numbers of CFU-GM (granulocyte-macrophage
differentiating series), BFU-E (erythroid burst forming unit), and
CFU-E mix (erythrocyte mixed differentiating series).
[0573] FIG. 4 shows the results from two-week co-culture of the
CD34 positive hematopoietic stem cells and AGM-s3 subclones A9, A7,
or D11. As a result of the co-culture, A9 and D11 subclones among
13 kinds of AGM-s3 subclones supported proliferation of all three
lineages of CFU-GM, BFU-E and CFU-E mix. Especially, although BFU-E
and CFU-E mix, that is, the precursor cells of an erythrocytes were
hardly to be supported in usual, they proliferated in the
co-culture system with A9 or D11 cells. The results showed that
proliferation or maintenance of the hematopoietic stem cells or the
hemopoietic precursor cells occurred in the co-culture with A9 or
D11 cells and the precursor cells of the erythrocyte were
continuously supplied. In contrast, although cellular morphology of
A7 was similar to that of A9, A7 did not support CFU-GM, BFU-E, and
CFU-E mix.
5) Comparison of an Activity to Support the Human Hematopoietic
Stem Cells Between A9 and a Stromal Cell Strain OP9 Derived from
Mouse Fetus
[0574] Comparison of activities to support the CD34 positive
hematopoietic stem cells derived from the human cord blood between
AGM-s3 subclones A9 and A7, and a stromal cell line OP9 derived
from mouse fetus were performed with CFU-GM, BFU-E, CFU-E and CFU-E
mix as indexes using the above described method. FIG. 5 shows the
results from the two-week co-culture. In the A7 cell culture
system, CFU-GM, BFU-E, and CFU-E were significantly decreased and
CFU-E mix was completely disappeared. In contrast, with OP9 cells,
a variety of blood cell precursor cells including CFU-E mix were
supported, although the supporting ability was less than that of A9
cells. Therefore, OP9 cells were clear to possess the activity to
support the hematopoietic stem cells.
(2) Assessment of Activities to Support the Hematopoietic Stem
Cells in a Variety of Cell Strains
[0575] The above described stromal cell lines(AGM-s3-A9, AGM-s3-A7,
and AGM-s3-G1), 3T3Swiss (ATCC), OP9 (RCB1124, RIKEN Cell
Development Bank), and NIH3T3 (ATCC) were seeded in a 24-well
culture dish. (Falcon) at 5.times.10.sup.4 cells/well. The cell
lines were cultured in MEM.alpha. medium (GIBCO BRL) containing
non-active 10% FCS (bovine fetal serum, Hyclone) for one day and
allowed to proliferated until the cells covered all over the bottom
surfaces of the wells. Then, the medium was replaced to one
milliliter of fresh medium, thirty cells of the mouse hematopoietic
stem cells (derived from C57BL/6-Ly5.1) obtained in Example 9 were
sorted on this cell layer, and co-culture was initiated.
[0576] On seventh day of the cultivation, the cells were
trypsinized (0.05% trypsin in PBS containing 0.5 mM EDTA (GIBCO
BRL) at 37.degree. C. for two to five minutes) and dispersed and
all the cells on the culture dish were recovered. The recovered
whole cells of each cell line and whole bone marrow cells at
200,000 cells (derived from C57BL/6-Ly5.2 mouse, Charles River)
were transplanted into the C57BL/6-Ly5.2 mice (eight weeks age and
male, Charles River) irradiated with X-ray at 8.5 Gy through the
tail vein. After the transplantation, the peripheral blood was
collected from the retro-orbital sinus at intervals, and calculated
the ratio of a cell number derived from the C57BL/6-Ly5.1 prep
mouse with FACS. The peripheral blood was analyzed according to the
usual method (S. Kouzu, Fundamental techniques for immunology,
YODOSHA, 1995). Three hundreds and fifty .mu.L of distilled water
was added to 50 .mu.L of the peripheral blood, allowed to stand for
30 sec. so as to lyze the erythrocytes. Then, PBS at twice
concentrations was added and centrifuged, so that the white blood
cells were recovered. After the cells were washed once using the
staining buffer (PBS containing 5% FCS and 0.05% NaN.sub.3),
anti-CD16 antibody, Ly5.1 (CD45.1) antibody labeled with FITC, Gr-1
and CD11c, antibodies labeled with phycoerythrin, and CD45R (B220)
antibody and CD90 (Thy1) antibody labeled with allophycocyanin (all
of these were purchased from Pharmingen) were added. After these
cells were allowed to stand for reaction in the ice bath for 30
min., they were washed with the staining buffer and FACS analysis
was performed.
[0577] Expansion in the number of cells capable of reconstitution
during the hematopoietic stem cell culture was assessed by
calculating the proportions of Ly5.1 positive cells in the Gr-1 or
CD11c positive cells (myeloid cells) or Ly5.1 positive cells in the
CD90 or CD45R positive cells (lymphoid cells) in the peripheral
blood at intervals post transplantation.
[0578] FIG. 6 shows the results. When the cells were co-cultured
with AGM-s3-A9, OP9, and 3T3Swiss cells, high chimerism of donor
cells were maintained after the transplantation. Therefore, these
stromal cells were considered to have a high activity to support
the hematopoietic stem cells. In contrast, when the cells were
co-cultured with AGM-s3-A7, AGM-s3-G1, and NIH3T3 cells, high
chimerism were not observed in the transplanted cells. Therefore,
these stromal cells had low activity to support the hematopoietic
stem cells or the hemopoietic progenitor cells.
Example 11
Isolation of Mouse SCR-1 Fragment
[0579] Total RNA was prepared from AGM-s3-A9 cells at
1.4.times.10.sup.8 cells dissolved in 20 mL of ISOGEN (Nippon gene,
Japan) according to the attachment. Messenger RNA was purified from
one milligram of the total RNA according to the protocol of the
mRNA purification kit (Amersham Pharmacia, U.S.A.). cDNA was
synthesized from this mRNA by oligo-dT primed with SuperScript
Plasmid System (GIBCO Lifetech, U.S.A.) and inserted into pSPORT1
(GIBCO Lifetech, U.S.A.). An AGM-s3-A9 cell specific cDNA clone was
obtained from this library with SBH method (Hyseq, U.S.A.). A
nucleotide sequence of the clone was determined using ABI377 DNA
sequencer (Perkin Elmer, U.S.A.). The obtained sequence was
analyzed by homology search, so that the gene was identified as a
novel gene SCR-1. The nucleotide sequence obtained was nucleotide
numbers 1032 to 1484 of SEQ ID NO: 31.
Example 12
Whole Cloning of Mouse SCR-1
[0580] Total RNA was prepared from AGM-s3-A9 cells at
1.4.times.10.sup.8 cells dissolved in 20 mL of ISOGEN (Nippon gene,
Japan) according to the attachment. Messenger RNA was purified from
one milligram of the total RNA according to the protocol of the
mRNA purification kit (Amersham Pharmacia, U.S.A.). cDNA library
was constructed from 2 mg of prepared mRNA using SMART cDNA library
construction kit (CLONTECH, U.S.A.) according to the attachment.
This library included about 400,000 kinds of independent clones in
total and divided into 15 fractions. The fraction containing SCR-1
cDNA clone was identified by PCR using the following
conditions.
[0581] The following primers were synthesized based on the gene
fragment sequence obtained in Example 11 PCR at 35 cycles was
performed using each fraction of AGM-s3-A9 cDNA library as a
template, one cycle being a step performed at 94.degree. C. for 30
seconds, at 55.degree. C. for 30 seconds, and 72.degree. C. for one
minute.
TABLE-US-00010 SCR-1F1: AGTACAAAGAAAGAAGTGTTC (SEQ ID NO: 35)
SCR-1R1: TGAGTCTACAGTAACCTCGCA (SEQ ID NO: 36)
[0582] The PCR products were subjected to a 2% agarose gel
electrophoresis, and the fraction in which a PCR product had an
expected size was identified. Two positive fractions were seeded on
petri dishes at a diameter of 15 cm at 50,000 plaque, each
fractions being seeded on two petri dishes. After incubating the
dishes at 37.degree. C. for 10 hours, each plaque was transferred
to a Biodyne nylon filter (Pall, U.S.A.). DNAs on the nylon filters
were immobilized according to the attachment. Screening was
performed using a .sup.32P labeled DNA probe.
[0583] The probe was prepared as follows. PCR at 35 cycles was
performed using SCR-1R1 and T7 primer (TAATACGACTCACTATAGGG) (SEQ
ID NO: 37), and a plasmid including the gene fragment obtained in
Example 11 as a template, one cycle being a step performed at
94.degree. C. for 30 seconds, at 55.degree. C. for 30 seconds, and
72.degree. C. for one minute. The PCR products were subjected to a
2% agarose gel electrophoresis, and the amplified fragment was
purified using JETSORB (GENOMED Ger.). .sup.32P labeled DNA probe
was prepared using Megaprime labeling kit (Amersham Pharmacia
U.S.A.) and 25 ng of purified PCR fragment as a template.
[0584] Hybridization using ExpressHybSolution (CLONTECH, U.S.A.)
and washing were performed according to the attachment. X-ray films
(Fuji Photo Film Co. Ltd., Japan) were exposed to the hybridized
nylon filters for one day and developed using a Fuji film
auto-developer apparatus. Based on the analyzed results, the plaque
which corresponded to the spot strongly exposed was scratched from
the petri dish. The plaque was again seeded on a petri dish at a
diameter of 10 cm so as to generate about 200 plaques. Screening
was performed again according to the method described above, so
that a single plaque was isolated. The obtained phage clone was
introduced into E. coli BM25.8 strain according to the attachment
of the SMART cDNA library constructing kit, so that it was excised
in vivo in E. coli BM25.8 strain. The infected E. coli was cultured
on LB agar medium added with 50 mg/mL of ampicillin until colonies
were formed. A single colony was seeded in three milliliters of LB
medium containing 50 mg/mL of ampicillin and cultured overnight at
30.degree. C. About 10 mg of plasmid was purified from the cultured
cells using RPM Kit (BIO101, U.S.A.). The sequence of the both ends
of the inserted fragment was determined using
.lamda.TriplEx5'LD-Insert Screening Amplimer
(CTCGGGAAGCGCGCCATTGTGTTGGT: CLONTECH, U.S.A; SEQ ID NO: 30.) by
ABI377 DNA sequencer. The clone was found to include CDNA which has
a nucleotide sequence beginning from 1 in SEQ ID NO: 31. After the
whole nucleotide sequence of the inserted cDNA was determined using
ABI377 DNA sequencer, the nucleotide sequence of SEQ ID NO: 31 was
confirmed. Amino acid sequences predicted from the above nucleotide
sequence were shown in SEQ ID NO: 31 and SEQ ID NO: 32.
[0585] The plasmid including DNA with the nucleotide sequence of
SEQ ID NO: 31 has been internationally deposited in National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology (Zip code 305-8566; Higashi 1-1-3, Tsukuba,
Ibaraki, Japan) on Jun. 26, 2000, and the registered number was
given to be FERM BP-7198.
Example 13
Cloning Of Human SCR-1
[0586] Based on the nucleotide sequence of mouse SCR-1, the
database of GenBank (NCBI, U.S.A.) was searched using Blast. A
homologous nucleotide sequence with mouse SCR-1 was found
(Accession Nos. A1872133 and AW316562). The following primers were
synthesized using this sequence derived from human.
TABLE-US-00011 h782F1: TCGCGGCGATGCCAGCCACCCCAG (SEQ ID NO: 38)
h782F2: AGCACGCCTATCGGATGTGAGAGGAGAAGT (SEQ ID NO: 39) h782R1:
CTATTAACAAATATATTTATTGTGGTGGCT (SEQ ID NO: 40) h782R2:
TGGTGGCTTTCTCCCCTACTAGATATACCT (SEQ ID NO: 41)
[0587] cDNA was synthesized from 3 .mu.g of mRNA derived from the
placenta and the skeletal muscle (CLONTECH, U.S.A.) using oligo-dT
primer and reverse transcriptase (SuperscriptII, GIBCO-BRL). PCR
was performed using this CDNA as a template; h782F1, h782F2,
h782R1, or h782R2 as a primer; and Platinum Pfx DNA Polymerase
(GIBCO Lifetech, U.S.A.). As a result, an amplified fragment was
obtained from each organ. Among them, the PCR fragment derived from
the placenta was ligated to pCR-Blunt vector (Invitrogen, U.S.A.),
and the gene was introduced into E. coli DH5a. Then, the
transferred E. coli was seeded on LB agar medium containing 100
mg/ml of ampicillin, so that colonies were formed. Each of isolated
16 colonies was added to 10 ml of PCR reaction solution, and
treated at 94.degree. C. for five minutes. Then, PCR at 35 cycles
was performed, one cycle being a step performed at 94.degree. C.
for 30 seconds, at 55.degree. C. for 30 seconds, and 72.degree. C.
for one minute. T7 primer or SP6 primer (GATTTTAGGTGACACTATAG) (SEQ
ID NO: 42) was used as a primer at a final concentration of 0.2 mM.
The PCR products were subjected to a 2% agarose gel
electrophoresis. After the amplified fragment was confirmed,
sequences of the three confirmed fragments were determined using
ABI377 DNA sequencer. A nucleotide sequence of the obtained cDNA
(SEQ ID NO: 33) was confirmed to be a human orthologous to that of
mouse SCR-1.
[0588] The plasmid including DNA with the nucleotide sequence of
SEQ ID NO: 33 has been internationally deposited in National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology (Zip code 305-8566; Higashi 1-1-3, Tsukuba,
Ibaraki, Japan) on Jun. 26, 2000, and the registered number was
given to be FERM BP-7197.
[0589] With respect to mouse SCR-1 and human SCR-1 , when the
established database was searched, a human gene sequence having
unknown function was found (WO98/49302). The homology of coding
regions of these genes is shown in Table 1. The comparison of the
homology was performed using a homology search function of
DNAIS-Mac version 3.7 and calculating with the default settings of
the software (nucleic acid Mode: Normal, Range: All (1-819 base),
Cutoff: 45, Ktup: 4, amino acid Range: All (1-273 a.a.), Cutoff:
45, Ktup: 2). In this method, since only high homologous regions
are used for calculation, low homologous regions, locating at an
end, are excluded for calculation.
TABLE-US-00012 TABLE 1 Mouse SCR-1 Gene having unknown function
Human SCR-1 88.5 98.5 (87.1) (100) Mouse SCR-1 87.2 (85.1) The
upper numbers show the homology in nucleotide level. The lower
numbers (parenthesize) show the homology in amino acid level.
[0590] Human SCR-1 and the gene having unknown function had the
same nucleotides from the initiation codon to the first nucleotide
of the 266th codon (nucleotide number 1054 in SEQ ID NO: 33) in the
coding region. However, the down stream sequences thereof were not
identical. The nucleotide sequence and the amino acid sequence of
this nonidentical region in this gene having unknown function were
shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively. The first
nucleotide in SEQ ID NO: 45 corresponded to the nucleotide number
1054 in SEQ ID NO: 33, and these were identical. One nucleotide was
nonidentical at the position corresponding to the 567th nucleotide
in SEQ ID NO: 33 in both genes.
Example 14
Study Of The Expression Region Of SCR-1
[0591] Northern blot analysis was performed using probes used in
Example 13. Hybridization was performed with respect to Northern
blots of Human MTN Blot I, II, III, Immune System II, Mouse MTN
Blot (CLONTECH, U.S.A.). The hybridization was performed using
ExpressHyb Hybridization Solution (CLONTECH, U.S.A.) according to
the supplier's instruction. After prehybridization at 68.degree. C.
for two hours, a labeled probe was added. Hybridization was further
performed at 68.degree. C. for 18 hours. Washing of the filter was
performed at room temperature in 2.times.SSC, 0.05% SDS solution
for 30 min and repeated once. Further washing was performed at
50.degree. C., in 0.1.times.SSC, 0.1% SDS solution for 30 min
twice. Analysis of the hybridization was performed by exposure to
an imaging plate (Fuji Photo film Co. Ltd., Japan) for three hours
using a bioimaging analyzer BAS2000 (Fuji Photo Fihn Co., Ltd.,
Japan). Northern blot technique analysis was performed using probes
used in Example 13 and MTN blot (CLONTECH, U.S.A.), so that the
expression thereof in human was examined. As a result, the
expression of mRNA at about 2.6 kb was confirmed in many organs
including the liver, the placenta, the skeletal muscle, and the
uterus. In mouse, the expression of mRNA at about 2.6 kb was
confirmed in similar organs.
Example 15
Expression Of Mouse SCR-1 In Stromal Cell
(1) Construction of Retrovirus Vector for Expression of Mouse
SCR-1
[0592] Only ORF sequence in SCR-1 gene (a nucleotide sequence from
nucleotides numbers 511 to 1350 in SEQ ID NO: 31) was inserted into
a retrovirus vector, so that a vector for expression in stromal
cells was constructed.
[0593] Messenger RNA was purified from one milligram of the total
RNA in AGM-s3-A-9 cell according to the protocol of the mRNA
purification kit (Amersham Pharmacia, U.S.A.). cDNA was synthesized
from this mRNA according to the conventional method. The following
primers were synthesized and PCR at 30 cycles was performed using
the above described cDNA as a template and Platinum Pfx DNA
Polymerase (GIBCO Lifetech, U.S.A.), one cycle being a step
performed at 94.degree. C., for 20 seconds, at 55.degree. C. for 30
seconds, and 68.degree. C. for one minute.
TABLE-US-00013 m782F2: CCGCTCGAGCCACCATGCACTTGCGACTGATTTC (SEQ ID
NO: 43) m782R2: ATTGAATTCCTAGTGTACAGTGCTGACTG (SEQ ID NO: 44)
[0594] An amplified fragment was digested with restriction enzymes
EcoRI and XhoI. After electrophoresis, a DNA fragment was purified
using JETSORB (Genomed, Germany). The purified DNA fragment was
ligated with pMX-IRES-GFP vector digested with EcoRI and XhoI (gift
form Professor T. Kitamura, TOKYO UNIV. INST. OF MEDICAL SCIENCE,
Japan). The pMX-IRES-GFP vector was a plasmid in which IRES GFP was
inserted into the retrovirus vector pMX. The obtained recombinant
vector was transferred into E. coli DH5a, and was seeded on LB agar
medium containing 100 mg/ml of ampicillin, so that independent
colonies were formed. After the isolated colony was cultured in 100
mL of LB medium containing 100 mg/ml of ampicillin, plasmid was
purified using QIAGENtip100 (QIAGEN, U.S.A.). The sequence of the
inserted gene was determined using conventional method, so that the
sequence was confirmed to be identical to the corresponding region
in SEQ ID NO: 31.
(2) Transfer of Mouse SCR-1 into Stromal Cell
[0595] Initially, BOSC23 cells at 2.times.10.sup.6 cells/dish were
seeded on a collagen type I coated 60 mm dish (Asahi technoglass),
and cultured in DMEM medium containing 10% FCS at 37.degree. C.,
under an atmosphere of 5% CO.sub.2, and at a humidity of 100%.
Twelve to 18 hours after the start of the culture, the medium was
replaced by two milliliters of OPTI MEM medium (GIBCO BRL).
[0596] About 3 .mu.g of plasmid inserted with SCR-1 into the above
described pMX-IRES-GFP was added to 18 .mu.L of LIPOFECTAMINE
Reagent (GIBCO BRL) diluted with 100 .mu.L of OPTI MEM medium, and
allowed to stand at room temperature for 30 min. The prepared DNA
solution was added to the above-prepared BOSC23 cell culture
solution. After about five hours, two milliliters of DMEM-medium
containing 20% FCS (GIBCO BRL) was added.
[0597] IRES (Internal Ribosome Entry Site) was determined by an
access of the ribosome to the internal site of the mRNA. Therefore,
two genes could be expressed from one mRNA caused by ligation of
upward and downward genes separated by IRES in one transcription
unit during the construction of an expression vector. With respect
to the above-described plasmid, cDNA of SCR-1 was inserted in
upward site and GFP (Green Fluorescence Protein) was inserted in
downward site. Thus, the expression of SCR-1 could be monitored by
detecting the expression of GFP using FACS.
[0598] After about 24 hours, the medium was replaced by 4 ml of
DMEM containing 10% FCS. Further, after about 48 hours, the culture
medium was harvested. After the culture medium was filtrated
through 0.45 .mu.m filter, the filtrate was centrifuged at 1,200 g
for 16 hours and the supernatant was removed, so that the virus
precipitation was obtained.
[0599] AGM-s3-A7 cells were cultured in one milliliter of
MEM.alpha. medium containing 10% FCS (GIBCO BRL) on a 24-well
culture dish (FALCON) at 1.times.10.sup.4 cells/well. After 12 to
18 hours, the virus precipitation was suspended in one milliliter
of MEM.alpha. medium containing 10% FCS, so that the stromal cell
culture medium and the virus suspension were replaced. Next,
POLYBRENE (Sigma, SEQUA-BRENE) was added to be 10 .mu.g/mL. After
the culture dish was centrifuged at 700 g for 45 min., the cells
were cultured at 37.degree. C., under an atmosphere of 5% CO.sub.2,
and at a humidity of 100%. After 48 hours, the medium was replaced
by one milliliter of MEM medium containing 10% FCS. After 24 hours,
the cells were passaged on a 6-well culture dish (FALCON) and
cultured in three milliliters of MEM medium containing 10% FCS.
Forty-eight hours after the passage, GFP expression in the stromal
cells was detected using a cell sorter (FACSVantage, Becton
Dickinson), so that it was indirectly confirmed that not less than
80% cells expressed SCR-1.
Example 16
Co-Culture Of Stromal Cells in Which SCR-1 Gene was Overexpressed
with Mouse Hematopoietic Stem Cells
[0600] AGM-s3-A9 cells, AGM-s3-A7 cells, or AGM-s3-A7 cells,
transduced with SCR-1 gene by retrovirus infections, were seeded in
a 24-well culture dish at a density of 1.times.10.sup.5 cells/well,
and were cultured in MEM.alpha. medium containing 10% FCS for one
day in order to allow the cells to proliferate to cover the whole
bottom surface of the culture dish.
[0601] Then, the medium was replaced by 1 ml of fresh medium and
thirty cells of the mouse hematopoietic stem cells (derived from
C57BL/6-Ly5.1) obtained in Example 9 were sorted on this cell layer
to initiate the co-cultures.
[0602] After 7 days of culture, all the cells in the co-culture
were harvested by trypsinization (0.05% trypsin in PBS containing
0.5 mM EDTA at 37.degree. C. for two to five minutes), and the
extent of the expansion of hematopoietic stem and/or progenitor
cells was analyzed in the following experiment.
Example 17
Transplantation of Hematopoietic Cells into Irradiated Recipient
Mice
[0603] Thirty freshly isolated hematopoietic stem cells, obtained
from C57BL/6-Ly5.1 mice by the procedure described above (CD 34
negative, Sca-1 positive, c-Kit positive, Lin negative cells, or
cells derived from C57BL/6-Ly5.1 pep mouse), or whole the cells
harvested on the 7th day of the co-culture, which was initiated
with 30 hematopoietic stem cells, were transplanted into the five
C57BL/6-Ly5.2 mice (eight weeks age and male, Charles River),which
were irradiated with X-ray at 8.5 Gy, through the tail vein
together with the 200,000 whole bone marrow cells derived from
C57BL/6-Ly5.2 mice (Charles River).
[0604] After the transplantation, the peripheral blood cells were
collected from the retro-orbital sinus over time, and analyzed for
the proportion of the cells that were derived from Ly5.1
hematopoietic cells. The peripheral blood was analyzed according to
the usual method (S. Kouzu, Fundamental techniques for immunology,
YODOSHA, 1995). In order to lyse erythrocytes, three hundreds and
fifty .mu.L of distilled water was added to 50 .mu.L of the
peripheral blood and 30 seconds after addition of distilled water,
the same amount of the twice concentrated PBS and centrifuged, so
that white blood cells were recovered. After the cells were washed
once using the staining buffer (PBS containing 5% FCS and 0.05%
NaN.sub.3), they were stained with anti-CD16 antibody,
FITC-anti-Ly5.1, PE-anti-myeloid cells (Gr-1 and CD11c) and
APC-anti-lymphoid cells (B220 and Thy1)(purchased from Pharmingen)
and incubated for 30 min on ice. Stained cells were washed using
the staining buffer and FACS analysis was performed.
[0605] Expansions in the number of the cells, that were capable of
reconstituting hematopoietic cells during the culture of the
hematopoietic stem cells were estimated by calculating proportions
of Ly5.1 positive cells in the Gr-1 or CD11c positive cells
(myeloid cells), or Ly5.1 positive cells in the CD90 or CD45R
positive cells (lymphoid cells) in the peripheral blood in the
transplanted mice at intervals.
[0606] FIG. 7 shows the results. When the cells co-cultured with
AGM-s3-A7 cells were transplanted, high chimerism derived from the
cultured Ly5.1 hematopoietic cells was not observed. From this
result, it was demonstrated that AGM-s3-A7 cells themselves showed
low activity to support the hematopoietic stem cells or hemopoietic
precursor cells. When cells co-cultured with AGM-s3-A7 cells in
which SCR-1 gene was overexpressed were transplanted, a significant
high proportion of cells derived from the cultured cells was
detected in both myeloid and lymphoid cells in the peripheral
blood. Therefore, it was clear that the hematopoietic stem cells
and the hemopoietic precursor cells, which could reconstitute the
hemopoietic system of the irradiated mice, were supported and
amplified on the A7 stromal cells in which SCR-1 gene was
overepressed.
[0607] As a result, it was evident that SCR-1 had a function to
give the stromal cells without an activity to support the survival
or the proliferation of the hematopoietic stem cells or the
hematopoietic progenitor cells the above described activity. From
these results, it was evident that SCR-1 had an activity to effect
on support the proliferation or the survival of the hematopoietic
stem cells or the hemopoietic precursor cells; or an activity to
effect the stromal cells so as to give them an activity to support
the hematopoietic stem cells.
Example 18
SCR-1 Transgenic Mice
[0608] The activity of mouse and human SCR-1 can be confirmed by
establishing genetically modified mice, such as transgenic mice.
Appropriate promoters are selected for expression of SEQ ID NOS: 31
and 33 which allows their activity for hematopoietic stem cell
growth or survival promotion to be confirmed. GATA-2 promoter
drives expression of genes in very early hematopoietic stem or
progenitor cell population. Expression of the SCR-1 gene (SEQ ID
NOS: 31 or 33) under the regulation of GATA-2 promoter in
transgenic mice will cause the hematopoietic stem or progenitor
cells to express the SCR-1 gene. This SCR-1 gene expression in
hematopoietic stein progenitor cells will lead to expansion of
hematopoietic stem or progenitor cells which will result in an
increase of hematopoietic cells in embryos, neonates or adult
mutant mice. GATA-2 promoters described by Minegishi et al may be
used for this purpose (J Biol Chem. 1998 Feb 6;273(6):3625-34).
[0609] The SCR-1 gene (SEQ ID NOS: 31 or 33) may also be expressed
under the control of CAG or other promoters that work in ubiquitous
tissues (Kiwaki et al. Gene Ther, 1996 May 1;7(7):821-30). This
will allow for determination of the effects of SCR-1 gene
expression in other tissue cell types together with hematopoietic
cells. Transgenic mice can be established accroding to the methods
described in "Manipulating the Mouse Embryo" (Brigid Hogan, Rosa
Beddington, Frank Costantini, Elizabeth Lacy, 1994, Cold Spring
Harbor Laboratory Press).
Sequence CWU 1
1
481301DNAHomo sapiens 1gcacgagacg aggaaaaaaa ggaagggaga ggaaaagaaa
aaaacctaat aaaggagaaa 60gtaaagaagc aatacctgac agcaaaagtc tggaatccag
caaagaaatc ccagagcaac 120gagaaaacaa acagcagcag aagaagcgaa
aagtccaaga taaacagaaa tcggtatcag 180tcagcactgt acactagagg
gttccatgag attattgtag actcatgatg ctgctatctc 240aaccagatgc
ccaggacagg tgctctagcc attaggacca caaatggaca tgtcagttat 300t
3012392DNAHomo sapiens 2tggaactcga tatccagata taaataagcg tacaaaatgc
aaagctgact gtgatacctg 60tttcaacaaa gatttctgca caaaatgtaa aagtggattt
tacttacacc ttggaaagtg 120ccttgacaat tgcccagaag ggttggaagc
caacaaccat actatggagt gtgtcagtat 180tgtgcactgt gaggtcagtg
aatggaatcc ttggagtcca tgcacgaaga agggaaaaac 240atgtggcttc
aaaagaggga ctgaaacacg ggtccgagaa ataatacagc atccttcagc
300aaagggtaac ctatgtcccc caacaaatga gacaagaaag tgtacagtgc
aaaggaagaa 360gtgtcagaag ggagaacgag gaaaataagg ag 3923475DNAHomo
sapiensmisc_feature(3)..(3)n = A, T, G, or C 3gtnagtaccc ccagggattt
cactgagngc ctggactgag gacccgtcna anngcnngan 60ccacgcgtnc gcccacgcgt
ccggagagga aaagaaaaaa acctaattta ggagaaagta 120aagaagcaat
acctgacagc ggaagtctgg aatggagcaa agaaatccca gagcaacgag
180aaaacaaaca gcagcagaag aagcgaaaag tccaagataa acagaaatcg
gtatcagtca 240gcactgtaca ctagagggtt ccatgagatt attgtagact
catgatgctg ctatctcaac 300cagatgccca ggacaggtgc tctagccatt
aggaccacaa atggacatgt cagttattgc 360tctgtctaaa caacattccc
agtagttgct atattcttca tacaagcata gttaacaaca 420aagagccaaa
agatcaaaga agggatactt tcagatggtt gtcttgtgtg cttcn 4754473DNAHomo
sapiensmisc_feature(7)..(10)n = A, T, G, or C 4tgggcannnn
aaanttttga nattcgatcc gcgctgcagg aattcggcac gagacgagga 60aaaaaaggaa
gggagaggaa aagaaaaaaa cctaataaag gagaaagtaa agaagcaata
120cctgacagca aaagtctgga atccagcaga gaaatcccag agcaacgaga
aaacaaacag 180cagcagaaga agcgaaaagt ccaagataaa cagaaatcgg
tatcagtcag cactgtacac 240tagagggttc catgagatta ttgtagactc
atgatgctgc tatctcaacc agatgcccag 300gacaggtgct ctagccatta
ggaccacaaa tggacatgtc agttattgct ctgtctaaac 360aacattccca
gtagttgcta tattcttcat acaagcatag ttaacaacaa agagccaaaa
420gatcaaagaa gggatacttt cagatggttg tcttgtgtgc ttctctgcat ttt
4735462DNAHomo sapiensmisc_feature(7)..(11)n = A, T, G, or C
5tgggagannn ntttgaaact gagatcgtcg canacncnac nangaataaa aggaagggag
60agggaaagaa aaaaacctaa taaaggagaa agtaaagaat caatttctga cagcaaaagt
120ctggaatcca tcaaagaaat cccatatcaa cgagaaaaca gacagcagca
caaaaagcga 180aaagtccaag ataaacagaa atcggtatca gtcagcactg
tacactagag ggttccatga 240gattattgta gactcatgat gctgctatct
caaccagatg cccaggacag gtgctctatc 300cattacgacc acaaatggac
atgtcagtta ttgctctgtc taaacaacat tcccagtagt 360tgctatattc
ttcatacaag catagttaac aacaaagagc caaaagatca aagaagggat
420actttcagat ggttgtcttg tgtgcttctc tgcattttta aa 4626384DNAHomo
sapiens 6aataatgtgt acaaaatgca aagctgactg tgatacctgt ttcaacaaaa
atttctgcac 60aaaatgtaaa agtggatttt acttacacct tggaaagtgc cttgacaatt
gcccagaagg 120gttggaagcc aacaaccata ctatggagtg tgtcagtatt
gtgcactgtg aggtcagtga 180atggaatcct tggagtccat gcacgaagaa
gggaaaaaca tgtggcttca aaagagggac 240tgaaacacgg gtccgagaaa
taatacagca tccttcagca aagggtaacc tatgtccccc 300aacaaatgag
acaagaaagt gtacagtgca aaggaagaag tgtcagaagg gagaacgagg
360aaaaaaagga agggagagga aaag 3847390DNAHomo
sapiensmisc_feature(390)..(390)n = A, T, G, or C 7cgttgctctg
ggatttcttt gctggattcc agacttttgc tgtcaggtat tgcttcttta 60ctttctcctt
tattaggttt ttttcttttc ctctcccttc ctttttttcc tcgttctccc
120ttctgacact tcttcctttg cactgtacac tttcttgtct catttgttgg
gggacatagg 180ttaccctttg ctgaaggatg ctgtattatt tctcggaccc
gtgtttcagt ccctcttttg 240aagccacatg tttttccctt cttcgtgcat
ggactccaag gattccattc actgacctca 300cagtgcacaa tactgacaca
ctccatagta tggttgttgg cttccaaccc ttctgggcaa 360ttgtcaaggc
actttccaag gtgtaagtan 39081345DNAHomo
sapiensmisc_feature(321)..(1235)similar to gi4519541 in the genpept
database release 114, Run with FASTXY3.3t00, default parameter
8gcggccgccc cggcggctcc tggaaccccg gttcgcggcg atgccagcca ccccagcgaa
60gccgccgcag ttcagtgctt ggataatttg aaagtacaat agttggtttc cctgtccacc
120cgccccactt cgcttgccat cacagcacgc ctatcggatg tgagaggaga
agtcccgctg 180ctcgggcact gtctatatac gcctaacacc tacatatatt
ttaaaaacat taaatataat 240taacaatcaa aagaaagagg agaaaggaag
ggaagcatta ctgggttact atgcacttgc 300gactgatttc ttggcttttt
atcattttga actttatgga atacatcggc agccaaaacg 360cctcccgggg
aaggcgccag cgaagaatgc atcctaacgt tagtcaaggc tgccaaggag
420gctgtgcaac atgctcagat tacaatggat gtttgtcatg taagcccaga
ctattttttg 480ctctggaaag aattggcatg aagcagattg gagtatgtct
catcttcatg tccaagtgga 540tattatggaa ctcgatatcc agatataaat
aatgtgtaca aaatgcaaag ctgactgtga 600tacctgtttc aacaaaaatt
tctgcacaaa atgtaaaagt ggattttact tacaccttgg 660aaagtgcctt
gacaattgcc cagaagggtt ggaagccaac aaccatacta tggagtgtgt
720cagtattgtg cactgtgagg tcagtgaatg gaatccttgg agtccatgca
cgaagaaggg 780aaaaacatgt ggcttcaaaa gagggactga aacacgggtc
cgagaaataa tacagcatcc 840ttcagcaaag ggtaacctat gtcccccaac
aaatgagaca agaaagtgta cagtgcaaag 900gaagaagtgt cagaagggag
aacgaggaaa aaaaggaagg gagaggaaaa gaaaaaaacc 960taataaagga
gaaagtaaag aagcaatacc tgacagcaaa agtctggaat ccagcaaaga
1020aatcccagag caacgagaaa acaaacagca gcagaagaag cgaaaagtcc
aagataaaca 1080gaaatcggta tcagtcagca ctgtacacta gagggttcca
tgagattatt gtagactcat 1140gatgctgcta tctcaaccag atgcccagga
caggtgctct agccattagg accacaaatg 1200gacatgtcag ttattgctct
gtctaaacaa cattcccagt agttgctata ttcttcatac 1260aagcatagtt
aacaacaaag agccaaaaga tcaaagaagg gatactttca gatggttgtc
1320ttgtgtgctt ctctgcattt ttaaa 134591343DNAHomo
sapiensCDS(291)..(1109) 9gcggccgccc cggcggctcc tggaaccccg
gttcgcggcg atgccagcca ccccagcgaa 60gccgccgcag ttcagtgctt ggataatttg
aaagtacaat agttggtttc cctgtccacc 120cgccccactt cgcttgccat
cacagcacgc ctatcggatg tgagaggaga agtcccgctg 180ctcgggcact
gtctatatac gcctaacacc tacatatatt ttaaaaacat taaatataat
240taacaatcaa aagaaagagg agaaaggaag ggaagcatta ctgggttact atg cac
296 Met His 1ttg cga ctg att tct tgg ctt ttt atc att ttg aac ttt
atg gaa tac 344Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe
Met Glu Tyr 5 10 15atc ggc agc caa aac gcc tcc cgg gga agg cgc cag
cga aga atg cat 392Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln
Arg Arg Met His 20 25 30cct aac gtt agt caa ggc tgc caa gga ggc tgt
gca aca tgc tca gat 440Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys
Ala Thr Cys Ser Asp35 40 45 50tac aat gga tgt ttg tca tgt aag ccc
aga cta ttt ttt gct ctg gaa 488Tyr Asn Gly Cys Leu Ser Cys Lys Pro
Arg Leu Phe Phe Ala Leu Glu 55 60 65aga att ggc atg aag cag att gga
gta tgt ctc tct tca tgt cca agt 536Arg Ile Gly Met Lys Gln Ile Gly
Val Cys Leu Ser Ser Cys Pro Ser 70 75 80gga tat tat gga act cga tat
cca gat ata aat aag tgt aca aaa tgc 584Gly Tyr Tyr Gly Thr Arg Tyr
Pro Asp Ile Asn Lys Cys Thr Lys Cys 85 90 95aaa gct gac tgt gat acc
tgt ttc aac aaa aat ttc tgc aca aaa tgt 632Lys Ala Asp Cys Asp Thr
Cys Phe Asn Lys Asn Phe Cys Thr Lys Cys 100 105 110aaa agt gga ttt
tac tta cac ctt gga aag tgc ctt gac aat tgc cca 680Lys Ser Gly Phe
Tyr Leu His Leu Gly Lys Cys Leu Asp Asn Cys Pro115 120 125 130gaa
ggg ttg gaa gcc aac aac cat act atg gag tgt gtc agt att gtg 728Glu
Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser Ile Val 135 140
145cac tgt gag gtc agt gaa tgg aat cct tgg agt cca tgc acg aag aag
776His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr Lys Lys
150 155 160gga aaa aca tgt ggc ttc aaa aga ggg act gaa aca cgg gtc
cga gaa 824Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val
Arg Glu 165 170 175ata ata cag cat cct tca gca aag ggt aac cta tgt
ccc cca aca aat 872Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys
Pro Pro Thr Asn 180 185 190gag aca aga aag tgt aca gtg caa agg aag
aag tgt cag aag gga gaa 920Glu Thr Arg Lys Cys Thr Val Gln Arg Lys
Lys Cys Gln Lys Gly Glu195 200 205 210cga gga aaa aaa gga agg gag
agg aaa aga aaa aaa cct aat aaa gga 968Arg Gly Lys Lys Gly Arg Glu
Arg Lys Arg Lys Lys Pro Asn Lys Gly 215 220 225gaa agt aaa gaa gca
ata cct gac agc aaa agt ctg gaa tcc agc aaa 1016Glu Ser Lys Glu Ala
Ile Pro Asp Ser Lys Ser Leu Glu Ser Ser Lys 230 235 240gaa atc cca
gag caa cga gaa aac aaa cag cag cag aag aag cga aaa 1064Glu Ile Pro
Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys Arg Lys 245 250 255gtc
caa gat aaa cag aaa tcg gta tca gtc agc act gta cac tag 1109Val Gln
Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265
270agggttccat gagattattg tagactcatg atgctgctat ctcaaccaga
tgcccaggac 1169aggtgctcta gccattagga ccacaaatgg acatgtcagt
tattgctctg tctaaacaac 1229attcccagta gttgctatat tcttcataca
agcatagtta acaacaaaga gccaaaagat 1289caaagaaggg atactttcag
atggttgtct tgtgtgcttc tctgcatttt taaa 134310272PRTHomo sapiens
10Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met1
5 10 15Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg
Arg 20 25 30Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala
Thr Cys 35 40 45Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu
Phe Phe Ala 50 55 60Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys
Leu Ser Ser Cys65 70 75 80Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro
Asp Ile Asn Lys Cys Thr 85 90 95Lys Cys Lys Ala Asp Cys Asp Thr Cys
Phe Asn Lys Asn Phe Cys Thr 100 105 110Lys Cys Lys Ser Gly Phe Tyr
Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125Cys Pro Glu Gly Leu
Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140Ile Val His
Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr145 150 155
160Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val
165 170 175Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys
Pro Pro 180 185 190Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys
Lys Cys Gln Lys 195 200 205Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg
Lys Arg Lys Lys Pro Asn 210 215 220Lys Gly Glu Ser Lys Glu Ala Ile
Pro Asp Ser Lys Ser Leu Glu Ser225 230 235 240Ser Lys Glu Ile Pro
Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250 255Arg Lys Val
Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265
27011819DNAHomo sapiens 11atgcacttgc gactgatttc ttggcttttt
atcattttga actttatgga atacatcggc 60agccaaaacg cctcccgggg aaggcgccag
cgaagaatgc atcctaacgt tagtcaaggc 120tgccaaggag gctgtgcaac
atgctcagat tacaatggat gtttgtcatg taagcccaga 180ctattttttg
ctctggaaag aattggcatg aagcagattg gagtatgtct ctcttcatgt
240ccaagtggat attatggaac tcgatatcca gatataaata agtgtacaaa
atgcaaagct 300gactgtgata cctgtttcaa caaaaatttc tgcacaaaat
gtaaaagtgg attttactta 360caccttggaa agtgccttga caattgccca
gaagggttgg aagccaacaa ccatactatg 420gagtgtgtca gtattgtgca
ctgtgaggtc agtgaatgga atccttggag tccatgcacg 480aagaagggaa
aaacatgtgg cttcaaaaga gggactgaaa cacgggtccg agaaataata
540cagcatcctt cagcaaaggg taacctatgt cccccaacaa atgagacaag
aaagtgtaca 600gtgcaaagga agaagtgtca gaagggagaa cgaggaaaaa
aaggaaggga gaggaaaaga 660aaaaaaccta ataaaggaga aagtaaagaa
gcaatacctg acagcaaaag tctggaatcc 720agcaaagaaa tcccagagca
acgagaaaac aaacagcagc agaagaagcg aaaagtccaa 780gataaacaga
aatcggtatc agtcagcact gtacactag 81912822DNAHomo
sapiensCDS(1)..(822) 12atg ggt cac ttg cga ctg att tct tgg ctt ttt
atc att ttg aac ttt 48Met Gly His Leu Arg Leu Ile Ser Trp Leu Phe
Ile Ile Leu Asn Phe1 5 10 15atg gaa tac atc ggc agc caa aac gcc tcc
cgg gga agg cgc cag cga 96Met Glu Tyr Ile Gly Ser Gln Asn Ala Ser
Arg Gly Arg Arg Gln Arg 20 25 30aga atg cat cct aac gtt agt caa ggc
tgc caa gga ggc tgt gca aca 144Arg Met His Pro Asn Val Ser Gln Gly
Cys Gln Gly Gly Cys Ala Thr 35 40 45tgc tca gat tac aat gga tgt ttg
tca tgt aag ccc aga cta ttt ttt 192Cys Ser Asp Tyr Asn Gly Cys Leu
Ser Cys Lys Pro Arg Leu Phe Phe 50 55 60gct ctg gaa aga att ggc atg
aag cag att gga gta tgt ctc tct tca 240Ala Leu Glu Arg Ile Gly Met
Lys Gln Ile Gly Val Cys Leu Ser Ser65 70 75 80tgt cca agt gga tat
tat gga act cga tat cca gat ata aat aag tgt 288Cys Pro Ser Gly Tyr
Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys 85 90 95aca aaa tgc aaa
gct gac tgt gat acc tgt ttc aac aaa aat ttc tgc 336Thr Lys Cys Lys
Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys 100 105 110aca aaa
tgt aaa agt gga ttt tac tta cac ctt gga aag tgc ctt gac 384Thr Lys
Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp 115 120
125aat tgc cca gaa ggg ttg gaa gcc aac aac cat act atg gag tgt gtc
432Asn Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val
130 135 140agt att gtg cac tgt gag gtc agt gaa tgg aat cct tgg agt
cca tgc 480Ser Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser
Pro Cys145 150 155 160acg aag aag gga aaa aca tgt ggc ttc aaa aga
ggg act gaa aca cgg 528Thr Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg
Gly Thr Glu Thr Arg 165 170 175gtc cga gaa ata ata cag cat cct tca
gca aag ggt aac cta tgt ccc 576Val Arg Glu Ile Ile Gln His Pro Ser
Ala Lys Gly Asn Leu Cys Pro 180 185 190cca aca aat gag aca aga aag
tgt aca gtg caa agg aag aag tgt cag 624Pro Thr Asn Glu Thr Arg Lys
Cys Thr Val Gln Arg Lys Lys Cys Gln 195 200 205aag gga gaa cga gga
aaa aaa gga agg gag agg aaa aga aaa aaa cct 672Lys Gly Glu Arg Gly
Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro 210 215 220aat aaa gga
gaa agt aaa gaa gca ata cct gac agc aaa agt ctg gaa 720Asn Lys Gly
Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu225 230 235
240tcc agc aaa gaa atc cca gag caa cga gaa aac aaa cag cag cag aag
768Ser Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys
245 250 255aag cga aaa gtc caa gat aaa cag aaa tcg gta tca gtc agc
act gta 816Lys Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser
Thr Val 260 265 270cac tag 822His13273PRTHomo sapiens 13Met Gly His
Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe1 5 10 15Met Glu
Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg 20 25 30Arg
Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr 35 40
45Cys Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe
50 55 60Ala Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser
Ser65 70 75 80Cys Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile
Asn Lys Cys 85 90 95Thr Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn
Lys Asn Phe Cys 100 105 110Thr Lys Cys Lys Ser Gly Phe Tyr Leu His
Leu Gly Lys Cys Leu Asp 115 120 125Asn Cys Pro Glu Gly Leu Glu Ala
Asn Asn His Thr Met Glu Cys Val 130 135 140Ser Ile Val His Cys Glu
Val Ser Glu Trp Asn Pro Trp Ser Pro Cys145 150 155 160Thr Lys Lys
Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg 165 170 175Val
Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro 180 185
190Pro Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln
195 200 205Lys Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys
Lys Pro 210 215 220Asn Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser
Lys Ser Leu Glu225 230 235 240Ser Ser Lys Glu Ile Pro Glu Gln Arg
Glu Asn Lys Gln Gln Gln Lys 245 250 255Lys Arg Lys
Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val 260 265
270His14160PRTHomo sapiens 14Cys Thr Lys Cys Lys Ala Asp Cys Asp
Thr Cys Phe Asn Lys Asn Phe1 5 10 15Cys Thr Lys Cys Lys Ser Gly Phe
Tyr Leu His Leu Gly Lys Cys Leu 20 25 30Asp Asn Cys Pro Glu Gly Leu
Glu Ala Asn Asn His Thr Met Glu Cys 35 40 45Val Ser Ile Val His Cys
Glu Val Ser Glu Trp Asn Pro Trp Ser Pro 50 55 60Cys Thr Lys Lys Gly
Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr65 70 75 80Arg Val Arg
Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys 85 90 95Pro Pro
Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys 100 105
110Gln Lys Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys
115 120 125Pro Asn Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys
Ser Leu 130 135 140Glu Ser Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn
Lys Gln Gln Gln145 150 155 1601521PRTHomo sapiens 15Met His Leu Arg
Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met1 5 10 15Glu Tyr Ile
Gly Ser 2016251PRTHomo sapiens 16Gln Asn Ala Ser Arg Gly Arg Arg
Gln Arg Arg Met His Pro Asn Val1 5 10 15Ser Gln Gly Cys Gln Gly Gly
Cys Ala Thr Cys Ser Asp Tyr Asn Gly 20 25 30Cys Leu Ser Cys Lys Pro
Arg Leu Phe Phe Ala Leu Glu Arg Ile Gly 35 40 45Met Lys Gln Ile Gly
Val Cys Leu Ser Ser Cys Pro Ser Gly Tyr Tyr 50 55 60Gly Thr Arg Tyr
Pro Asp Ile Asn Lys Cys Thr Lys Cys Lys Ala Asp65 70 75 80Cys Asp
Thr Cys Phe Asn Lys Asn Phe Cys Thr Lys Cys Lys Ser Gly 85 90 95Phe
Tyr Leu His Leu Gly Lys Cys Leu Asp Asn Cys Pro Glu Gly Leu 100 105
110Glu Ala Asn Asn His Thr Met Glu Cys Val Ser Ile Val His Cys Glu
115 120 125Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr Lys Lys Gly
Lys Thr 130 135 140Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val Arg
Glu Ile Ile Gln145 150 155 160His Pro Ser Ala Lys Gly Asn Leu Cys
Pro Pro Thr Asn Glu Thr Arg 165 170 175Lys Cys Thr Val Gln Arg Lys
Lys Cys Gln Lys Gly Glu Arg Gly Lys 180 185 190Lys Gly Arg Glu Arg
Lys Arg Lys Lys Pro Asn Lys Gly Glu Ser Lys 195 200 205Glu Ala Ile
Pro Asp Ser Lys Ser Leu Glu Ser Ser Lys Glu Ile Pro 210 215 220Glu
Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys Arg Lys Val Gln Asp225 230
235 240Lys Gln Lys Ser Val Ser Val Ser Thr Val His 245
2501723PRTHomo sapiens 17Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn
Phe Cys Thr Lys Cys Lys1 5 10 15Ser Gly Phe Tyr Leu His Leu
201846PRTHomo sapiens 18Ile Asn Lys Cys Thr Lys Cys Lys Ala Asp Cys
Asp Thr Cys Phe Asn1 5 10 15Lys Asn Phe Cys Thr Lys Cys Lys Ser Gly
Phe Tyr Leu His Leu Gly 20 25 30Lys Cys Leu Asp Asn Cys Pro Glu Gly
Leu Glu Ala Asn Asn 35 40 451920PRTHomo sapiens 19Met His Pro Asn
Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys1 5 10 15Ser Asp Tyr
Asn 202037PRTHomo sapiens 20Ile Val His Cys Glu Val Ser Glu Trp Asn
Pro Trp Ser Pro Cys Thr1 5 10 15Lys Lys Gly Lys Thr Cys Gly Phe Lys
Arg Gly Thr Glu Thr Arg Val 20 25 30Arg Glu Ile Ile Gln
352110PRTHomo sapiens 21Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys1 5
102242PRTHomo sapiens 22Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys
Gly Glu Arg Gly Lys1 5 10 15Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro
Asn Lys Gly Glu Ser Lys 20 25 30Glu Ala Ile Pro Asp Ser Lys Ser Leu
Glu 35 402314PRTHomo sapiens 23Thr Cys Phe Asn Lys Asn Phe Cys Thr
Lys Cys Lys Ser Gly1 5 102420PRTHomo sapiens 24Cys Glu Val Ser Glu
Trp Asn Pro Trp Ser Pro Cys Thr Lys Lys Gly1 5 10 15Lys Thr Cys Gly
2025229PRTMus musculus 25Val Gly Ser Arg Gly Ile Lys Gly Lys Arg
Gln Arg Arg Ile Ser Ala1 5 10 15Glu Gly Ser Gln Ala Cys Ala Lys Gly
Cys Glu Leu Cys Ser Glu Val 20 25 30Asn Gly Cys Leu Lys Cys Ser Pro
Lys Leu Phe Ile Leu Leu Glu Arg 35 40 45Asn Asp Ile Arg Gln Val Gly
Val Cys Leu Pro Ser Cys Pro Pro Gly 50 55 60Tyr Phe Asp Ala Arg Asn
Pro Asp Met Asn Lys Cys Ile Lys Cys Lys65 70 75 80Ile Glu His Cys
Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys 85 90 95Gln Glu Ala
Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro 100 105 110Glu
Gly Ser Thr Ala Ala Asn Ser Thr Met Glu Cys Gly Ser Pro Ala 115 120
125Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys
130 135 140Arg Lys Leu Cys Gly Phe Arg Lys Gly Ser Glu Glu Arg Thr
Arg Arg145 150 155 160Val Leu His Ala Pro Gly Gly Asp His Thr Thr
Cys Ser Asp Thr Lys 165 170 175Glu Thr Arg Lys Cys Thr Val Arg Arg
Thr Pro Cys Pro Glu Gly Gln 180 185 190Lys Arg Arg Lys Gly Gly Gln
Gly Arg Arg Glu Asn Ala Asn Arg His 195 200 205Pro Ala Arg Lys Asn
Ser Lys Glu Pro Arg Ser Asn Ser Arg Arg His 210 215 220Lys Gly Gln
Gln Gln22526265PRTHomo sapiens 26Met His Leu Arg Leu Ile Ser Trp
Leu Phe Ile Ile Leu Asn Phe Met1 5 10 15Glu Tyr Ile Gly Ser Gln Asn
Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30Met His Pro Asn Val Ser
Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45Ser Asp Tyr Asn Gly
Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60Leu Glu Arg Ile
Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys65 70 75 80Pro Ser
Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95Lys
Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105
110Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn
115 120 125Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys
Val Ser 130 135 140Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp
Ser Pro Cys Thr145 150 155 160Lys Lys Gly Lys Thr Cys Gly Phe Lys
Arg Gly Thr Glu Thr Arg Val 165 170 175Arg Glu Ile Ile Gln His Pro
Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190Thr Asn Glu Thr Arg
Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205Gly Glu Arg
Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220Lys
Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser225 230
235 240Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys
Lys 245 250 255Arg Lys Val Gln Asp Lys Gln Lys Ser 260
265278PRTHomo sapiens 27Ser Val Ser Val Ser Thr Val His 1
5287PRTHomo sapiens 28Val Ser Val Ser Thr Val His 1 52927PRTHomo
sapiens 29Gly Ile Glu Val Thr Leu Ala Glu Gly Leu Thr Ser Val Ser
Gln Arg 1 5 10 15Thr Gln Pro Thr Pro Cys Arg Arg Arg Tyr Leu 20
253030DNAArtificial SequenceDescription of Artificial Sequence PCR
primer 30ctcgggaaga agcgcgccat ttgtgttggt 30312384DNAMus
musculusCDS(511)..(1347)misc_feature(2367)..(2367)n = A, T, G, or C
31ggagcggctc ctgctcagaa cgccagaagc agctcgggtc tctccagcgc cccttgacca
60tggctgcggt acccacggcg tccgcttccc tgcgctcccg gggtccctgc cacagccgca
120gccgctgcag cctctgagcc ccaggggcca ctgctcgcct ggattccgcc
cgcagccgcc 180gctgctgtgc aaccgaggct aacctgcggc cagccaggag
gctcctgcaa ccttcgctcg 240cggcgatgac agccacccca gagcagccgg
ctgtgttcgg acaatttgag aatgcaattg 300ttggtttccc ggtccacccg
tcccgcttcg cttgccatca cagcacgcct gttggatctc 360agtggagaag
tcccgctgct ctggtttttc tactcttcgt atagactcgc ctaacaccta
420catacatatt tttctttaaa aaaaaacatt aaatataact aacagtgaaa
agaaaaagga 480gagaaaaaag ggaaacatta cagggttact atg cac ttg cga ctg
att tct tgt 534 Met His Leu Arg Leu Ile Ser Cys 1 5ttt ttt atc att
ttg aac ttt atg gaa tac att ggc agc caa aac gcc 582Phe Phe Ile Ile
Leu Asn Phe Met Glu Tyr Ile Gly Ser Gln Asn Ala 10 15 20tcc cga gga
agg cgc cag cga aga atg cat cct aat gtc agt caa ggc 630Ser Arg Gly
Arg Arg Gln Arg Arg Met His Pro Asn Val Ser Gln Gly 25 30 35 40tgc
caa gga ggc tgt gca acg tgt tca gat tac aat ggc tgt ttg tca 678Cys
Gln Gly Gly Cys Ala Thr Cys Ser Asp Tyr Asn Gly Cys Leu Ser 45 50
55tgt aag ccc aga ctg ttt ttt gtt ctg gaa agg att ggc atg aag cag
726Cys Lys Pro Arg Leu Phe Phe Val Leu Glu Arg Ile Gly Met Lys Gln
60 65 70ata gga gtg tgt ctc tct tcg tgt cca agt gga tat tac gga act
cga 774Ile Gly Val Cys Leu Ser Ser Cys Pro Ser Gly Tyr Tyr Gly Thr
Arg 75 80 85tat cca gat ata aat aaa tgt aca aaa tgc aaa gtt gac tgt
gat acc 822Tyr Pro Asp Ile Asn Lys Cys Thr Lys Cys Lys Val Asp Cys
Asp Thr 90 95 100tgt ttc aac aaa aat ttc tgc aca aag tgt aaa agt
gga ttt tac tta 870Cys Phe Asn Lys Asn Phe Cys Thr Lys Cys Lys Ser
Gly Phe Tyr Leu105 110 115 120cac ctt gga aag tgc ctt gac agt tgc
cca gaa ggg tta gaa gcc aac 918His Leu Gly Lys Cys Leu Asp Ser Cys
Pro Glu Gly Leu Glu Ala Asn 125 130 135aat cat act atg gaa tgt gtc
agt att gta cac tgt gag gcc agt gaa 966Asn His Thr Met Glu Cys Val
Ser Ile Val His Cys Glu Ala Ser Glu 140 145 150tgg agt cca tgg agt
cca tgt atg aag aaa gga aaa aca tgt ggc ttc 1014Trp Ser Pro Trp Ser
Pro Cys Met Lys Lys Gly Lys Thr Cys Gly Phe 155 160 165aaa agg ggg
act gaa aca cgg gtc cga gat ata cta cag cat cct tca 1062Lys Arg Gly
Thr Glu Thr Arg Val Arg Asp Ile Leu Gln His Pro Ser 170 175 180gcc
aag ggt aag ggt aac ctg tgc ccc cca acc agc gag aca aga act 1110Ala
Lys Gly Lys Gly Asn Leu Cys Pro Pro Thr Ser Glu Thr Arg Thr185 190
195 200tgt ata gta caa aga aag aag tgt tca aag gga gag cga gga aaa
aag 1158Cys Ile Val Gln Arg Lys Lys Cys Ser Lys Gly Glu Arg Gly Lys
Lys 205 210 215gga aga gag aga aaa cga aaa aaa ctg aat aaa gaa gaa
aga aag gaa 1206Gly Arg Glu Arg Lys Arg Lys Lys Leu Asn Lys Glu Glu
Arg Lys Glu 220 225 230aca agc tcc tcc tct gac agc aaa ggt ttg gag
tcc agc att gag acc 1254Thr Ser Ser Ser Ser Asp Ser Lys Gly Leu Glu
Ser Ser Ile Glu Thr 235 240 245cca gac cag cag gaa aac aaa gag agg
cag cag cag cag aag aga aga 1302Pro Asp Gln Gln Glu Asn Lys Glu Arg
Gln Gln Gln Gln Lys Arg Arg 250 255 260gcc cga gac aag caa cag aaa
tcg gta tca gtc agc act gta cac 1347Ala Arg Asp Lys Gln Gln Lys Ser
Val Ser Val Ser Thr Val His265 270 275tagagggtcc tgcgaggtta
ctgtagactc atgatgctgc tatctcaacc agatgtccag 1407gacaggtgtt
ctagccatta gaaccacaaa tggacaacac atcagttacc actctgtcta
1467aacaacattc ctaatagttg ctatattctt catacaaaca tagtaaacag
caaagagcca 1527aatgttcaaa gaagggatac tttcagatgg ttatcttatg
tgcttctgtg tatttttaaa 1587agatgagaaa atttgtacat aattatcaat
aagctataag atatcctcaa tgtaatgacg 1647acagctggac aagaatcatc
ttttctttat aaaaaaatta ttcttcgaat aattgtcttt 1707aagaagcaaa
aggtaattct gcaacttcaa aaatgcagtg tccctcaaaa ccaagatttg
1767tcaggggaga gaatcatggc tccatgtaca gggtggattt gtcccggaga
actagtgaat 1827gctcagaatt agggcctggc attttgaatc ctagagttaa
tcatcacaga agcaagtggt 1887ttaggattgc ttcggttgcc ctcctctgca
agaaactgaa catgcataat agagttaaat 1947atattgtgtg gagttggaat
aaggcaagct gtggaagaaa tcatagagct ggagaccatc 2007ttgtgctttc
cagaaccgtg aggggttttg gtcacctgga acagggctcc aatctatatt
2067agcactgtgt ggttgatctt ccactactcc ttggtttata taagtctgta
aacatgtacc 2127tgtacctttc ttccaaaagt aaaaccatac ttactagaag
aaaattctaa ctttatggaa 2187aacaaaagtg taagaagaat gtgacatgtt
tgcaaagttg agtgttttct ttctgaaatg 2247aggggaaaac tattttatta
cctgcctatg ggtccacctg gaactaaagg gatactactt 2307tctaacaagg
tgtatctagt aggagagaaa gccaccacaa taaatatatt tgttaatagn
2367taaaaaaaaa aaaaaaa 238432279PRTMus musculus 32Met His Leu Arg
Leu Ile Ser Cys Phe Phe Ile Ile Leu Asn Phe Met 1 5 10 15Glu Tyr
Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30Met
His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40
45Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Val
50 55 60Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser
Cys 65 70 75 80Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn
Lys Cys Thr 85 90 95Lys Cys Lys Val Asp Cys Asp Thr Cys Phe Asn Lys
Asn Phe Cys Thr 100 105 110Lys Cys Lys Ser Gly Phe Tyr Leu His Leu
Gly Lys Cys Leu Asp Ser 115 120 125Cys Pro Glu Gly Leu Glu Ala Asn
Asn His Thr Met Glu Cys Val Ser 130 135 140Ile Val His Cys Glu Ala
Ser Glu Trp Ser Pro Trp Ser Pro Cys Met145 150 155 160Lys Lys Gly
Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175Arg
Asp Ile Leu Gln His Pro Ser Ala Lys Gly Lys Gly Asn Leu Cys 180 185
190Pro Pro Thr Ser Glu Thr Arg Thr Cys Ile Val Gln Arg Lys Lys Cys
195 200 205Ser Lys Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg
Lys Lys 210 215 220Leu Asn Lys Glu Glu Arg Lys Glu Thr Ser Ser Ser
Ser Asp Ser Lys225 230 235 240Gly Leu Glu Ser Ser Ile Glu Thr Pro
Asp Gln Gln Glu Asn Lys Glu 245 250 255Arg Gln Gln Gln Gln Lys Arg
Arg Ala Arg Asp Lys Gln Gln Lys Ser 260 265 270Val Ser Val Ser Thr
Val His 275332101DNAHomo sapiensCDS(259)..(1074) 33tcgcggcgat
gccagccacc ccagcgaagc cgccgcagtt cagtgcttgg ataatttgaa 60agtacaatag
ttggtttccc tgtccacccg ccccacttcg cttgccatca cagcacgcct
120atcggatgtg agaggagaag tcccgctgct cgggcactgt ctatatacgc
ctaacaccta 180catatatttt aaaaacatta aatataatta acaatcaaaa
gaaagaggag aaaggaaggg 240aagcattact gggttact atg cac ttg cga ctg
att tct tgg ctt ttt atc 291 Met His Leu Arg Leu Ile Ser Trp Leu Phe
Ile 1 5 10att ttg aac ttt atg gaa tac atc ggc agc caa aac gcc tcc
cgg gga 339Ile Leu Asn Phe Met Glu Tyr Ile Gly Ser Gln Asn Ala Ser
Arg Gly 15 20 25agg cgc cag cga aga atg cat cct aac gtt agt caa ggc
tgc caa gga 387Arg Arg Gln Arg Arg Met His Pro Asn Val Ser Gln Gly
Cys Gln Gly 30 35 40ggc tgt gca aca tgc tca gat tac aat gga tgt ttg
tca tgt aag ccc 435Gly Cys Ala Thr Cys Ser Asp Tyr Asn Gly Cys Leu
Ser Cys Lys Pro 45 50 55aga cta ttt ttt gct ctg gaa aga att ggc atg
aag cag att gga gta 483Arg Leu Phe Phe Ala Leu Glu Arg Ile Gly Met
Lys Gln Ile Gly Val 60 65 70 75tgt ctc tct tca tgt cca agt gga tat
tat gga act cga tat cca gat 531Cys Leu Ser Ser Cys Pro Ser Gly Tyr
Tyr Gly Thr Arg Tyr Pro Asp 80 85 90ata aat aag tgt aca aaa tgc aaa
gct gac
tgt gat acc tgt ttc aac 579Ile Asn Lys Cys Thr Lys Cys Lys Ala Asp
Cys Asp Thr Cys Phe Asn 95 100 105aaa aat ttc tgc aca aaa tgt aaa
agt gga ttt tac tta cac ctt gga 627Lys Asn Phe Cys Thr Lys Cys Lys
Ser Gly Phe Tyr Leu His Leu Gly 110 115 120aag tgc ctt gac aat tgc
cca gaa ggg ttg gaa gcc aac aac cat act 675Lys Cys Leu Asp Asn Cys
Pro Glu Gly Leu Glu Ala Asn Asn His Thr 125 130 135atg gag tgt gtc
agt att gtg cac tgt gag gtc agt gaa tgg aat cct 723Met Glu Cys Val
Ser Ile Val His Cys Glu Val Ser Glu Trp Asn Pro140 145 150 155tgg
agt cca tgc acg aag aag gga aaa aca tgt ggc ttc aaa aga ggg 771Trp
Ser Pro Cys Thr Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly 160 165
170act gaa aca cgg gtc cga gaa ata ata cag cat cct tca gca aag ggt
819Thr Glu Thr Arg Val Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly
175 180 185aac cta tgt ccc cca aca aat gag aca aga aag tgt aca gtg
caa agg 867Asn Leu Cys Pro Pro Thr Asn Glu Thr Arg Lys Cys Thr Val
Gln Arg 190 195 200aag aag tgt cag aag gga gaa cga gga aaa aaa gga
agg gag agg aaa 915Lys Lys Cys Gln Lys Gly Glu Arg Gly Lys Lys Gly
Arg Glu Arg Lys 205 210 215aga aaa aaa cct aat aaa gga gaa agt aaa
gaa gca ata cct gac agc 963Arg Lys Lys Pro Asn Lys Gly Glu Ser Lys
Glu Ala Ile Pro Asp Ser220 225 230 235aaa agt ctg gaa tcc agc aaa
gaa atc cca gag caa cga gaa aac aaa 1011Lys Ser Leu Glu Ser Ser Lys
Glu Ile Pro Glu Gln Arg Glu Asn Lys 240 245 250cag cag cag aag aag
cga aaa gtc caa gat aaa cag aaa tcg gta tca 1059Gln Gln Gln Lys Lys
Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser 255 260 265gtc agc act
gta cac tagagggttc catgagatta ttgtagactc atgatgctgc 1114Val Ser Thr
Val His 270tatctcaacc agatgcccag gacaggtgct ctagccatta ggaccacaaa
tggacatgtc 1174agttattgct ctgtctaaac aacattccca gtagttgcta
tattcttcat acaagcatag 1234ttaacaacaa agagccaaaa gatcaaagaa
gggatacttt cagatggttg tcttgtgtgc 1294ttctctgcat ttttaaaaga
caagacattc ttgtacatat tatcaatagg ctataagatg 1354taacaacgaa
atgatgacat ctggagaaga aacatctttt ccttataaaa atgtgttttc
1414aagctgttgt tttaagaagc aaaagatagt tctgcaaatt caaagataca
gtatcccttc 1474aaaacaaata ggagttcagg gaagagaaac atccttcaaa
ggacagtgtt gttttgaccg 1534ggagatctag agagtgctca gaattagggc
ctggcatttg gaatcacagg atttatcatc 1594acagaaacaa ctgttttaag
attagttcca tcactctcat cctgtatttt tataagaaac 1654acaagagtgc
ataccagaat tgaatatacc atatgggatt ggagaaagac aaatgtggaa
1714gaaatcatag agctggagac tacttttgtg ctttacaaaa ctgtgaagga
ttgtggtcac 1774ctggaacagg tctccaatct atgttagcac tatgtggctc
agcctctgtt accccttgga 1834ttatatatca acctgtaaac atgtgcctgt
aacttacttc caaaaacaaa atcatactta 1894ttagaagaaa attctgattt
tatagaaaaa aaatagagca aggagaatat aacatgtttg 1954caaagtcatg
tgttttcttt ctcaatgagg gaaaaacaat tttattacct gcttaatggt
2014ccacctggaa ctaaaaggga tactattttc taacaaggta tatctagtag
gggagaaagc 2074caccacaata aatatatttg ttaatag 210134272PRTHomo
sapiens 34Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn
Phe Met 1 5 10 15Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg
Arg Gln Arg Arg 20 25 30Met His Pro Asn Val Ser Gln Gly Cys Gln Gly
Gly Cys Ala Thr Cys 35 40 45Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys
Pro Arg Leu Phe Phe Ala 50 55 60Leu Glu Arg Ile Gly Met Lys Gln Ile
Gly Val Cys Leu Ser Ser Cys 65 70 75 80Pro Ser Gly Tyr Tyr Gly Thr
Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95Lys Cys Lys Ala Asp Cys
Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110Lys Cys Lys Ser
Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125Cys Pro
Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135
140Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys
Thr145 150 155 160Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr
Glu Thr Arg Val 165 170 175Arg Glu Ile Ile Gln His Pro Ser Ala Lys
Gly Asn Leu Cys Pro Pro 180 185 190Thr Asn Glu Thr Arg Lys Cys Thr
Val Gln Arg Lys Lys Cys Gln Lys 195 200 205Gly Glu Arg Gly Lys Lys
Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220Lys Gly Glu Ser
Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser225 230 235 240Ser
Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250
255Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His
260 265 2703521DNAArtificial SequenceDescription of Artificial
Sequence PCR primer 35agtacaaaga aagaagtgtt c 213621DNAArtificial
SequenceDescription of Artificial Sequence PCR primer 36tgagtctaca
gtaacctcgc a 213720DNAArtificial SequenceDescription of Artificial
Sequence PCR primer 37taatacgact cactataggg 203824DNAArtificial
SequenceDescription of Artificial Sequence PCR primer 38tcgcggcgat
gccagccacc ccag 243930DNAArtificial SequenceDescription of
Artificial Sequence PCR primer 39agcacgccta tcggatgtga gaggagaagt
304030DNAArtificial SequenceDescription of Artificial Sequence PCR
primer 40ctattaacaa atatatttat tgtggtggct 304130DNAArtificial
SequenceDescription of Artificial Sequence PCR primer 41tggtggcttt
ctcccctact agatatacct 304220DNAArtificial SequenceDescription of
Artificial Sequence PCR primer 42gattttaggt gacactatag
204334DNAArtificial SequenceDescription of Artificial Sequence PCR
primer 43ccgctcgagc caccatgcac ttgcgactga tttc 344429DNAArtificial
SequenceDescription of Artificial Sequence PCR primer 44attgaattcc
tagtgtacag tgctgactg 294584DNAHomo sapiensDescription of Artificial
Sequence PCR primer 45ggg att gaa gtc acc cta gct gaa ggc ctc acc
agt gtt tca cag agg 48Gly Ile Glu Val Thr Leu Ala Glu Gly Leu Thr
Ser Val Ser Gln Arg 1 5 10 15aca cag ccc acc cct tgc agg agg agg
tat ctc tga 84Thr Gln Pro Thr Pro Cys Arg Arg Arg Tyr Leu 20
254627PRTHomo sapiens 46Gly Ile Glu Val Thr Leu Ala Glu Gly Leu Thr
Ser Val Ser Gln Arg 1 5 10 15Thr Gln Pro Thr Pro Cys Arg Arg Arg
Tyr Leu 20 25471436DNAHomo sapiens 47cccggcggct cctggaaccc
cggttcgcgg cgatgccagc caccccagcg aagccgccgc 60agttcagtgc ttggataatt
tgaaagtaca atagttggtt tccctgtcca cccgccccac 120ttcgcttgcc
atcacagcac gcctatcgga tgtgagagga gaagtcccgc tgctcgggca
180ctgtctatat acgcctaaca cctacatata ttttaaaaac attaaatata
attaacaatc 240aaaagaaaga ggagaaagga agggaagcat tactgggtta
ctatgcactt gcgactgatt 300tcttggcttt ttatcatttt gaactttatg
gaatacatcg gcagccaaaa cgcctcccgg 360ggaaggcgcc agcgaagaat
gcatcctaac gttagtcaag gctgccaagg aggctgtgca 420acatgctcag
attacaatgg atgtttgtca tgtaagccca gactattttt tgctctggaa
480agaattggca tgaagcagat tggagtatgt ctctcttcat gtccaagtgg
atattatgga 540actcgatatc cagatataaa taagtgtaca aaatgcaaag
ctgactgtga tacctgtttc 600aacaaaaatt tctgcacaaa atgtaaaagt
ggattttact tacaccttgg aaagtgcctt 660gacaattgcc cagaagggtt
ggaagccaac aaccatacta tggagtgtgt cagtattgtg 720cactgtgagg
tcagtgaatg gaatccttgg agtccatgca cgaagaaggg aaaaacatgt
780ggcttcaaaa gagggactga aacacgggtc cgagaaataa tacagcatcc
ttcagcaaag 840ggtaacctgt gtcccccaac aaatgagaca agaaagtgta
cagtgcaaag gaagaagtgt 900cagaagggag aacgaggaaa aaaaggaagg
gagaggaaaa gaaaaaaacc taataaagga 960gaaagtaaag aagcaatacc
tgacagcaaa agtctggaat ccagcaaaga aatcccagag 1020caacgagaaa
acaaacagca gcagaagaag cgaaaagtcc aagataaaca gaaatcgggg
1080attgaagtca ccctagctga aggcctcacc agtgtttcac agaggacaca
gcccacccct 1140tgcaggagga ggtatctctg agtgtgcagc acagaatcgc
atgacccacc ttaaccttcc 1200tgttgtcatg gaaggatgca cggctgctct
gtccactgtg attcctagcc ctctcaagat 1260cactgctttc tgaagaattt
gcaatgactc tggcttctgg ctgcttatct ctggacaccc 1320gttctccacc
agttgtacag ttcatgtaat ctacttggct taattgattt tccacttctc
1380tcttcctctt ctaagatata aacattttaa atgatttaaa aaaaaaaaaa aaaaaa
143648292PRTHomo sapiens 48Met His Leu Arg Leu Ile Ser Trp Leu Phe
Ile Ile Leu Asn Phe Met 1 5 10 15Glu Tyr Ile Gly Ser Gln Asn Ala
Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30Met His Pro Asn Val Ser Gln
Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45Ser Asp Tyr Asn Gly Cys
Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60Leu Glu Arg Ile Gly
Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80Pro Ser Gly
Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95Lys Cys
Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105
110Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn
115 120 125Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys
Val Ser 130 135 140Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp
Ser Pro Cys Thr145 150 155 160Lys Lys Gly Lys Thr Cys Gly Phe Lys
Arg Gly Thr Glu Thr Arg Val 165 170 175Arg Glu Ile Ile Gln His Pro
Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190Thr Asn Glu Thr Arg
Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205Gly Glu Arg
Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220Lys
Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser225 230
235 240Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys
Lys 245 250 255Arg Lys Val Gln Asp Lys Gln Lys Ser Gly Ile Glu Val
Thr Leu Ala 260 265 270Glu Gly Leu Thr Ser Val Ser Gln Arg Thr Gln
Pro Thr Pro Cys Arg 275 280 285Arg Arg Tyr Leu 290
* * * * *
References