Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of provisional application Ser. No.
60/072,134, filed January 22,1998, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
2 0 Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs 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
2 5 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
cloning
techniques, have advanced the state of the art by making available large
numbers of
3 0 DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 734 to nucleotide 1873;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cs756_2 deposited under accession
number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cs756 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cs756_2 deposited under accession number
ATCC 98636;
(g) a polynucleatide encoding a mature protein encoded by the cDNA
insert of clone cs756 2 deposited under accession number ATCC 98636;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:2;
2 5 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
{l) a polynucleotide that hybridizes under stringent conditions to any
3 0 one of the polynucleotides specified in {a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873; the nucleotide sequence of SEQ ID
NO:1
from nucleotide 1403 to nucleotide 1873; the nucleotide sequence of the full-
length
protein coding sequence of clone cs756_2 deposited under accession number ATCC
98636;
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or the nucleotide sequence of a mature protein coding sequence of clone
cs756_2
deposited under accession number ATCC 98636. In other preferred embodiments,
the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cs756 2 deposited under accession number ATCC 98636. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:2 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:2, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 185
to amino acid 194 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
2 0 (aa) SEQ ID N0:1, but excluding the poly(A) tail at the
3' end of SEQ ID NO:1; and
(ab) the nucleotide sequence of the cDNA insert of clone
cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
2 5 conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID NO:1; and
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(bb) the nucleotide sequence of the cDNA insert of clone
cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
{iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:1, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
NO:1 to
a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1 , but
excluding the
poly{A) tail at the 3' end of SEQ ID NO:1. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:1 from nucleotide 734 to nucleotide 1873, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide
734 to
nucleotide 1873. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, and extending contiguously from
a
2 0 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from
nucleotide 1403 to nucleotide 1873, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID NO:1 from nucleotide 1403 to nucleotide 1873.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 5 consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) fragments of the amino acid sequence of SEQ ID N0:2, each
fragment comprising eight consecutive amino acids of SEQ ID N0:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone
3 0 cs756 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
preferably
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comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:2, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:2 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 185 to amino acid 194 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 26 to nucleotide 1738;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ew150_1 deposited under accession
number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ew150_1 deposited under accession number
2 0 ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ew150_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 26 to nucleotide 1738; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 140 to nucleotide 1738; the nucleotide sequence of the full-length
protein
coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
or the
nucleotide sequence of a mature protein coding sequence of clone ew150_1
deposited
under accession number ATCC 98636. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ew150_1 deposited under accession number ATCC 98636. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino
acid
166. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:4
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:4, or a
polynucleotide encoding a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:4 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 280 to amino acid 289 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
2 0 Further embodiments of the invention provide isolated polynucleotides
produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
2 5 consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the
3' end of SEQ ID N0:3; and
(ab) the nucleotide sequence of the cDNA insert of clone
ew150_1 deposited under accession number ATCC 98636;
3 0 (ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
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(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:3, but excluding the poiy(A) tail at the
3' end of SEQ ID N0:3; and
(bb) the nucleotide sequence of the cDNA insert of clone
ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:3 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:3 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:3. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, and
extending
2 0 contiguously from a nucleotide sequence corresponding to the 5' end of
said sequence of
SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 26
to
nucleotide 1738. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
2 5 N0:3 from nucleotide 140 to nucleotide 1738, and extending contiguously
from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from
nucleotide 140 to nucleotide 1738, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID N0:3 from nucleotide 140 to nucleotide 1738.
In other embodiments, the present invention provides a composition comprising
3 0 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 108 to
amino acid 166;
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(c) fragments of the amino acid sequence of SEQ ID N0:4, each
fragment comprising eight consecutive amino acids of SEQ ID N0:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone
ew150_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid
sequence
of SEQ ID N0:4 from amino acid 108 to amino acid 166. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
ands of SEQ ID N0:4, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:4 having biological activity, the fragment comprising the amino acid
sequence from amino acid 280 to amino acid 289 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910;
2 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1920;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone gg894_13 deposited under accession
number ATCC 98636;
2 5 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone gg894_13 deposited under accession number
ATCC 98636;
3 0 {g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gg894_13 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
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(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynudeotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910; the nucleotide sequence of SEQ
ID N0:5
from nucleotide 1260 to nucleotide 1910; the nucleotide sequence of the full-
length
protein coding sequence of clone gg894_13 deposited under accession number
ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone
gg894_13
deposited under accession number ATCC 98636. In other preferred embodiments,
the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone gg894_13 deposited under accession number ATCC 98636. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:6 having
biological
2 0 activity, the fragment preferably comprising eight (more preferably
twenty, most
preferably thirty) consecutive amino acids of SEQ ID N0:6, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 130
to amino acid 139 of SEQ ID N0:6.
2 5 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:5.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:5, but excluding the poly(A) tail at the
3' end of SEQ ID N0:5; and
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(ab) the nucleotide sequence of the cDNA insert of clone
gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucieotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:5, but excluding the poly(A) tail at the
3' end of SEQ ID N0:5; and
(bb) the nucleotide sequence of the cDNA insert of clone
gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
2 0 Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:5 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide
isolated
2 5 according to the above process comprises a nucleotide sequence
corresponding to the
cDNA sequence of SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide
1101 to
3 0 nucleotide 1910. Also preferably the polynucleotide isolated according to
the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1910, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from
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nucleotide 1260 to nucleotide 1910, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID N0:5 from nucleotide 1260 to nucleotide 1910.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6, each
fragment comprising eight consecutive amino acids of SEQ ID N0:6; and
(c) the amino acid sequence encoded by the cDNA insert of clone
gg894_13 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
ands of SEQ ID N0:6, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:6 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 130 to amino acid I39 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an
2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102;
2 5 (c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone it217 2 deposited under accession
number
ATCC 98636;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
3 0 (e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone it217_2 deposited under accession number ATCC
98636;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone it217_2 deposited under accession number ATCC 98636;
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(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:8;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ; and
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102; the nucleotide sequence of the
full-length
protein coding sequence of clone it217 2 deposited under accession number ATCC
98636;
or the nucleotide sequence of a mature protein coding sequence of clone
it217_2 deposited
under accession number ATCC 98636. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone it217_2 deposited under accession number ATCC 98636. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
2 0 comprising a fragment of the amino acid sequence of SEQ ID N0:8 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:8, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8
having
biological activity, the fragment comprising the amino acid sequence from
amino and 103
2 5 to amino acid 112 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
3 0 (a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
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(aa) SEQ ID N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
(ab) the nucleotide sequence of the cDNA insert of clone
it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
(bb) the nucleotide sequence of the cDNA insert of done
it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:7 to
2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
3 0 SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, to a nucleotide
sequence
corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide
452 to
nucleotide 1102.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8, each
fragment comprising eight consecutive amino acids of SEQ ID N0:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone
it217 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:8. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
ands of SEQ ID N0:8, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:8 having biological activity, the fragment comprising the amino acid
sequ~ce
from amino acid 103 to amino acid 112 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387;
2 5 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone m1235_2 deposited under accession
number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
3 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone m1235_2 deposited under accession number
ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone m1235 2 deposited under accession number ATCC 98636;
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(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:10 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 172 to nucleotide 387; the nucleotide sequence of the full-length
protein coding
sequence of clone m1235_2 deposited under accession number ATCC 98636; or the
nucleotide sequence of a mature protein coding sequence of clone m1235_2
deposited
under accession number ATCC 98636. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone m1235 2 deposited under accession number ATCC 98636. In further
preferred
2 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID NO:10 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID NO:20, or a
polynucleotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10
having
2 5 biological activity, the fragment comprising the amino acid sequence from
amino acid 38
to amino acid 47 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced
3 0 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
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(aa) SEQ ID N0:9, but excluding the poly(A) tail at the
3' end of SEQ ID N0:9; and
(ab) the nucleotide sequence of the cDNA insert of clone
m1235 2 deposited under accession number A TCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotfde primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:9, but excluding the poly(A) tail at the
3' end of SEQ ID N0:9; and
(bb) the nucleotide sequence of the cDNA insert of clone
m1235 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 9:X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:9 to
2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:9 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:9. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:9 from nucleotide 127 to nucleotide 387, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
3 0 SEQ ID N0:9 from nucleotide 127 to nucleotide 387, to a nucleotide
sequence
corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide
127 to
nucleotide 387. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:9 from nucleotide 172 to nucleotide 387, and extending contiguously from a
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nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from
nucleotide 172 to nucleotide 387, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:9 from nucleotide 172 to nucleotide 387.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10, each
fragment comprising eight consecutive amino acids of SEQ ID NO:10; and
(c} the amino acid sequence encoded by the cDNA insert of clone
m1235 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID NO:10 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID NO:10, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:10 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 38 to amino acid 47 of SEQ ID NO:10.
2 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:11 from nucleotide 147 to nucleotide 1163;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone mt24 2 deposited under accession
3 0 number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
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(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone mt24_2 deposited under accession number
ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone mt24_2 deposited under accession number ATCC 98636;
(h) a polynudeotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:12;
(j) a polynucleotide which is an allelic variant of a polynucleotfde of
(ar(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 147 to nucleotide 1163; the nucleotide sequence of SEQ
ID N0:11
from nucleotide 273 to nucleotide 1163; the nucleotide sequence of the full-
length protein
2 0 coding sequence of clone mt24 2 deposited under accession number ATCC
98636; or the
nucleotide sequence of a mature protein coding sequence of clone mt24 2
deposited under
accession number ATCC 98636. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone mt24 2
deposited under accession number ATCC 98636. In further preferred embodiments,
the
2 5 present invention provides a polynucleotide encoding a protein comprising
a fragment
of the amino acid sequence of SEQ ID N0:12 having biological activity, the
fragment
preferably comprising eight (more preferably twenty, most preferably thirty)
consecutive
amino acids of SEQ ID N0:12, or a polynucleotide encoding a protein comprising
a
fragment of the amino acid sequence of SEQ ID N0:12 having biological
activity, the
3 0 fragment comprising the amino acid sequence from amino acid 164 to amino
acid 173 of
SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:11.
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Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
{i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:11, but excluding the poly(A) tail at the
3' end of SEQ ID N0:11; and
(ab) the nucleotide sequence of the cDNA insert of clone
mt24 2 deposited under accession number ATCC 98636;
(ii} hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
{iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
2 0 (ba) SEQ ID N0:11, but excluding the poly(A) tail at the
3' end of SEQ ID NO:11; and
(bb) the nucleotide sequence of the cDNA insert of clone
mt24 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
2 5 conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:11, and
3 0 extending contiguously from a nucleotide sequence corresponding to the 5'
end of SEQ
ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:11
, but
excluding the poly(A) tail at the 3' end of SEQ ID NO:11. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 147 to
nucleotide
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1163, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
of said sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1163, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from
nucleotide
147 to nucleotide 1163. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from
nucleotide 273 to nucleotide 1163, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID N0:11 from nucleotide 273 to nucleotide 1163.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
{a) the amino acid sequence of SEQ ID N0:12;
(b) fragments of the amino acid sequence of SEQ ID N0:12, each
fragment comprising eight consecutive amino acids of SEQ ID N0:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
mt24_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12. In further
preferred
2 0 embodiments, the present invention provides a protein comprising a
fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:12, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:12 having biological activity, the fragment comprising the amino
acid
2 5 sequence from amino acid 164 to amino acid 173 of SEQ ID N0:12.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone pe584_2 deposited under accession
number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone pe584 2 deposited under accession number
ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pe584 2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:14;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
2 0 (1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a~(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681; the nucleotide sequence of SEQ
ID N0:13
from nucleotide 437 to nucleotide 1681; the nucleotide sequence of the full-
length protein
2 5 coding sequence of clone pe584_2 deposited under accession number ATCC
98636; or the
nucleotide sequence of a mature protein coding sequence of clone pe584_2
deposited
under accession number ATCC 98636. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone pe584 2 deposited under accession number ATCC 98636. In further
preferred
3 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:14 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:14, or a
polynucleotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14
having
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biological activity, the fragment comprising the amino acid sequence from
amino acid 222
to amino acid 231 of SEQ ID N0:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:13, but excluding the poly(A) tail at the
3' end of SEQ ID N0:13; and
(ab) the nucleotide sequence of the cDNA insert of clone
pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
2 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:13, but excluding the poly(A) tail at the
2 5 3' end of SEQ ID N0:13; and
(bb) the nucleotide sequence of the cDNA insert of clone
pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
3 0 (iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
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ID N0:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:13
, but
excluding the poly(A) tail at the 3' end of SEQ ID N0:13. Also preferably .the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:13 from nucleotide 320 to
nucleotide
1681, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
of said sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from
nucleotide
320 to nucleotide 1681. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:13 from
nucleotide 437 to nucleotide 1681, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID N0:13 from nucleotide 437 to nucleotide 1681.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) fragments of the amino acid sequence of SEQ ID N0:14, each
fragment comprising eight consecutive amino acids of SEQ ID N0:14; and
2 0 (c) the amino acid sequence encoded by the cDNA insert of clone
pe584 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:14. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
2 5 amino acid sequence of SEQ ID N0:14 having biological activity, the
fragment preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:14, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:14 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 222 to amino acid 231 of SEQ ID N0:14.
3 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502;
- (c} a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone pj323_2 deposited under accession
number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone pj323 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone pj323 2 deposited under accession number
ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pj323_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:16;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:16 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:16;
2 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
a polynucleotide that hybridizes under stringent conditions to any
2 5 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502; the nucleotide sequence of SEQ ID
N0:15
from nucleotide 564 to nucleotide 1502; the nucleotide sequence of the full-
length protein
coding sequence of clone pj323 2 deposited under accession number ATCC 98636;
or the
3 0 nucleotide sequence of a mature protein coding sequence of clone pj323_2
deposited
under accession number ATCC 98636. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone pj323 2 deposited under accession number ATCC 98636. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
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comprising the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino
acid
145. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:16
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:16, or a
polynucleotide encoding a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:16 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:15.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:15, but excluding the poly(A) tail at the
3' end of SEQ ID N0:15; and
(ab} the nucleotide sequence of the cDNA insert of clone
2 0 pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
2 5 and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
3 0 (ba) SEQ ID NO:IS, but excluding the poly(A) tail at the
3' end of SEQ ID N0:15; and
(bb) the nucleotide sequence of the cDNA insert of clone
pj323_2 deposited under accession number ATCC 98636;
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(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
ID N0:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:15
, but
excluding the poly(A) tail at the 3' end of SEQ ID N0:15. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:15 from nucleotide 78 to
nucleotide
1502, and extending contiguously from a nucleotide sequence corresponding to
the 5' ~d
of said sequence of SEQ ID N0:15 from nucleotide 78 to nucleotide 1502, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ TD N0:15 from
nucleotide
78 to nucleotide 1502. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:15 from
nucleotide 564 to nucleotide 1502, to a nucleotide sequence corresponding to
the 3' end
2 0 of said sequence of SEQ ID N0:15 from nucleotide 564 to nucleotide 1502.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:16;
2 5 (b) the amino acid sequence of SEQ ID N0:16 from amino acid 54 to
amino acid 145;
(c) fragments of the amino acid sequence of SEQ ID N0:16, each
fragment comprising eight consecutive amino acids of SEQ ID N0:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone
3 0 pj323 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid
sequence
of SEQ ID N0:16 from amino acid 54 to amino acid 145. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
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amino acid sequence of SEQ ID N0:16 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:16, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:16 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone yb24_1 deposited under accession
number
ATCC 98636;
{e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 0 protein coding sequence of clone yb24_1 deposited under accession number
ATCC
98636;
{g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb24_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
2 5 sequence of SEQ ID N0:18;
(i) a polynucleofide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:18 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (ar(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294; the nucleotide sequence of SEQ ID
N0:17
from nucleotide 241 to nucleotide 294; the nucleotide sequence of the full-
length protein
coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
or the
nucleotide sequence of a mature protein coding sequence of clone yb24_1
deposited under
accession number ATCC 98636. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone yb24_1
deposited under accession number ATCC 98636. In further preferred embodiments,
the
present invention provides a polynucleotide encoding a protein comprising a
fragment
of the amino acid sequence of SEQ ID N0:18 having biological activity, the
fragment
preferably comprising eight (more preferably twenty, most preferably thirty)
consecutive
amino acids of SEQ ID N0:18, or a polynucleotide encoding a protein comprising
a
fragment of the amino acid sequence of SEQ ID N0:18 having biological
activity, the
fragment comprising the amino acid sequence from amino acid 22 to amino acid
31 of SEQ
ID N0:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
2 0 {a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:17, but excluding the poly(A) tail at the
2 5 3' end of SEQ ID N0:17; and
(ab) the nucleotide sequence of the cDNA insert of clone
yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
3 0 (iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
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(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected_from
the group consisting of:
(ba) SEQ ID N0:17, but excluding the poly(A) tail at the
3' end of SEQ ID N0:17; and
(bb) the nucleotide sequence of the cDNA insert of clone
yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
ID N0:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:17
, but
excluding the poly(A) tail at the 3' end of SEQ ID N0:17. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:17 from nucleotide 130 to
nucleotide
294, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
2 0 of said sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294, to
a nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from
nucleotide
130 to nucleotide 294. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:17 from nucleotide 241 to nucleotide 294, and extending contiguously from a
2 5 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:17 from
nucleotide 241 to nucleotide 294, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:17 from nucleotide 241 to nucleotide 294.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:18;
(b) fragments of the amino acid sequence of SEQ ID N0:18, each
fragment comprising eight consecutive amino acids of SEQ ID N0:18; and
29
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(c) the amino acid sequence encoded by the cDNA insert of clone
yb24_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:18. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:18 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:18, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:18 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 22 to amino acid 31 of SEQ ID N0:18.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 580 to nucleotide 1707;
(d) a polynucleotide comprising the nucleotide sequence of the full-
2 0 length protein coding sequence of clone yb44_1 deposited under accession
number
ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 5 protein coding sequence of clone yb44_1 deposited under accession number
ATCC
98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb44_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid
3 0 sequence of SEQ ID N0:20;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:20 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:20;
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(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707; the nucleotide sequence of SEQ
ID N0:19
from nucleotide 580 to nucleotide 1707; the nucleotide sequence of the full-
length protein
coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
or the
nucleotide sequence of a mature protein coding sequence of clone yb44_1
deposited under
accession number ATCC 98636. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone yb44_1
deposited under accession number ATCC 98636. In further preferred embodiments,
the
present invention provides a polynucleotide encoding a protein comprising a
fragment
of the amino acid sequence of SEQ ID N0:20 having biological activity, the
fragment
preferably comprising eight (more preferably twenty, most preferably thirty)
consecutive
amino acids of SEQ ID N0:20, or a polynucleotide encoding a protein comprising
a
fragment of the amino acid sequence of SEQ ID N0:20 having biological
activity, the
2 0 fragment comprising the amino acid sequence from amino acid 194 to amino
acid 203 of
SEQ ID N0:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
Further embodiments of the invention provide isolated polynucleotides produced
2 5 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
3 0 (aa) SEQ ID N0:19, but excluding the poly(A) tail at the
3' end of SEQ ID N0:19; and
(ab) the nucleotide sequence of the cDNA insert of clone
yb44_1 deposited under accession number ATCC 98636;
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(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:19, but excluding the poly(A) tail at the
3' end of SEQ ID N0:19; and
(bb) the nucleotide sequence of the cDNA insert of clone
yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at b5 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii.).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19, and
2 0 extending contiguously from a nucleotide sequence corresponding to the 5'
end of SEQ
ID N0:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:19
, but
excluding the poly(A) tail at the 3' end of SEQ ID N0:19. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:19 from nucleotide 514 to
nucleotide
2 5 1707, and extending contiguously from a nucleotide sequence corresponding
to the 5' end
of said sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 1707, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from
nucleotide
514 to nucleotide 1707. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
3 0 N0:19 from nucleotide 580 to nucleotide 1707, and extending contiguously
from a
nucleotide sequence corresponding to the.5' end of said sequence of SEQ ID
N0:19 from
nucleotide 580 to nucleotide 1707, to a nucleotide sequence corresponding to
the 3' end
of said sequence of SEQ ID N0:19 from nucleotide 580 to nucleotide 1707.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:20;
(b) fragments of the amino acid sequence of SEQ ID N0:20, each
fragment comprising eight consecutive amino acids of SEQ ID N0:20; and
(c) the amino acid sequence encoded by the cDNA insert of clone
yb44_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:20. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:20 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:20, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:20 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 194 to amino acid 203 of SEQ ID N0:20.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
2 0 Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
2 5 polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention.
Protein compositions of the present invention may further comprise a
3 0 pharmaceutically acceptable carrier. Compositions comprising an antibody
which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
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effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
acid sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 0 As used herein a "seae~ed" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "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
2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "cs756 2"
A polynucleotide of the present invention has been identified as clone
"cs756_2".
cs756_2 was isolated from a human fetal brain cDNA library using methods which
are
3 0 selective for cDNAs encoding secreted proteins {see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. cs756_2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"cs756 2 protein").
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VIrO 99/37674 PCTNS99/01404
The nucleotide sequence of cs756_2 as presently determined is reported in SEQ
ID
NOa, and includes a poly(A) tail. What applicants presently believe to be the
proper
reading frame and the predicted amino acid sequence of the cs756 2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:2.
Amino
acids 211 to 223 of SEQ ID N0:2 are a predicted leader/signal sequence, with
the
predicted mature amino acid sequence beginning at amino acid 224. Due to the
hydrophobic nature of the predicted leader/signal sequence, it is likely to
act as a
transmembrane domain should the predicted leader/signal sequence not be
separated
from the remainder of the cs756 2 protein. The TopPredII computer program
predicts a
potential transmembrane domain within the cs756_2 protein sequence of SEQ ID
N0:2,
centered around amino acid 15 of SEQ ID N0:2; amino acids 2 to 14 of SEQ ID
N0:2 are
also a possible leader/signal sequence, with the predicted mature amino acid
sequence
in that case beginning at amino acid 15.
Another possible cs756 2 reading frame and predicted amino acid sequence,
encoded by base pairs 385 to 825 of SEQ ID NO:1, is reported in SEQ ID N0:30;
the
TopPredII computer program predicts a potential transmembrane domain centered
around amino acid 100 of SEQ ID N0:30. The open reading frames corresponding
to SEQ
ID N0:30 and SEQ ID N0:2 could be joined if a frameshift were introduced into
the
nucleotide sequence of SEQ ID N0:1.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cs756_2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for cs756 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cs756_2 demonstrated at least some similarity with
sequences
2 5 identified as AA398077 (zt58c03.s1 Soares testis NHT Homo Sapiens cDNA
clone 726532
3'), AA541286 (nf97e03.s1 NCI_CGAP_Co3 Homo Sapiens cDNA clone 1MAGE:927868),
W28620 (49c2 Human retina cDNA randomly primed sublibrary Homo Sapiens cDNA),
and W47601 (zc35g08.r1 Soares senescent fibroblasts NbHSF Homo sapiens cDNA
clone
324350 5'). The predicted amino acid sequence disclosed herein for SEQ ID
N0:30 was
3 0 searched against the GenPept and GeneSeq amino acid sequence databases
using the
BLASTX search protocol. The predicted SEQ ID N0:30 protein demonstrated at
least
some similarity to sequences identified as L76938 (Werner syndrome gene,
complete cds
[Homo sapiensJ). "Werner's syndrome (WS) is an inherited disease with clinical
symptoms resembling premature aging ... [the] predicted protein is 1432 amino
acids in
CA 02318303 2000-07-13
WO 99/376'14 PCTNS99/01404
length and shows significant similarity to DNA helicases" (Yu et al., 1996,
Science
272(5259):258-262, which is incorporated by reference herein). Based upon
sequence
similarity, cs756 2 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of cs756 2 indicates that it may contain one
or more of
the following repetitive elements: MIR, MER.
Clone "ew150 1"
A polynucleotide of the present invention has been identified as clone
"ew150_1".
ew150_1 was isolated from a human adult placenta cDNA library using methods
which
are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. evV150_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ew150_1 protein').
The nucleotide sequence of ew150_1 as presently determined is reported in SEQ
ID N0:3, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the ew150_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:4.
Amino
acids 26 to 38 of SEQ ID N0:4 are a predicted leader/signal sequence, with the
predicted
2 0 mature amino acid sequence beginning at amino acid 39. Due to the
hydrophobic nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
ew150_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 ew150_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for ew150_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ew150_1 demonstrated at least some similarity with
sequences
identified as AA563938 (nk23b12.s1 NCI_CGAP Col1 Homo sapiens cDNA clone IMAGE
3 0 1014335), D63209 (Human placenta cDNA 5'-end GEN-506F01), M90423
(Bacteriphage
US3 lytic-enzyme), W23461 (zb33cOl.r1 Soares parathyroid tumor NbHPA Homo
sapiens
cDNA clone 305376 5'), and 256916 (H.sapiens CpG DNA, clone 153b7, forward
read
cpg153b7.ftla). In the region around position 1514 of SEQ ID N0:3, ew150_1
also
demonstrated at least some similarity with sequences encoding a mitochondria)
energy-
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WO 99/37674 PCTNS99/01404
transfer proteins signature motif which is found in mitochondrial and other
membrane
proteins. Based upon sequence similarity, ew150_1 proteins and each similar
protein or
peptide may share at least some activity. The TopPredII computer program
predicts ten
potential transmembrane domains within the ew150_1 protein sequence, which are
centered around amino acids 70,106,133, 200, 314, 349, 387, 457, 504, and 527
of SEQ ID
N0:4, respectively.
lone "g~894 1~"
A polynucleotide of the present invention has been identified as clone
"gg894_13".
gg894_13 was isolated from a human fetal kidney cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. gg894_13 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "gg894_13 protein").
The nucleotide sequence of gg894_13 as presently determined is reported in SEQ
ID N0:5, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the gg894_13 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino
2 0 acids 41 to 53 of SEQ ID N0:6 are a predicted leader/signal sequence, with
the predicted
mature amino acid sequence beginning at amino acid 54. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
gg894_13 protein. Another possible gg894_13 reading frame and predicted amino
acid
2 5 sequence, encoded by base pairs 602 to 1129 of SEQ ID N0:5, is reported in
SEQ ID
N0:31. The open reading frames corresponding to SEQ ID N0:31 and SEQ ID N0:6
could
be joined if a frameshift were introduced into the nucleotide sequence of SEQ
ID N0:5.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
gg894_13 should be approximately 2400 bp.
3 0 The nucleotide sequence disclosed herein for gg894_13 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. gg894_13 demonstrated at least some similarity with
sequences
identified as H57424 (yr13a10.s1 Homo sapiens cDNA clone 205146 3'), T23885
(Human
gene signature HUMGS05820), and W80358 (zh49a07.s1 Soares fetal liver spleen
1NFLS
37
CA 02318303 2000-07-13
WO 99!37674 PCT/US99/01404
Sl Homo sapiens cDNA clone 415380 3'). Based upon sequence similarity,
gg894_13
proteins and each similar protein or peptide may share at least some activity.
The
TopPredII computer program predicts a potential transmembrane domain within
the
gg894_13 protein sequence centered around amino acid 115 of SEQ ID N0:6. The
nucleotide sequence of gg894_13 indicates that it may contain a RBMI
repetitive element.
Clone "it217 2"
A polynucleotide of the present invention has been identified as clone
"it217_2".
it217 2 was isolated from a human adult brain (thalamus) cDNA library using
methods
which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or
was identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. it217 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"it217_2 protein").
The nucleotide sequence of it217 2 as presently determined is reported in SEQ
ID
N0:7, and includes a poly(A) tail. What applicants presently believe to be the
proper
reading frame and the predicted amino acid sequence of the it217_2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
Another
possible it217 2 reading frame and predicted amino acid sequence, encoded by
base pairs
2 0 45 to 311 of SEQ ID N0:7, is reported in SEQ ID N0:32. Amino acids 36 to
48 of SEQ ID
N0:32 are a predicted leader/signal sequence, with the predicted mature amino
acid
sequence beginning at amino acid 49. Due to the hydrophobic nature of the
predicted
leader/signal sequence, it is likely to act as a transmembrane domain should
the predicted
leader/signal sequence not be separated from the remainder of the it217_2
protein. The
2 5 open reading frames corresponding to SEQ ID N0:32 and SEQ ID N0:8 could be
joined
if at least one frameshift were introduced into the nucleotide sequence of SEQ
ID N0:7.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
it217_2 should be approximately 2250 bp.
The nucleotide sequence disclosed herein for it217 2 was searched against the
3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. it217_2 demonstrated at least some similarity with
sequences
identified as AA242969 (zr65h09.r1 Soares NhHMPu S1 Homo sapiens cDNA clone
668321 5' similar to SW SCC2_HUMAN P48594 SQUAMOUS CELL CARCINOMA
ANTIGEN 2 ;contains Alu repetitive element), B44876 (HS-1060-Al-G06-MR.abi CTT
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i~NO 99/37674 PGT/US99/01404
Human Genomic Sperm Library C Homo sapien genomic clone Plate CT 782 Col 11
Row
M), H82168 (yv78d08.r1 Homo sapiens cDNA clone), 566896 (squamous cell
carcinoma
antigen), U19556 (Human squamous cell carcinoma antigen 1 (SCCAl) mRNA,
complete
cds), U19557 (Human squamous cell carcinoma antigen 2 (SCCA2) mRNA, complete
cds),
S and U35459 (Human bomapin mRNA, complete cds). The predicted amino acid
sequence
disclosed herein for it217_2 was searched against the GenPept and GeneSeq
amino acid
sequence databases using the BLASTX search protocol. The predicted it217_2
protein
demonstrated at least some similarity to sequences identified as L40377
(cytoplasmic
antiproteinase 2 [Homo sapiens]), M34352 (ovalbumin (callus gallus]), M91161
(serpin
[Equus caballus]), 825276 (SCC antigen), 848379 (Human megakaryocyte
differentiation
factor), 566896 (squamous cell carcinoma antigen, SCC antigen serine protease
inhibitor
[human, Peptide, 390 aa] [Homo sapiens]), U19568 (squamous cell carcinoma
antigen
[Homo sapiens]), and U19576 (squamous cell carcinoma antigen [Homo sapiens]).
Human bomapin may play a role in the regulation of protease activities during
hematopoiesis (Riewald et al.,1995, J. Biol. Chem. 270: 26754, which is
incorporated by
reference herein). Serpins are SERine Proteinase IlVhibitors and are
considered
extracellular in localization. Human squamous cell carcinoma antigen (SSCA) is
a
member of the serpin family of proteinase inhibitors, purified from sera of
cancer patients.
Based upon sequence similarity, it217 2 proteins and each similar protein or
peptide may
2 0 share at least some activity.
Clong "m1235 2"
A polynucleotide of the present invention has been identified as clone "m1235
2".
m1235_2 was isolated from a human adult brain (caudate nucleus) cDNA library
using
2 5 methods which are selective for cDNAs encoding secreted proteins (see U.S.
Pat. No.
5,536,637), or was identified as encoding a secreted or transmembrane protein
on the basis
of computer analysis of the amino acid sequence of the encoded protein. m1235
2 is a full-
length clone, including the entire coding sequence of a secreted protein (also
referred to
herein as "m1235 2 protein').
3 0 The nucleotide sequence of m1235 2 as presently determined is reported in
SEQ
ID N0:9, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the m1235 2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:10. Amino
acids 3 to 15 of SEQ ID NO:10 are a predicted leader/signal sequence, with the
predicted
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mature amino acid sequence beginning at amino acid 16. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
m1235 2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
m1235 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for m1235 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. m1235_2 demonstrated at least some similarity with
sequences
identified as AA160887 (zo79b05.s1 Stratagene pancreas (#937208} Homo sapiens
cDNA
clone 593073 3'), 814349 (yf79f12.r1 Homo sapiens cDNA clone 28451 5'), and
854256
(yg74f07.r1 Homo sapiens cDNA clone 39059 5'). Based upon sequence similarity,
m1235 2 proteins and each similar protein or peptide may share at least some
activity.
The TopPredII computer program predicts a potential transmembrane domain
within the
m1235_2 protein sequence centered around amino acid 25 of SEQ ID N0:10.
Clone "mt24 2"
A polynudeotide of the present invention has been identified as clone "mt24
2".
mt24_2 was isolated from a human adult testes cDNA library using methods which
are
2 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. mt24 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"mt24 2 protein').
2 5 The nucleotide sequence of mt24 2 as presently determined is reported in
SEQ ID
N0:11, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the mt24 2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:12. Amino
acids 30 to 42 of SEQ ID N0:12 are a predicted leader/signal sequence, with
the predicted
3 0 mature amino acid sequence beginning at amino acid 43. Due to the
hydrophobic nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
mt24 2 protein.
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The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
mt24 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for mt24_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. mt24 2 demonstrated at least some similarity with
sequences
identified as AA062589 (zf68f04.r1 Soares pineal gland N3HPG Homo sapiens cDNA
clone 3821115') and T19332 (b08016t Testis 1 Homo sapiens cDNA clone b08016 5'
end).
Based upon sequence similarity, mt24 2 proteins and each similar protein or
peptide may
share at least some activity. The TopPredII computer program predicts four
potential
transmembrane domains within the mt24_2 protein sequence centered around amino
acids 38,153,167, and 232 of SEQ ID N0:12, respectively.
Clone"pe584 2"
A polynucleotide of the present invention has been identified as clone
"pe584_2".
pe584_2 was isolated from a human adult blood (chronic myelogenous leukemia
K5)
cDNA library using methods which are selective for cDNAs encoding secreted
proteins
(see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or
transmembrane
protein on the basis of computer analysis of the amino acid sequence of the
encoded
protein. pe584 2 is a full-length clone, including the entire coding sequence
of a secreted
2 0 protein (also referred to herein as "pe584 2 protein").
The nucleotide sequence of pe584 2 as presently determined is reported in SEQ
ID N0:13, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the pe584_2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:14. Amino
2 5 acids 27 to 39 of SEQ ID N0:14 are a predicted leader/signal sequence,
with the predicted
mature amino acid sequence beginning at amino acid 40. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
pe584_2 protein.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
pe584 2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for pe584_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. pe584 2 demonstrated at least some similarity with
sequences
41
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WO 99/37674 PCT/US99/01404
identified as AA303149 (EST13039 Uterus tumor I), AA405004 (zt06e03.s1
NCI_CGAP_GCBl Homo Sapiens cDNA clone IMAGE 712348 3'), AA481230 (aa34gOl.r1
NCI_CGAP GCB1 Homo sapiens cDNA clone 815184 5' similar to SW TCR2_ECOLI
P02981 TETRACYCLINE RESISTANCE PROTEIN), D88315 (Mouse mRNA for
tetracycline transporter-like protein, complete cds), and T10077 (seq1295 Homo
sapiens
cDNA clone b4HB3MA-COT8-HAP-Ft109 5'}. The predicted amino acid sequence
disclosed herein for pe584 2 was searched against the GenPept and GeneSeq
amino acid
sequence databases using the BLASTX search protocol. The predicted pe584_2
protein
demonstrated at least some similarity to sequences identified as D88315
(tetracycline
transporter-like protein [Mus musculus]). Mouse tetracycline transporter-like
protein is
a sugar transporter (Matsuo et al.,1997, Biochem. Biophys. Res. Comm. 238: 126-
192, which
is incorporated by reference herein). Based upon sequence similarity, pe584 2
proteins
and each similar protein or peptide may share at least some activity. The
TopPredII
computer program predicts eleven potential transmembrane domains within the
pe584 2
protein sequence, which are centered around amino acids 32, 55, 78, 114,142,
196, 235,
264, 287, 332, and 375 of SEQ ID N0:14, respectively.
Clony '~i3,~_'
A polynucleotide of the present invention has been identified as clone "pj323
2".
2 0 pj323 2 was isolated from a human fetal carcinoma (NTD2 cells treated with
retinoic acid
for 23 days) cDNA library using methods which are selective for cDNAs encoding
secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding
a secreted or
transmembrane protein on the basis of computer analysis of the amino acid
sequence of
the encoded protein. pj323 2 is a full-length clone, including the entire
coding sequence
2 5 of a secreted protein (also referred to herein as "pj323 2 protein").
The nucleotide sequence of pj323_2 as presently determined is reported in SEQ
ID
N0:15, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the pj323 2 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:16. Amino
3 0 acids 150 to 162 of SEQ ID N0:16 are a predicted leader/signal sequence,
with the
predicted mature amino acid sequence beginning at amino acid 163. Due to the
hydrophobic nature of the predicted leader/signal sequence, it is likely to
act as a
transmembrane domain should the predicted leader/signal sequence not be
separated
from the remainder of the pj323_2 protein.
42
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WO 99/37674 PCT/US99/01404
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
pj323_2 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for pj323_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. pj323 2 demonstrated at least some similarity with
sequences
identified as AA160454 (zo74g05.r1 Stratagene pancreas (#937208) Homo sapiens
cDNA
clone 592664 5'), AA398257 (zt60a08.s1 Scares testis NHT Homo Sapiens cDNA
clone
726710 3'), and T47284 (yb64g11.s1 Homo Sapiens cDNA clone 76004 3'). The
predicted
amino acid sequence disclosed herein for pj323 2 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted pj323 2 protein demonstrated at least some similarity to human
integral nuclear
envelope protein, lamin B receptors from several species, and sterol
reductases from
several species. Lamin B receptors have hydrophobic carboxy terminal portions
and
hydrophilic amino terminal portions. Antibodies to lamin B receptors have been
found
in patients with primary biliary cirrhosis. Sterol reductases demonstrate
sequence
similarity to the hydrophobic portions of lamin B receptors. Based upon
sequence
similarity, pj323_2 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts six potential transmembrane
domains
within the pj323 2 protein sequence, which are centered around amino acids
47,106,164,
2 0 187, 341, and 432 of SEQ ID N0:16, respectively.
pj323 2 protein was expressed in a COS cell expression system, and an
expressed
protein band of approximately 46 kDa was detected in membrane fractions using
SDS
polyacrylamide gel electrophoresis.
2 S Clone "vb24 1"
A polynucleotide of the present invention has been identified as clone
"yb24_1".
yb24_1 was isolated from a human fetal brain cDNA library and was identified
as
encoding a secreted or transmembrane protein on the basis of computer analysis
of the
amino acid sequence of the encoded protein. yb24_1 is a full-length clone,
including the
3 0 entire coding sequence of a secreted protein (also referred to herein as
"yb24_1 protein").
The nucleotide sequence of yb24_1 as presently determined is reported in SEQ
ID
N0:17, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the yb24_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:18. Amino
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WO 99/37674 PGTNS99/01404
acids 25 to 37 of SEQ ID N0:18 are a predicted leader/signal sequence, with
the predicted
mature amino acid sequence beginning at amino acid 38. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
yb24_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
yb24_1 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for yb24_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. yb24_1 demonstrated at least some similarity with
sequences
identified as AA149807 (z147c09.s1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 505072 3') and AB003515 (Rat mRNA for GEF-2, complete cds). Based upon
sequence similarity, yb24_1 proteins and each similar protein or peptide may
share at least
some activity.
A polynucleotide of the present invention has been identified as clone
"yb44_1".
yb44_1 was isolated from a human fetal brain cDNA library and was identified
as
encoding a secreted or transmembrane protein on the basis of computer analysis
of the
2 0 amino acid sequence of the encoded protein. yb44_1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"yb44_1 protein").
The nucleotide sequence of yb44_1 as presently determined is reported in SEQ
ID
N0:19, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the yb44_1 protein
2 5 corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:20. Amino
acids 10 to 22 of SEQ ID N0:20 are a predicted leader/signal sequence, with
the predicted
mature amino acid sequence beginning at amino acid 23. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
3 0 yb44_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
yb44_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for yb44_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
44
CA 02318303 2000-07-13
WO 99/37674 PGT/US99/01404
FASTA search protocols. yb44_1 demonstrated at least some similarity with
sequences
identified as AC000016 {*** SEQUENCING IN PROGRESS *** EPMl /APECED region of
chromosome 21, BAC clone B4P3; HTGS phase 1,10 unordered pieces). The
predicted
amino acid sequence disclosed herein for yb44_1 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted yb44_1 protein demonstrated at least some similarity to sequences
identified
as 872377 (Human auxiliary cytochrome P450 species 2D6 variant 2 protein) and
U44753
(cytochrome P450 [Drosophila melanogaster]). Based upon sequence similarity,
yb44_1
proteins and each similar protein or peptide may share at least some activity.
The
TopPredII computer program predicts three additional potential transmembrane
domains
within the yb44_1 protein sequence, which are centered around amino acids
82,128, and
361 of SEQ ID N0:20, respectively. The nucleotide sequence of yb44_1 indicates
that it
may contain one or more of the following repetitive elements: Alu, AT, TATACA,
MER44A, TACA.
Den osit of ~,lones
Clones cs756_2, ew150_l, gg894_13, it217_2, m1235_2, mt24_2, pe584 2, pj323 2,
yb24_l, and yb44_1 were deposited on January 22,1998 with the American Type
Culture
Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.)
as an
2 0 original deposit under the Budapest Treaty and were given the accession
number ATCC
98636, from which each clone comprising a particular polynucleotide is
obtainable. All
restrictions on the availability to the public of the deposited material will
be irrevocably
removed upon the granting of the patent, except for the requirements specified
in 37
C.F.R. ~ 1.808(b), and the term of the deposit will comply with 37 C.F.R. ~
1.806.
2 5 Each clone has been transfected into separate bacterial cells (E. coh~ in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pEDb or
pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector
3 0 ("pED6") was derived from pED6dpc1 by insertion of a new polylinker to
facilitate
cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 4485-4490); the pNOTs
vector
was derived from pMT2 (Kaufman et al.,1989, Mol. Cell. Biol. 9: 946-958) by
deletion of
the DHFR sequences, insertion of a new polylinker, and insertion of the M13
origin of
replication in the CIaI site. In some instances, the deposited clone can
become "flipped"
CA 02318303 2000-07-13
'WO 99/37674 PCT/US99/01404
(i.e., in the reverse orientation) in the deposited isolate. In such
instances, the cDNA insert
can still be isolated by digestion with EcoRI and NotI. However, NotI will
then produce
the 5' site and EcoRI will produce the 3' site for placement of the cDNA in
proper
orientation for expression in a suitable vector. The cDNA may also be
expressed from the
vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of an
oligonudeotide probe that was used to isolate or to sequence each full-length
clone is
identified below, and should be most reliable in isolating the clone of
interest.
Clone Probe Sequence
cs756_2 SEQ ID N0:21
ew150_1 SEQ ID N0:22
gg894_13 SEQ ID N0:23
it217_2 SEQ ID N0:24
m1235 2 SEQ ID N0:25
2 0 mt24_2 SEQ ID N0:26
pe584_2 ~ SEQ ID N0:27
pj323_2 SEQ ID N0:28
yb24_1 SEQ ID N0:29
2 5 In the sequences listed above which include an N at position 2, that
position is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as, for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
3 0 The design of the oligonucleotide probe should preferably follow these
parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
46
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WO 99/37674 PCTNS99/01404
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with y 32P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 Ixl of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
2 0 colonies to nitrocellulose filters and lyre, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ~g/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
3 0 filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
47
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WO 99/37674 PCT/US99/01404
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
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 1~0 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc. ~~, 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.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature
forms) of such protein may be obtained by expression of the disclosed full-
length
2 0 polynucleotide (preferably those deposited with ATCC) in a suitable
mammalian cell or
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
2 5 are transcribed to produce the mRNAs from which cDNA polynucleotide
sequences are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
3 0 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. An "isolated gene" is
a gene that
48
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
The chromosomal location corresponding to the polynucleotide sequences
disclosed herein may also be determined, for example by hybridizing
appropriately
labeled polynucleotides of the present invention to chromosomes in situ. It
may also be
possible to determine the corresponding chromosomal location for a disclosed
polynucleotide by identifying significantly similar nucleotide sequences in
public
databases, such as expressed sequence tags (ESTs), that have already been
mapped to
particular chromosomal locations. For at least some of the polynucleotide
sequences
disclosed herein, public database sequences having at least some similarity to
the
polynucleotide of the present invention have been listed by database accession
number.
Searches using the GenBank accession numbers of these public database
sequences can
then be performed at an Internet site provided by the National Center for
Biotechnology
Information having the address http://www.ncbi.nlm.nih.gov/UniGene/, in order
to
identify "UniGene clusters" of overlapping sequences. Many of the "UniGene
clusters"
so identified will already have been mapped to particular chromosomal sites.
Organisms that have enhanced, reduced, or modified expression of the genes)
corresponding to the polynucleotide sequences disclosed herein are provided.
The
desired change in gene expression can be achieved through the use of aniisense
2 0 polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed
from the
gene (Albert and Morris,1994, Trends Phurmacol. Sci.15(~: 250-254; Lavarosky
et al.,1997,
Biochem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. Mol.
Biol. 58:1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
2 5 herein, preferably produced by transformation of cells with genetic
constructs that are
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 B1, incorporated by reference
herein).
3 0 In addition, organisms are provided in which the genes) corresponding to
the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
49
CA 02318303 2000-07-13
VNO 99/37674 PCTNS99/01404
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. LISA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
LISA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present inv~tion is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms, part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
For
example, the TopPredII computer program can be used to predict the location of
transmembrane domains in an amino acid sequence, domains which are described
by the
2 0 location of the center of the transmsmbrane domain, with at least ten
transmembrane
amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
2 5 identity (more preferably, at least 75% identity; most preferably at least
90% or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while mi~aimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
3 0 (more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
In particular, sequence identity may be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
version 1.4, which in turn is based on the public domain NCBI-BLAST version
1.4
(Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods
in Enzymology
266: 460-480; Altschul et al., 1990, Basic local alignment search tool,
Journal of
Molecular Biology 2I5: 403-410; Gish and States, 1993, Identification of
protein coding
regions by database similarity search, Nature Genetics 3: 266-272; Karlin and
Altschul,
1993, Applications and statistics for multiple high-scoring segments in
molecular
sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are
incorporated by
reference herein). WU-BLAST version 2.0 executable programs for several UNIX
platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The
complete
suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is
provided at that site, in addition to several support programs. WU-BLAST 2.0
is
copyrighted and may not be sold or redistributed in any form or manner without
the
express written consent of the author; but the posted executables may
otherwise be freely
used for commercial, nonprofit, or academic purposes. In all search programs
in the suite
-- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX -- the gapped alignment
routines are integral to the database search itself, and thus yield much
better sensitivity and
selectivity while producing the more easily interpreted output. Gapping can
optionally be
fumed off in all of these programs, if desired. The default penalty (Q) for a
gap of length
one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to
any
2 0 integer value including zero, one through eight, nine, ten, eleven, twelve
through twenty,
twenty-one through fifty, fifty-one through one hundred, etc. The default per-
residue
penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for
BLASTN,
but may be changed to any integer value including zero, one, two, three, four,
five, six,
seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through
fifty, fifty-one
2 5 through one hundred, etc. Any combination of values for Q and R can be
used in order to
align sequences so as to maximize overlap and identity while minimizing
sequence gaps.
The default amino acid comparison matrix is BLOSUM62, but other amino acid
comparison matrices such as PAM can be utilized.
Species homologues of the disclosed polynucleotides and proteins are also
3 0 provided by the present invention. As used herein, a "species homologue"
is a protein or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
51
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
the given polynudeotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues 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. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hylobates
concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus
norvegicus,
CricetuIus griseus, Fells catus, Mustela visor, Canis familiaris, O~yctolagus
cuniculus, Bos taurus,
Ovis aries, Sus scrota, and Equus caballus, for which genetic maps have been
created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
(O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; OBrien et al., 1993,
Nature
2 0 Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et
al.,1997, Nature
Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
1997, Genome Research 7:1123-1137; all of which are incorporated by reference
herein).
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotides
2 5 which also encode proteins which are identical or have significantly
similar sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
60% sequence identity (more preferably, at least 75% identity; most preferably
at least 90%
identity} with the given polynudeotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
3 0 overlap and identity while minimizing sequence gaps. Allelic variants may
be isolated and
identified by making suitable probes or primers from the sequences provided
herein and
screening a suitable nucleic acid source from individuals of the appropriate
species.
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CA 02318303 2000-07-13
VlrO 99/37674 PCTNS99/01404
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highiy stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
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WO 99/37674 PCT/US99/01404
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)= Buffer' and Buffers
_
A DNA:DNA z 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC Te*; lxSSC
C DNA:RNA z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, SO% formamide
F RNA:RNA <50 TF*; lxSSC TF*; ixSSC
G DNA:DNA 2 SO 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
1 H DNA:DNA <50 T"*; 4xSSC TH'; 4xSSC
O
I DNA:RNA z 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA 2 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~'; 2xSSC T~'; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA a 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 509 formamide
P DNA:RNA <50 TP*; 6xSSC TP'; 6xSSC
Q RNA:RNA a 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50~ formamide
2 R RNA:RNA <50 TR'; 4xSSC TR*; 4xSSC
0
~: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynudeotides. When
hybridizing a polynucleotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynudeotides and identifying the region
or regions of optimai sequence complementarily.
t: SSPE (lxSSPE is 0.15M NaCI, IOmM NaH2P0" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is O.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "Ts - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined aaording to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log~~[Na'j) + 0.41(~°G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'j is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na') for lxSSC = 0.165 M).
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CA 02318303 2000-07-13
WO 99/37674 PCT/US99/41404
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
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
ICaufman et al., Nucleic Acids Res. 12, 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.
ICaufman, Methods in Enzymology ~$~, 537-566 (1990). As defined herein
"operably
2 0 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.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from i~ vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 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
CA 02318303 2000-07-13
WO 99/37674 PCTNS99/01404
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.
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, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Ae~ricultural
Experiment
Station Bulletin No. 1555 (198, incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
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
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 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). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 Invitrogen Corporation (Carlsbad, CA), 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") is commercially available from the Eastman
Kodak
Company (New Haven, CT).
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CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
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."
The protein of the invention may also be expressed as a product of transgeruc
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.
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 struchlral 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.
2 0 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,
2 5 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. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
3 0 insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
expected to retain profiein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
57
CA 02318303 2000-07-13
VVO 99/37674 PCT/US99/01404
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
USES AND BIOLOGICAL ACTIVL~
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 below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
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 Southern gels; as chromosome markers
or tags
2 0 (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
2 5 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
3 0 polynucleotide can also be used in interaction trap assays (such as, for
example, those
described in Gyuris et al.,1993, Cell 75: 791-803 and in Rossi et al.,1997,
Proc. Natl. Acad.
Sci. LISA 94: 8405-8410, all of which are incorporated by reference herein) to
identify
polynucleotides encoding the other protein with which binding occurs or to
identify
inhibitors of the binding interaction.
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CA 02318303 2000-07-13
CVO 99/37674 PGT/US99/01404
The proteins provided by the present invention can similarly be used in assay
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.
Any or alI of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
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
2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
ICimmel eds.,1987.
Nutritional Uses
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
2 5 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
3 0 can be added to the medium in or on which the microorganism is cultured.
~,tr,k;np and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or intu'biting) or cell differentiation (either inducing or
inhibiting) activity or may
59
CA 02318303 2000-07-13
CVO 99/37674 PCTNS99/01404
induce production of other cytokines in certain cell populations. 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 a protein 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, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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., J. Immunol. 149:3778-3783,1992; Bowman
et al., J.
Immunol. 152: 1756-1761,1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
2 0 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 Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto.1994.
2 5 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
3 0 336:690-692, 1988; Greenberger et al., Proc. Natl. 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.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, john Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. 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
CA 02318303 2000-07-13
WO 99/3?6?4 PCT/US99/01404
in Immunology. j.E.e.a. 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.e.a. Coiigan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto.1991.
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.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Su~ressing, Activity
A protein 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 protein may be useful in the treatment of various
immune
2 0 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
2 5 diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. C~f course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
3 0 treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
inv~tion 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,
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CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to regulate immune
responses in a number of ways. 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.
Down regulating or preventing one or more antigen functions (including without
2 0 limitation B lymphocyte antigen functions (such as , for example, B7)),
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
2 5 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
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
3 0 1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
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anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents 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, marine 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.
Blocking antigen function 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
2 0 activation of autoreactive T cells may reduce or eliminate disease
symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens 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
2 5 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 marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/Ipr mice or NZB hybrid
mice,
3 0 marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
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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 through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
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 with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
2 0 carcinoma) transfected with a nucleic acid encoding at least one peptide
of the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell ran be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-2-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
3 0 lymphocyte antigens) on the surface of the tumor cell provides the
necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
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cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
(iZ
microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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.
2 0 USA 78:248&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;
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 ai., 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.
Assays for T-cell-dependent immunoglobuiin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
3 0 in: Maliszewski, J. Imrnunol.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.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
ICruisbeek,
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. 140:508-5I2, 1988; Bertagnolli et al., J. Immunol.149:3778-
3783,1992.
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; Macatorua 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 67:4062-4069, 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.
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:
Darzynkiewicz
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,
2 0 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.
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-I17, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et
al., Blood
2 5 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-
7551,1991.
Hematopoiesis Reg~,, ' a ActivitX
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
3 0 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 progenitox 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
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precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
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 thrombocytoperua, 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.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
2 0 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.
2 5 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.L Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
3 0 hematopoietic colony forming cells with high proliferative potential,
McNiece, LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.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. l-21, Wiley-Liss, lnc.., New York,
NY.1994; Long
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WO 99/37674 PCT/US99/01404
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, NY.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, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces 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.
Such a
preparation employing a protein 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 osteogeruc agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
2 0 A protein of this invention may also be used in the treatment of
periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, 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.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
3 0 invention, which induces 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
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WO 99/37674 PCT/US99/01404
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 nova tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congerutai, 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 an environment to attract
tendon- ar
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 viva for return in viva 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.
The protein 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 protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
2 0 localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson s disease, Huntingtori 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
2 5 from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins 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.
3 0 It is expected that a protein of the present invention may also exhibit
activity for
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. Part of the desired effects may be by inhibition or
modulation
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WO 99!37674 PCT/US99/01404
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogeruc activity.
A protein 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.
A protein 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.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. WCr91/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
2 0 Activin /Inhibin ActivitX
A protein of the present invention may also exhibit activin- or inhibin-
related
activities. 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
2 5 invention, alone or in heterodimers with a member of the inhibin a 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-
3 0 ~ 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,
United States Patent 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 cows, sheep and
pigs.
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WO 99/37674 PCTNS99/01404
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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.
Chemotactic/Chemokinetic ActivitX
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
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.
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
2 0 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.
The activity of a protein of the invention may, among other means, be measured
2 5 by the following methods:
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
3 0 include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. ICruisbeek, D.H. Margulies, 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.
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WO 99/37674 PCT/US99/01404
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.
HemoStatic and Thrombolvtic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to 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 protein 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).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474,1988.
2 0 Receptor/Ligand,~, Acrivitv
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
2 5 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
3 0 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.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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VlrO 99/37674 PCTNS99/01404
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.
Anti-Inflammatory Activity
Proteins 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. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
2 0 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. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasio~,Su~~ressor ActivitX
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss
or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
3 0 tumor growth and metastasis. Cadherin malfunction is also implicated in
other human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
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extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynudeotides into cancer cells can reduce or eliminate the cancerous changes
observed
2 0 in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
2 5 inappropriately expressed cadherins, restoring normal cell adhesive
properties and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
3 0 the adhesion of inappropriately expressed tumor-cell cadherins, preventing
the cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
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WO 99/37674 PCT/US99/01404
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-
1038,1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
antibody-dependent cell-mediated cytotoxicity (ADCC)). A protein may exhibit
its tumor
inhibitory activity by acting on tumor tissue or tumor precursor tissue, by
inhibiting
formation of tissues necessary to support tumor growth (such as, for example,
by
2 0 inhibiting angiogenesis), by causing production of other factors, agents
or cell types which
inhibit tumor growth, or by suppressing, eliminating or inhibiting factors,
agents or cell
types which promote tumor growth.
9~her Activities
2 5 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
3 0 or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic 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, cofactors or other nutritional factors or
component(s);
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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.
ADMINISTRATION ANA DOSI_NG
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein 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
2 0 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, TNFO, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
2 5 cell factor, and erythropoietin. The pharmaceutical composition may
further contain other
agents which either enhance the activity of the protein 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 of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
3 0 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.
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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
muitimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) 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 NiHC genes on host cells will
serve to
present the peptide antigens) 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
immunolgobulin
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
2 0 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, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
2 5 art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent
No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
3 0 sufficient to show a meaningful patient benefit, i.e., 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, the term refers to that ingredient alone. When
applied to
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a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein 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 cytolcines,
lympholcines
or other hematopoietic factors. When co-administered with one or more
cytolcines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytolcine(s), lympholcine(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 of the present invention in combination with
cytolcine(s), lympholcine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein 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.
2 0 Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein 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
2 5 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid Garner 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
3 0 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
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
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When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein 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 of the present invention, an isotonic vehicle
such as Sodium
Chloride Injeckion, Ringei 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.
The amount of protein 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 of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein 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
2 0 increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 lzg
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 Ilg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
2 5 present invention will vary, depending on the severity of the disease
being treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
3 0 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
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WO 99/37674 PG"T/US99/01404
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. $,~, 2149-2154 (1963); J.L. Krstenansky, et
al., FEBS Lett.
21~, 10 (1987). 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.
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 of the invention
which may also
2 0 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
composition to the site of bone and/or cartilage damage, providing a structure
for the
2 5 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.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
3 0 application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, 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
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WO 99/37674 PCTNS99/01404
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, 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
tricalciumphosphate. 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.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), 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, polyethylene glycol), polyoxyethyiene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
2 0 wt%, preferably 1-10 wt% based on total formulation weight, which
represents the
amount necessary to prevent desorbtion 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.
2 5 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
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor {PDGF), transforming growth factors (TGF-
a and
TGF (i), and insulin-like growth factor (IGF).
3 0 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 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
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WO 99/37674 PCT/US99/01404
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.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
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.
2 0 Patent and literature references cited herein are incorporated by
reference as if
fully set forth.
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SEQUENCE LISTING
<110> Jacobs, Kenneth
MCCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Merberg, David
Treacy, Maurice
Agostino, Michael J.
Steininger II, Robert J.
Wong, Gordon G.
Clark, Hilary
Fechtel, Kim
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6062A
<140>
<141>
<160> 32
<170> PatentIn Ver. 2.0
<210> 1
<211> 3149
<212> DNA
<213> Homo sapiens
<400> 1
ggtctttaac gtgagcccgc tgcaggtgtg cggcccagtc cgagacagca gatgaggaga 60
ctgtccttcc tgtttcgcag atgaggaaac tgaggcttag agaagtttgg caaattggct 120
aagttcctac agctaccaca gcagaaagtg ctgggcagta gagagctgcc ccctccagaa 180
gatgatcagc tgcactccag tgcccccaga tcctcgtgga aggaacggat ccttaaagca 240
aaggtggtga cggtgtctca ggaggcagar tgggatcaaa tcgagccctt gcttagaagt 300
gaattagaag attttccagt acttggaatt gactgtgagt gggtaaattt ggaaggcaaa 360
gcctgccctc tgtcacttct acaaatggcc tccccaagtg gcctgtgtgt cttggttcgc 420
ctgcccaagc taatctgtgg aggaaaaaca ctaccaagaa cgttattgga tattttggca 480
gatggcacca ttttgaaagt tggagtggga tgctcagaag atgccagcaa gcttctgcag 540
gattatggcc tcgttgttag ggggtgcctg gacctccgat acctagccat gcggcagaga 600
aacaatttgc tctgtaatgg gcttagcctg aagtccctcg ctgagactgt tttgaacttt 660
ccccttgaca agtcccttct acttcgttgc agcaactggg atgctgagac tctcacagag 720
gaccaggtaa tttatgctgc cagggatgcc cagatttcag tggctctctt tcttcatctt 780
cttggatacc ctttctctag gaattcacct ggagaaaaaa aacgatgacc acagtagctg 840
gagaaaagtc ttggasaaat gccagggtgt ggtcgacatc ccatttcgaa gcaaaggaat 900
gagcagattg ggagaagagg ttaatgggga agcaacagaa tctcagcaga agccaagaaa 960
taagaagtct aagatggatg ggatggtgcc aggcaaccac caagggagag accccagaaa 1020
acataaaaga aagcctctgg gggtgggcta ttctgccaga aaatcacctc tttatgataa 1080
ctgctttctc catgctcctg atggacagcc cctctgcact tgtgatagaa gaaaagctca 1140
gtggtacctg gacaaaggca ttggtgagct ggtgagtgaa gagccctttg tggtgaagct 1200
gcggtttgaa cctgcaggaa ggcccgaatc tcctggagac tattacttga tggttaaaga 1260
gaacctgtgt gtagtgtgtg gcaagagaga ctcctacatt cggaagaacg tgattccaca 1320
tgagtaccgg aagcacttcc ccatcgagat gaaggaccac aactcccacg atgtgctgct 1380
gctctgcacc tcctgccatg ccatttccaa ctactatgac aaccatctga agcagcagct 1440
ggccaaggag ttccaggccc ccatcggctc tgaggagggc ttgcgcctgc tggaagatcc 1500
tgagcgccgg caggtgcgtt ctggggccag ggccctgctc aacgcggaga gcctgcctac 1560
tcatcgaaag gaggagctgc tgcaagcact cagagagttt tataacacag acgtggtcac 1620
agaggagatg cttcaagagg ctgccagcct ggagaccaga atctccaatg aaaactatgt 1680
1
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
tcctcacggg ctgaaggtgg tgcagtgtca cagccagggt ggcctgcgct ccctcatgca 1740
gctggagagc cgctggcgtc agcacttcct ggactccatg cagcccaagc acctgcccca 1800
gcagtggtca gtggaccaca accatcagaa gctgctccgg aaattcgggg aagatcttcc 1860
catccagctg tcttgatagc tgctttcctc ccagttagga caagtgggaa gctggagcca 1920
aggttgaaga gtcacctctt cccattttag tacatcatta attgtcaaag cctgtgtgac 1980
acaactcaga atactaacct agactaatcc caggatgctt ctgctggagc aaagatattg 2040
tttgaaggag agtttatggt tttggatttt aaacgggcag ggtctttttt cctctcattt 2100
ttgtggacaa gagaggcctt cgcctttatt tttactctcc ctcttctgct gtccctgtgc 2160
agaggaaaaa tgaagaattc tcccagaagt gacttgtcaa gacttaaaaa aaatgttttt 2220
aatgcatttc ttccttgtct agtgcctcgg tttatctcta acaggggctg tccagtatat 2280
cggtcctgtt aggaggggag aaaaagttct tccaaaggct ggagaagtga acaaggagtc 2340
aaatttattt tcccaattca acttcataat tatcatttct ttggcttcat gctctcccgt 2400
aactcatgtg gttgggatcc atcccatctg ggtcacttca gtctacttca cgtacttgaa 2460
aaggctttcc tttacacttc caggaccaaa cagcaacttc ctgccacaca cttccaccct 2520
atcactggga gaaatccttt tctggacatg agcctttgac ctgggtgggg cagaaagaac 2580
cacaaactcc atctcccaat agaactttga aattcactca gcttttcctt tcatgctgtt 2640
tgttgcctgc ttgttgcact cctcctgccc cagaactgca agatttttag cttcacccct 2700
ttctgagagt aatgttatct tttatcagaa tcagtatcag ttcccctgta ttctgtgctt 2760
catcgaattt gcaagactga cctcttttaa gcatttaatt cactcccaga gtcatctggt 2820
caggttgcaa tatgaggact tctctgtctc ctctgaagcc tgggacactg agcttactta 2880
atacattaga tgttcaaaag aggagcgttg tttcatcttt caaaatgtta ggccattact 2940
ttgaatataa aatcgactta ttaatgatta gtaatttttc taaagtattg ggaaaacttt 3000
cttattttat aagatcttaa caagcttaaa aaagaatttt atgaccagaa tccaacaaga 3060
gctctatttt ggaattgtgc ccaagttggt gatgtttact ctaaaattaa taataaaact 3120
acttgtaagc aaaaaaaaaa aaaaaaaaa 3149
<210> 2
<211> 380
<212> PRT
<213> Homo sapiens
<400> 2
Met Leu Pro Gly Met Pro Arg Phe Gln Trp Leu Ser Phe Phe Ile Phe
1 5 10 15
Leu Asp Thr Leu Ser Leu Gly Ile His Leu Glu Lys Lys Asn Asp Asp
20 25 30
His Ser Ser Trp Arg Lys Val Leu Glu Lys Cys Gln Gly Val Val Asp
35 40 45
Ile Pro Phe Arg Ser Lys Gly Met Ser Arg Leu Gly Glu Glu Val Asn
50 55 60
Gly Glu Ala Thr Glu Ser Gln Gln Lys Pro Arg Asn Lys Lys Ser Lys
65 70 75 80
Met Asp Gly Met Val Pro Gly Asn His Gln Gly Arg Asp Pro Arg Lys
85 90 95
His Lys Arg Lys Pro Leu Gly Val Gly Tyr Ser Ala Arg Lys Ser Pro
100 105 110
Leu Tyr Asp Asn Cys Phe Leu His Ala Pro Asp Gly Gln Pro Leu Cys
115 120 125
Thr Cys Asp Arg Arg Lys Ala Gln Trp Tyr Leu Asp Lys Gly Ile Gly
130 135 140
Glu Leu Val Ser Glu Glu Pro Phe Val Val Lys Leu Arg Phe Glu Pro
2
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
145 150 155 160
Ala G1y Arg Pro Glu Ser Pro Gly Asp Tyr Tyr Leu Met Val Lys Glu
165 170 175
Asn Leu Cys Val Val Cys Gly Lys Arg Asp Ser Tyr Ile Arg Lys Asn
leo le5 190
Val Ile Pro His Glu Tyr Arg Lys His Phe Pro Ile Glu Met Lys Asp
195 200 205
His Asn Ser His Asp Val Leu Leu Leu Cys Thr Ser Cys His Ala Ile
210 215 220
Ser Asn Tyr Tyr Asp Asn His Leu Lys Gln Gln Leu Ala Lys Glu Phe
225 230 235 240
Gln Ala Pro Ile Gly Ser Glu Glu Gly Leu Arg Leu Leu Glu Asp Pro
245 250 255
Glu Arg Arg Gln Val Arg Ser Gly Ala Arg Ala Leu Leu Asn Ala Glu
260 265 270
Ser Leu Pro Thr His Arg Lys Glu Glu Leu Leu Gln Ala Leu Arg Glu
275 280 285
Phe Tyr Asn Thr Asp Val Val Thr Glu Glu Met Leu Gln Glu Ala Ala
290 295 300
Ser Leu Glu Thr Arg Ile Ser Asn Glu Asn Tyr Val Pro His Gly Leu
305 310 315 320
Lys Val Val Gln Cys His Ser Gln Gly Gly Leu Arg Ser Leu Met Gln
325 330 335
Leu Glu Ser Arg Trp Arg Gln His Phe Leu Asp Ser Met Gln Pro Lys
340 345 350
His Leu Pro Gln Gln Trp Ser Val Asp His Asn His Gln Lys Leu Leu
355 360 365
Arg Lys Phe Gly Glu Asp Leu Pro Ile Gln Leu Ser
370 375 380
<210> 3
<211> 1861
<212> DNA
<213> Homo sapiens
<400> 3
agagccaggg gggtcgcgta gtgtcatgac cagggcggga gatcacaacc gccagagagg 60
atgctgtgga tccttggcgg actacctgac ctctgcaaaa ttccttctct accttggtca 120
ttctctctct acttggggag atcggatgtg gcactttgcg gtgtctgtgt ttctggtaga 180
gctctatgga aacagcctcc ttttgacagc agtctacggg ctggtggtgg cagggtctgt 240
tctggtcctg ggagccatca tcggtgactg ggtggacaag aatgctagac ttaaagtggc 300
ccagacctcg ctggtggtac agaatgtttc agtcatcctg tgtggaatca tcctgatgat 360
ggttttctta cataaacatg agcttctgac catgtaccat ggatgggttc tcacttcctg 420
ctatatcctg atcatcacta ttgcaaatat tgcaaatttg gccagtactg ctactgcaat 480
cacaatccaa agggattgga ttgttgttgt tgcaggagaa gacagaagca aactagcaaa 540
3
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
tatgaatgcc acaatacgaa ggattgacca gttaaccaac atcttagccc ccatggctgt 600
tggccagatt atgacatttg gctccccart catcggctgt ggctttattt cgggatggaa 660
cttggtatcc atgtgcgtgg agtacgttct gctctggaag gtttaccaga aaaccccagc 720
tctagctgtg aaagctggtc ttaaagaaga ggaaactgaa ttgaaacagc tgaatttaca 780
caaagatact gagccaaaac ccctggaggg aactcatcta atgggtgtga aagactctaa 840
catccatgag cttgaacatg agcaagagcc tacttgtgcc tcccagatgg ctgagccctt 900
ccgtaccttc cgagatggat gggtctccta ctacaaccag cctgtgtttc tggctggcat 960
gggtcttgct ttcctttata tgactgtcct gggctttgac tgcatcacca cagggtacgc 1020
ctacactcag ggactgagtg gttccatcct cagtattttg atgggagcat cagctataac 1080
tggaataatg ggaactgtag cttttacttg gctacgtcga aaatgtggtt tggttcggac 1140
aggtctgatc tcaggattgg cacagctttc ctgtttgatc ttgtgtgtga tctctgtatt 1200
catgcctgga agccccctgg acttgtccgt ttctcctttt gaagatatcc gatcaaggtt 1260
cattcaagga gagtcaatta cacctaccaa gatacctgaa attacaactg aaatatacat 1320
gtctaatggg tctaattctg ctaatattgt cccggagaca agtcctgaat ctgtgcccat 1380
aatctctgtc agtctgctgt ttgcaggcgt cattgctgct agaatcggtc tttggtcctt 1440
tgatttaact gtgacacagt tgctgcaaga aaatgtaatt gaatctgaaa gaggcattat 1500
aaatggtgta cagaactcca tgaactatct tcttratctt ctgcatttca tcatggtcat 1560
cctggctcca aatcctgaag cttttggctt gctcgtattg atttcagtct cctttgtggc 1620
aatgggccac attatgtatt tccgatttgc ccaaaatact ctgggaaaca agctctttgc 1680
ttgcggtcct gatgcaaaag aagttaggaa ggaaaatcaa gcaaatacat ctgttgtttg 1740
agacagttta actgttgcta tcctgttact agattatata gagcacatgt gcttattttg 1800
tactgcagaa ttccaataaa tggctgggtg tr_ttgctctg tttttaaaaa aaaaaaaaaa 1860
a
1861
<210> 4
<211> 571
<212> PRT
<213> Homo sapiens
<220>
<221> UNSURE
<222> (202)
<220>
<221> UNSURE
<222> (504)
<400> 4
Met Thr Arg Ala Gly Asp His Asn Arg Gln Arg Gly Cys Cys Gly Ser
1 5 10 15
Leu Ala Asp Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His
20 25 30
Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val
35 40 45
Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr
50 55 60
Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly
65 70 75 80
Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu
85 90 95
Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met
100 105 110
Val Phe Leu His Lys His Glu Leu Leu Thr Met Tyr His Gly Trp Val
4
CA 02318303 2000-07-13
WO 99/376'74 PCTNS99/01404
115 120 125
Leu Thr Ser Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn
130 135 140
Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val
145 150 155 160
Val Val Ala Gly Glu Asp Arg Ser Lys Leu Ala Asn Met Asn Ala Thr
165 170 175
Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Als Pro Met Ala Val
180 185 190
Gly Gln Ile Met Thr Phe Gly Ser Pro Xaa Ile Gly Cys Gly Phe Ile
195 200 205
Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Val Leu Leu Trp
210 215 220
Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Gly Leu Lys
225 230 235 240
Glu Glu Glu Thr Glu Leu Lys Gln Leu Asn Leu His Lys Asp Thr Glu
245 250 255
Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Val Lys Asp Ser Asn
260 265 270
Ile His Glu Leu Glu His Glu Gln Glu Pro Thr Cys Ala Ser Gln Met
275 280 285
Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn
290 295 300
Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr
305 310 315 320
Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly
325 330 335
Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr
340 345 350
Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly
355 360 365
Leu Val Arg Thr Gly Leu Ile Ser Gly Leu Ala Gln Leu Ser Cys Leu
370 375 380
Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu
385 390 395 400
Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu
405 410 415
Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr Thr Glu Ile Tyr Met
420 425 430
Ser Asn Gly Ser Asn Ser Ala Asn Ile Val Pro Glu Thr Ser Pro Glu
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
435 440 445
Ser Val Pro Ile hle Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala
450 455 460
Ala Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu
465 470 475 480
Gln Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln
485 490 495
Asn Ser Met Asn Tyr Leu Leu Xaa Leu Leu His Phe Ile Met Val Ile
500 505 510
Leu Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val
515 520 525
Ser Phe Val Ala Met Gly His Ile Met Tyr Phe Arg Phe Ala Gln Asn
530 535 540
Thr Leu Gly Asn Lys Leu Phe Ala Cys Gly Pro Asp Ala Lys Glu Val
545 550 555 560
Arg Lys Glu Asn Gln Ala Asn Thr Ser Val Val
565 570
<210> 5
<211> 2157
<212> DNA
<213> Homo Sapiens
<400> 5
ctctctttaa tatcttcacc tctaccatgt gtctttcttt taatatagtt ataattttcc 60
aaccacgtag atcaatattt actcatcatg accataaaat gcagtttagc catatagaaa 120
actatgatta cttttcttta taatttccct tcagttaata cttattttat tttctgtttt 180
tatcatctag tcaactcgca aacttccagc atttgtctaa atctactcaa tatattccag 240
tacatcagat aatatatcag tttcatcctc ctgaaaaact cttttccagt gtatcctgac 300
ctgctctaat tttgacttga tgctttctgt atctggtgca cagctgttac cttggaatct 360
tcccttcatc attattcaga gtgtttctgt agtttttctc ttgcattgga ttttgtgctt 420
cctgaatccc tctctCtctt tttttttttt tttttacttg gcttactcct tgctttgatg 480
gatctcaggc tccagtagct tccttggaaa gagtgtttgg aagttgcttc tgcaggaagc 540
ctttttggtg gcatggtcct caagaagttc ctaaaaggtt gatgaaaagc ccagaacctt 600
gatgacagat tgtctggtta taaagcattt tttacgtaaa atcatcatgg tgcaccctaa 660
ggtcagattt catttcagtg taaaggtaaa tggaatcctc tccacagaga tctttggggt 720
ggagaatgaa cccactttga accttgggaa tggaattgct cttttggtcg actcccagca 780
ttatgtgagt agaccaaatt ttggtacaat tgaatcacac tgcagcagaa ttcaccctgt 840
gctaggacat ccagtaatgc ttttcatccc tgaagacgtg gctggcatgg acttgttggg 900
agaactgata ctgactccag cagctgcact gtgccccagc ccaaaggttt cttccaacca 960
gcttaacagg atttcttcag tttccatatt tctatatgga cctttgggtc tgcctctgat 1020
attgtcaact tgggagcagc cgatgactac tttcttcaaa gatacctctt ctttagttga 1080
ctggaaaata ccatttgtgt atgataccca atttggatct caatttggat agagatttgg 1140
tgcttccaga tgtgagttat caggtggaat ccagtgagga ggatcagtct cagactatgg 1200
atcctcaagg acasactctg ctgctttttc tctttgtgga tttccacagt gcatttccag 1260
tccagcaaat ggaaatctgg ggagtctata ctttgctcac aactcatctc aatgccatcc 1320
ttgtggagag ccacagtgta gtgcaaggtt ccatccaatt cactgtggac aaggtcttgg 1380
agcaacatca ccaggctgcc aaggctcagc agaaactaca ggcctcactc tcagtggctg 1440
tgaactccat catgagtatt ctgactggaa gcactaggag cagcttccga aagatgtgtc 1500
tccagaccct tcaagcagct gacacacaag agttcaggac caaactgcac aaagtatttc 1560
gtgagatcac ccaacaccaa tttcttcacc actgctcatg tgaggtgaag cagctaaccc 1620
6
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
tagaaaaaaaggactcagcc cagggcactg tgataacagcagcctggagc1680
aggacgcacc
tcctagcagtgcttaaacag ccttcccagc aggggtacagcagctctcac1740
ccacagcagc
attcagtcactagcagagat gccagatacc cagaaaacaagaggctcaag1800
agcgggcaag
aggggcagcccccgcataga ggagatgcga ctgccagggccccgagcccg1860
gctctgcgct
tcagaggccgccccgcgccg cccggaagcc ccctcactcytagaggaagg1920
accgcggccc
gagcaccgcgaggctcacgg cagggccctg gggcgagcctcggaagccgc1980
gcgccgggca
ctggaggacgtcctgtggct gcaggaggtc cagagtggctgagtcccagc2040
tccaacctgt
cctgggccctgagccgggtc cccttccgca gatccggaggctgcgggcag2100
agcgcccacc
ccgttatcccgtggtttaat aaagctgccg aaaaaaaaaaaaaaaaa 2157
cgcgctcacc
<210>
6
<211>
270
<212>
PRT
<213> Sapiens
Homo
<400>
6
Met Ile Asn Leu Asp Leu Asn Leu Asp Leu Leu Pro
Pro Asp Arg Val
1 S 10 15
Asp Val Tyr Gln Val Glu Ser Ser Asp Gln Gln Thr
Ser Glu Glu Ser
20 25 30
Met Asp Gln Gly Gln Thr Leu Leu Leu Phe Asp Phe
Pro Leu Phe Val
35 40 45
His Ser Phe Pro Val Gln Gln Met Trp Gly Tyr Thr
Ala Glu Ile Val
50 55 60
Leu Leu Thr His Leu Asn Ala Ile Glu Ser Ser Val
Thr Leu Val His
65 70 75 80
Val Gln Ser Ile Gln Phe Thr Val Val Leu Gln His
Gly Asp Lys Glu
85 90 95
His Gln Ala Lys Ala Gln Gln Lys Ala Ser Ser Val
Ala Leu Gln Leu
100 105 110
Ala Val Ser Ile Met Ser Ile Leu Ser Thr Ser Ser
Asn Thr Gly Arg
115 120 125
Phe Arg Met Cys Leu Gln Thr Leu Ala Asp Gln Glu
Lys Gln Ala Thr
130 135 140
Phe Arg Lys Leu His Lys Val Phe Ile Thr His Gln
Thr Arg Glu Gln
145 150 155 160
Phe Leu His Cys Ser Cys Glu Val Leu Thr Glu Lys
His Lys Gln Leu
165 170 175
Lys Asp Ala Gln Gly Thr Glu Asp Asp Asn Ser Leu
Ser Ala Pro Ser
180 185 190
Glu Leu Ala Val Leu Lys Gln Pro Pro Thr Ala Gly
Leu Ser Gln Ala
195 200 205
Val Gln Leu Ser His Ser Val Thr Asp Ala Tyr Gln
Gln Ser Arg Arg
210 215 220
Arg Ala Arg Lys Gln Glu Ala Gln Gln Pro His Arg
Ser Glu Gly Pro
225 230 235 240
7
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
Gly Asp Ala Ser Ser Ala Leu Cys Gln Gly Pro GIu Pro Val Arg Gly
245 250 255
Arg Pro Ala Pro Pro Gly Ser His Arg Gly Pro Pro His Ser
260 265 270
<210> 7
<211> 1607
<212> DNA
<213> Homo Sapiens
<400> 7
gtgaacttca ctactggaaa gcaacaaagg cagtcggcat aaaaatgggt tctctcagca 60
cagctaacgt tgaattttgc cttgatgtgt tcaaagagct gaacagtaac aacataggag 120
ataacatctt cttttcttcg ctgagtctgc tttatgctct aagcatggtc ctccttggtg 180
ccaggggaga gactgcagag caattggaga aggtgcttca ttttagtcat actgtagact 240
cattaaaacc agggttcaag gactcaccta agtgcagcca agctggaaga attcattccg 300
agtttggtgt ctaattctct caaatcaacc agccagactc taactgtacc ctcagcattg 360
ccaacaggct ctacgggaca aagacgatgg catttcatca ggaaaagtcg caaatctctt 420
tggaaagagc acaattgacc cttcatctgt aatggtcctg gtgaatacca tatatttcaa 480
aggacaatgg caaaataaat ttcaagtaag agagacagtt aaaagtcctt ttcagctaag 540
tgagggtaaa aatgtaactg tggaaatgat gtatcaaatt ggaacattta aactggcctt 600
tgtaaaggag ccgcagatgc aagttcttga gctgccctac gttaacaaca aattaagcat 660
gattattctg cttccagtag gcatagctaa tctgaaacag atagaaaagc agctgaattc 720
ggggacgttt catgagtgga caagctcttc taacatgatg gaaagagaag ttgaagtaca 780
cctccccaga ttcaaacttg aaattaagta tgagctaaat tccctgttaa aacctctagg 840
ggtgacagat ctcttcaacc aggtcaaagc tgatctttct ggaatgtcac caaccaaggg 900
cctatattta tcaaaagcca tccacaagtc atacctggat gtcagcgaag agggcacgga 960
ggcagcagca gccactgggg acagcatcgc tgtaaaaagc ctaccaatga gagctcagtt 1020
caaggcgaac caccccttcc tgttctttat aaggcacact cataccaaca cgatcctatt 1080
ctgtggcaag cttgcctctc cctaatcaga tggggttgag taaggctcag agttgcagat 1140
gaggtgcaga gacaatcctg tgactttccc acggccaaaa agctgttcac acctcacaca 1200
cctctgtgcc tcagtttgct catctgcaaa ataggtctag gatttcttcc aaccatttca 1260
tgagttgtga agctaaggct ttgttaatca tggaaaaagg tagacttatg cagaaagcct 1320
ttctggcttt cttatctgtg gtgtctcatt tgagtgctgt ccagtgacat gatcasgtca 1380
atgagtaaaa ttttaaggga ttagattttc ttgacttgta kgtatctgtg agatcttgaa 1440
taagtgacct gacatctctg cttaaagaaa accagctgaa gggcttcaac tttgcttgga 1500
tttttaaata ttttccttgc atatgtaaat agaatgtggt gagttttagt tcaasattct 1560
ctgttgagaa taataaatgc atgaaatacc ttaaaaaaaa aaaaaaa 1607
<210> 8
<211> 217
<212> PRT
<213> Homo Sapiens
<400> 8
Met Val Leu Val Asn Thr Ile Tyr Phe Lys Gly Gln Trp Gln Asn Lys
1 5 10 15
Phe Gln Val Arg Glu Thr Val Lys Ser Pro Phe Gln Leu Ser GIu Gly
20 25 30
Lys Asn Val Thr Val Glu Met Met Tyr Gln Ile Gly Thr Phe Lys Leu
35 40 45
Ala Phe Val Lys Glu Pro Gln Met Gln Val Leu Glu Leu Pro Tyr Val
50 55 60
8
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
Asn Asn Lys Leu Ser Met Ile Ile Leu Leu Pro Val Gly Ile Ala Asn
65 70 75 80
Leu Lys Gln Ile Glu Lys Gln Leu Asn Ser Gly Thr Phe His Glu Trp
85 90 95
Thr Ser Ser Ser Asn Met Met Glu Arg Glu Val Glu Val His Leu Pro
100 105 I10
Arg Phe Lys Leu Glu Ile Lys Tyr Glu Leu Asn Ser Leu Leu Lys Pro
115 120 125
Leu Gly Val Thr Asp Leu Phe Asn Gln Val Lys Ala Asp Leu Ser Gly
130 135 140
Met Ser Pro Thr Lys Gly Leu Tyr Leu Ser Lys Ala Ile His Lys Ser
145 150 155 160
Tyr Leu Asp Val Ser Glu Glu Gly Thr Glu Ala Ala Ala Ala Thr Gly
165 170 175
Asp Ser Ile Ala Val Lys Ser Leu Pro Met Arg Ala Gln Phe Lys Ala
180 185 190
Asn His Pro Phe Leu Phe Phe Ile Arg His Thr His Thr Asn Thr Ile
195 200 205
Leu Phe Cys Gly Lys Leu Ala Ser Pro
210 215
<210> 9
<211> 1537
<212> DNA
<213> Homo sapiens
<400> 9
gtaggatttg gggatgtgga tatttaagac aatttctttt ttcttttggt ttaatagggg 60
cgggtatagg gaccaactgg gaccgagtgc ccagggggcc gagcacggtc atgctggccg 120
gcctgcatgc atgcgtgtgc cgggctgggc tgggcggccg gcggtcgtgg ggcagggttg 190
ggggtctgtg ctcagctgat aactgccatg cactgtactg cacacgtccc tagagcctac 240
cgggacccga cgcttttcag ggcatttctc cctccagcca gggcccaact cccacctgcc 300
tgggcgaatc tcctccaagg aagtcccagg aggatgggga ccaggaaggc tgtggacccc 360
catctccagg gggccttccc agcctgatcc ctgtcctcca agttctggag gaggccgctg 420
tagggtctgg ctgagcttcc cacccacttt ccctggtccc aatcctttct tgtcctatac 480
ccagctgggg ttgctgccct gaacgaactg cgtgtggggc cggcacatcc tagcaggcag 540
cccctggcgc ctgctgcctc agggatgctc caaccaccct cgttctcctc gcagtggccc 600
tggctcccac ctcccgcccc agcctgccgt ggggcccgtc agcctggtcc cacccccatg 660
gagaacccaa agtcttactg tatataactc caggtgacgt ttctatattt atagcagtgt 720
tgaaaaccca cgtgttttac acagaaccac cctctccaac ccctcccttc ccgaccccaa 780
caaaacgttt tcaaacccct tacagttcct ggggcaggcg gaaacaggct cacagattgt 840
gtgtcggctg cagcagtgat tccaacaagc agctattggg ggggaaacac agcatttaaa 900
aagatcatca ttaaaaaaca agatttatac aacaattact taggatgttt gtgatctgcc 960
gaccttgcta tagatgccat gttaccaatg atttcctgtg gtgggggctt gccattgttt 1020
actctcttat ttaccaactt ctggcctagg catgacagtg ggcaccttcc cccagccctg 1080
gctgggccca gcgcctgtgt tytgtgttag aaaggtttta tatatatata aaattacata 1140
tatakgtaga aatatatgta attttggggg ccctgttcct tgcacatttt acagttacct 1200
catttttccc atgtatgtat ttgagaaaat gctaatatat agagaaaaaa atggttctta 1260
aaacttaaat gtgtggtttt ttccattcca tgggattcac attggtttgt agcatttaac 1320
ataactagta tgttgtatta tatatatgtg tatactgatt gaaattttta acagatttgt 1380
9
CA 02318303 2000-07-13
WO 99/37674 PC"TNS99/01404
acttttttta aaatgaaagt tgctagttct gcttgaccaa gtagtgcaat cattattttt 1440
tttaatattg ttgctgattt cagagggata ttcactaata aatgtatgat gtatacccac 1500
graaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1537
<210> 10
<211> 86
<212> PRT
<213> Homo sapiens
<400> 10
Met His Ala Cys Ala Gly Leu Gly Trp Ala Ala Gly Gly Arg Gly Ala
1 5 10 15
Gly Leu Gly Val Cys Ala Gln Leu Ile Thr Ala Met His Cys Thr Ala
20 25 30
His Val Pro Arg Ala Tyr Arg Asp Pro Thr Leu Phe Arg Ala Phe Leu
35 40 45
Pro Pro Ala Arg Ala Gln Leu Pro Pro Ala Trp Ala Asn Leu Leu Gln
50 55 60
Gly Ser Pro Arg Arg Met Gly Thr Arg Lys Ala Val Asp Pro His Leu
65 70 75 80
Gln Gly Ala Phe Pro Ala
<210> 11
<211> 1302
<212> DNA
<213> Homo sapiens
<400> 11
cttcatggcc tacacacacc accttacccc tctgctggca agaggggacc tgattcatcc 60
tcacgctaaa cactcattct acccaactga ttgagacaga acagaagata aactgaaact 120
tctctgcctt cccgctgcaa gagtgaatga gcgatccctc tcaactgact caaaatgttt 180
gcctcaccca ggagatggag ctctcgaagg ccttctctgg ccagcggaca ctcctatctg 240
ccatcctcag catgctatca ctcagcttct ccacaacatc cctgctcagc aactactggt 300
ttgtgggcac acagaaggtg cccaagcccc tgtgcgagaa aggtctggca gccaagtgct 360
ttgacatgcc agtgtccctg gatggagata ccaacacatc cacccaggag gtggtacaat 420
acaactggga gactggggat gaccggttct ccttccggag cttccggagt ggcatgtggc 480
tatcctgtga ggaaactgtg gaagaaccag gggagaggtg ccgaagtttc attgaactta 540
caccaccagc caagagagaa atcctatggt tatccctggg aacgcagatc acctacatcg 600
gacttcaatt catcagcttc ctcctgctac taacagactt gctactcact gggaaccctg 660
cctgtgggct caaactgagc gcctttgctg ctgtttcctc tgtcctgtca ggtctcctgg 720
ggatggtggc ccacatgatg tattcacaag tcttccaagc gactgtcaac ttgggtccag 780
aagactggag accacatgtt tggaattatg gctgggcctt ctacatggcc tggctctcct 840
tcacctgctg catggcgtcg gctgtcacca ccttcaacac gtacaccagg atggtgctgg 900
agttcaagtg caagcatagt aagagcttca aggaaaaccc gaactgccta ccacatcacc 960
atcagtgttt ccctcggcgg ctgtcaagtg cagcccccac cgtgggtcct ttgaccagct 1020
accaccagta tcataatcag cccatccact ctgtctctga gggagtcgac ttctactccg 1080
agctgcggaa caagggattt caaagagggg ccagccagga gctgaaagaa gcagttaggt 1140
catctgtaga ggaagagcag tgttaggagt taagcgggtt tggggagtag gcttgagccc 1200
taccttacac gtctgctgat tatcaacatg tgcttaagcc aaaaaaaaaa aaaaaaaaaa 1260
aaseaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa as 1302
<210> 12
<211> 339
lU
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
<212> PRT
<213> Homo sapiens
<400> 12
Met Ser Asp Pro Ser Gln Leu Thr Gln Asn Val Cys Leu Thr Gln Glu
1 5 10 15
Met Glu Leu Ser Lys Ala Phe Ser Gly Gln Arg Thr Leu Leu Ser Ala
20 25 30
Ile Leu Ser Met Leu Ser Leu Ser Phe Ser Thr Thr Ser Leu Leu Ser
35 40 45
Asn Tyr Trp Phe Val Gly Thr Gln Lys Val Pro Lys Pro Leu Cys Glu
50 55 60
Lys Gly Leu Ala Ala Lys Cys Phe Asp Met Pro Val Ser Leu Asp Gly
65 70 75 80
Asp Thr Asn Thr Ser Thr Gln Glu Val Val Gln Tyr Asn Trp Glu Thr
B5 90 95
Gly Asp Asp Arg Phe Ser Phe Arg Ser Phe Arg Ser Gly Met Trp Leu
100 105 110
Ser Cys Glu Glu Thr Val Glu Glu Pro Gly Glu Arg Cys Arg Ser Phe
115 120 125
Ile Glu Leu Thr Pro Pro Ala Lys Arg Glu Ile Leu Trp Leu Ser Leu
130 135 140
Gly Thr Gln Ile Thr Tyr Ile Gly Leu Gln Phe I1e Ser Phe Leu Leu
145 150 155 160
Leu Leu Thr Asp Leu Leu Leu Thr Gly Asn Pro Ala Cys Gly Leu Lys
165 170 175
Leu Ser Ala Phe Ala Ala Val Ser Ser Val Leu Ser Gly Leu Leu Gly
180 185 190
Met Val Ala His Met Met Tyr Ser Gln Val Phe Gln Ala Thr Val Asn
195 200 205
Leu Gly Pro Glu Asp Trp Arg Pro His Val Trp Asn Tyr Gly Trp Ala
210 215 220
Phe Tyr Met Ala Trp Leu Ser Phe Thr Cys Cys Met Ala Ser Ala Val
225 230 235 240
Thr Thr Phe Asn Thr Tyr Thr Arg Met Val Leu Glu Phe Lys Cys Lys
245 250 255
His Ser Lys Ser Phe Lys Glu Asn Pro Asri Cys Leu Pro His His His
260 265 270
Gln Cys Phe Pro Arg Arg Leu Ser Ser Ala Ala Pro Thr Val Gly Pro
275 280 285
Leu Thr Ser Tyr His Gln Tyr His Asn Gln Pro Ile His Ser Val Ser
290 295 300
11
CA 02318303 2000-07-13
VlrO 99/37674 PCT/US99/01404
Glu Gly Val Asp Phe Tyr Ser Glu Leu Arg Asn Lys Gly Phe Gln Arg
305 310 315 320
Gly Ala Ser Gln Glu Leu Lys Glu Ala Val Arg Ser Ser Val Glu Glu
325 330 335
Glu Gln Cys
<210> 13
<211> 3184
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (1644)
<400> 13
gtgcatgctt gtaatcgcag ctacttcgga gcctgagaga ctccttcagg gtgagcaaag 60
gcctggaaaa acctgtatgc agataaagaa aaggaaagaa agagataatc agtgcatgca 120
gttgtcagct ggctgggacc tgaggagagt cacttgtgga ggcaactggt ctttatcccc 180
attgtccggt acaaggcagg cattaatcct gtgatcctta tctgaagctc agctacaagg 240
ctttggccga ccaagtgtgt accatgctgc tattgtcatc ttccttgaat tctttgcgtg 300
gggcctgttg acaactccaa tgttgactgt tctacatgaa acattttctc aacacacatt 360
cctcatgaat ggtctcattc aaggtgtaaa gggcctgctc tcttttttga gtgccccact 420
cattggtgcc ctgtctgatg tgtgggggag gaagcccttt ctcctcggca ctgtattctt 480
tacctgcttc ccaatcccac tgatgaggat cagcccatgg tggtattttg cgatgatttc 540
tgtgtctgga gtcttctcgg tcacgttttc tgttatattt gcctatgtag ctgatgtcac 600
tcaggagcac gagcgaagta cagcttatgg atgggtctca gccacctttg cggctagtct 660
tgtcagcagc ccggccattg gagcatatct ttctgccagt tacggagaca gcctcgttgt 720
gctggtggcc acagtggtgg ctcttctgga catctgcttc atcttagtgg ctgttccaga 780
atctctgcct gagaaaatga gaccggtttc ctggggagct cagatttctt ggaaacaagc 840
agaccctttt gcgtcgttga agaaagttgg aaaagattct actgtcttac taatctgcat 900
caccgtgttt ctttcatacc ttcctgaagc tggacagtat tcaagttttt ttctctatct 960
caggcaggtc ataggttttg gatctgttaa aattgcagca ttcatagcta tggtaggaat 1020
tctgtctatt gtggctcaga cggcctttct tagcatcttg atgagatcat taggaaataa 1080
gaatactgtc ctccttggct tgggcttcca gatgctccag ttagcctggt acggttttgg 1140
atcacaggcc tggatgatgt gggcagcagg gaccgtggct gccatgtcca gcatcacgtt 1200
tccggcaatc agtgccctcg tctctcggaa tgcagagtca gatcagcaag gagttgccca 1260
ggggatcata actggaataa gaggactatg caatggcctg gggccagcac tgtatggctt 1320
catattctac atgttccatg tggaactgac tgagttgggc ccgaaattga attctaacaa 1380
cgttcccctg cagggagctg tcatcccagg cccgccgttt ttatttgggg catgtatagt 1440
ccttatgtct tttctggttg ccttattcat tcctgaatac agtaaagcca gtggagttca 1500
aaaacacagt aacagcagca gcggcagcct gaccaacacc ccagaacggg gcagtgatga 1560
ggacattgag ccactactgc aagacagcag catctgggag ctctcttcat ttgaggagcc 1620
tgggaatcag tgcactgagc tgtnaactcg gcagaaagtg ggattctgca tacgccatct 1680
ctgagagcca tggagggagc cacacccctg gtgacttcat ggtgctggat gggagacgct 1740
agcggcatcc ttcagggcca agtttgataa ataccaccgc catcattctg ctcatcctcc 1800
tcctgttttt tttttttctc ttacattctt tttttttttc ctgtttatac attagaacaa 1860
gataagattt gaaatacttc cttgcaaata atgtgcaact cccaaggtga aactcaaata 1920
gaaaaagtca tctctctggt agaaaggatg gctttcctgt aatgactata gagtaagagt 1980
ggcagcaatc tttccatgcc cttttcagca gaaggcacag aacagtagcg ggactgccat 2040
ctctggcaag atttcaggta aagaatctct tcttaatttc taccttcctg tttctctgaa 2100
tcagcccata ggtgttgatg agtggccact cttaaagagt cactcagtat cagggatcta 2160
ctgtctttgt tcaaaggtca aataaaaacc tagtctcctt ttattctact ttctattctt 2220
agctagaatg aaactcagca tatatacact tctggacata ataatattga atagtaatta 2280
cctttactag atgaaagaaa ttttcattac aaacttasat catgtaaaac tcaacaactc 2340
12
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
agattcctgg acctggtgtc ctggttgggt ccaaggtgat tttacagaag aaaaaaacaa 2900
ctcaagcatt ctggtggcaa catagagatt gtaggctgct tctaagaaag ttattaacaa 2460
tttggaaatt cctaagtagg atgagagtta gtaactggat acgagtgaag tttatatcca 2520
agttcagact caaaggcatt attatgattt gcttcttccc atgtcttcca tgtcctgctt 2580
ctcaaagttt ttctcatcca tcacactcct gccttaactg ctctgagtat gcatttgttt 2640
tcaattcatc tttatttcaa tctgtttaac ttttgaatcg catgggaata cgcacattaa 2700
gttcctttct aaaataaggt tttatgaagc tgagtttcac gataagtgtc ttgctatttt 2760
ttgagatgtt ttatggacaa agaaaacttt acagatttat atgtattttg ctgcaccagt 2820
aaatggacca ttaactaggg cccaccttta acagagcacc cctttgaaag ttttataggt 2880
atgaaatata tgtagatatt tgtaaagggt tttaattttt tttttttgat ggggtgctgt 2940
gtaaatcttg tatttataaa tgtaatgaag gtattgacag aaaaaaatat atacaacttt 3000
tataaaggat tgtgtactga ctgaatacat ttaaaagaaa atatattttg aaacctgttc 3060
tgctatgaac agagataaca tatcttttta ctatgctatt ggtttttagg ttaagcttcc 3120
taatgcataa taaatttaca gtggttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180
aaaa 3184
<210> 14
<211> 454
<212> PRT
<213> Homo Sapiens
<220>
<221> UNSURE
<222> (442)
<400> 14
Met Leu Thr Val Leu His Glu Thr Phe Ser Gln His Thr Phe Leu Met
1 5 10 15
Asn Gly Leu Ile Gln Gly Val Lys Gly Leu Leu Ser Phe Leu Ser Ala
20 25 30
Pro Leu Ile Gly Ala Leu Ser Asp Val Trp Gly Arg Lys Pro Phe Leu
35 40 45
Leu Gly Thr Val Phe Phe Thr Cys Phe Pro Ile Pro Leu Met Arg Ile
50 55 60
Ser Pro Trp Trp Tyr Phe Ala Met Ile Ser Val Ser Gly Val Phe Ser
65 70 75 80
Val Thr Phe Ser Val Ile Phe Ala Tyr Val Ala Asp Val Thr Gln Glu
85 90 95
His Glu Arg Ser Thr Ala Tyr Gly Trp Val Ser Ala Thr Phe Ala Ala
100 105 110
Ser Leu Val Ser Ser Pro Ala Ile Gly Ala Tyr Leu Ser Ala Ser Tyr
115 120 125
Gly Asp Ser Leu Val Val Leu Val Ala Thr Val Val Ala Leu Leu Asp
130 135 140
Ile Cys Phe Ile Leu Val Ala Val Pro Glu Ser Leu Pro Glu Lys Met
145 150 155 160
Arg Pro Val Ser Trp Gly Ala Gln Ile Ser Trp Lys Gln Ala Asp Pro
165 170 175
Phe Ala Ser Leu Lys Lys Val Gly Lys Asp Ser Thr Val Leu Leu Ile
13
CA 02318303 2000-07-13
V110 99/37674 PCT/US99/01404
lao las 190
Cys Ile Thr Val Phe Leu Ser Tyr Leu Pro Glu Ala Gly Gln Tyr Ser
195 200 205
Ser Phe Phe Leu Tyr Leu Arg Gln Val Ile Gly Phe Gly Ser Val Lys
210 215 220
Ile Ala Ala Phe Ile Ala Met Val Gly Ile Leu Ser Ile Val Ala Gln
225 230 235 240
Thr Ala Phe Leu Ser Ile Leu Met Arg Ser Leu Gly Asn Lys Asn Thr
245 250 255
Val Leu Leu Gly Leu Gly Phe Gln Met Leu Gln Leu Ala Trp Tyr Gly
260 265 270
Phe Gly Ser Gln Ala Trp Met Met Trp Ala Ala Gly Thr Val Ala Ala
275 280 285
Met Ser Ser Ile Thr Phe Pro Ala Ile Ser Ala Leu Val Ser Arg Asn
290 295 300
Ala Glu Ser Asp Gln Gln Gly Val Ala Gln Gly Ile Ile Thr Gly Ile
305 310 315 320
Arg Gly Leu Cys Asn Gly Leu Gly Pro Ala Leu Tyr Gly Phe Ile Phe
325 330 335
Tyr Met Phe His Val Glu Leu Thr Glu Leu Gly Pro Lys Leu Asn Ser
340 345 350
Asn Asn Val Pro Leu Gln Gly Ala Val Ile Pro Gly Pro Pro Phe Leu
355 360 365
Phe Gly Ala Cys Ile Val Leu Met Ser Phe Leu Val Ala Leu Phe Ile
370 375 380
Pro Glu Tyr Ser Lys Ala Ser Gly Val Gln Lys His Ser Asn Ser Ser
385 390 395 400
Ser Gly Ser Leu Thr Asn Thr Pro Glu Arg Gly Ser Asp Glu Asp Ile
405 410 415
Glu Pro Leu Leu Gln Asp Ser Ser Ile Trp Glu Leu Ser Ser Phe Glu
420 425 430
Glu Pro Gly Asn Gln Cys Thr Glu Leu Xaa Thr Arg Gln Lys Val Gly
435 440 445
Phe Cys Ile Arg His Leu
450
<210> 15
<211> 2481
<212> DNA
<213> Homo sapiens
<400> 15
14
CA 02318303 2000-07-13
WO 99/37674 PC1'/US99/01404
aggtctagaa ttcaatcggg aagaaggaaa agttcccttc tgctgtgaaa ctatttggca 60
agaggctgga gggcccaatg gctgcaaaat cgcaacccaa cattcccaaa gccaagagtc 120
tagatggcgt caccaatgac agaaccgcat ctcaagggca gtggggccgt gcctgggagg 180
tggactggtt ttcactggcg agcgtcatct tcctactgct gttcgccccc ttcatcgtct 240
actacttcat catggcttgt gaccaataca gctgcgccct gaccggccct gtggtggaca 300
tcgtcaccgg acatgctcgg ctctcggaca tctgggccaa gactccacct ataacgagga 360
aagccgccca gctctatacc ttgtgggtca ccttccaggt gcttctgtac acgtctctcc 420
ctgacttctg ccataagttt ctacccggct acgtaggagg catccaggag ggggccgtga 480
ctcctgcagg ggttgtgaac aagtatcaga tcaacggcct gcaagcctgg ctcctcacgc 540
acctgctctg gtttgcaaac gctcatctcc tgtcctggtt ctcgcccacc atcatcttcg 600
acaactggat cccactgctg tggtgcgcca acatccttgg ctatgccgtc tccaccttcg 660
ccatggtcaa gggctacttc ttccccacca gcgccagaga ctgcaaattc acaggcaatt 720
tcttttacaa ctacatgatg ggcatcgagt ttaaccctcg gatcgggaag tggtttgact 780
tcaagctgtt cttcaatggg cgccccggga tcgtcgcctg gaccctcatc aacctgtcct 840
tcgcagcgaa gcagcgggag ctccacagcc atgtgaccaa tgccatggtc ctggtcaacg 900
tcctgcaggc catctacgtg attgacttct tctggaacga aacctggtac ctgaagacca 960
ttgacatctg ccatgaccac ttcgggtggt acctgggctg gggcgactgt gtctggctgc 1020
cttatcttta cacgctgcag ggtctgtact tggtgtacca ccccgtgcag ctgtccaccc 1080
cgcacgccgt gggcgtcctg ctgctgggcc tggtgggcta ctacatcttc cgggtggcca 1140
accaccagaa ggacctgttc cgccgcacgg atgggcgctg cctcatctgg ggcaggaagc 1200
ccaaggtcat cgagtgctcc tacacatccg ccgacgggca gaggcaccac agcaagctgc 1260
tggtgtcggg cttctggggc gtggcccgcc acttcaacta cgtcggcgac ctgatgggca 1320
gcctggccta ctgcctggcc tgtggcggtg gccacctgct gccctacttc tacatcatct 1380
acatggccat cctgctgacc caccgctgcc tccgggacga gcaccgctgc gccagcaagt 1440
acggccggga ctgggagcgc tacaccgccg cagtgcctta ccgcctgctg cctggaatct 1500
tctaagggca cgccctaggg agaagccctg tggggctgtc aagagcgtgt tctgccaggt 1560
ccatgggggc tggcatccca gctccaactc gaggagcctc agtttcctca tctgtaaact 1620
ggagagagcc cagcacttgg caggtgtcca gtacctaatc acgctctgtt ccttgctttt 1680
gccttcaagg gaattccgag tgtccagcac tgccgtattg ccagcacaga cggattttct 1740
ctaatcagtg tccctggggc aggaggatga cccagtcacc tttactagtc ctttggagac 1800
aatttacctg tattaggagc ccaggccacg ctacactctg cccacactgg tgagcaggag 1860
gtcttcccac gccctgtcat taggctgcat ttactcttgc taaataaaag tgggagtggg 1920
gcgtgcgcgt tatccatgta ttgcctttca gctctagatc cccctcccct gcctgctctg 1980
cagtcgtggg tggggcccgt gcgccgtttc tccttggtag cgtgcacggt gttgaactgg 2040
gacactgggg agaaaggggc tttcatgtcg tttccttcct gctcctgctg macagctgcc 2100
aggagtgctc tgcctggagt ctgcagacct cagagaggtc ccagcactgg ctgtggcctt 2160
tcaggtgtag gcaggtgggc tctgcttccc gattccctgt gagcgcccac cctctcgaaa 2220
gaattttctg cttgccctgt gactgtgcag actctggctc gagcaacccg gggaacttca 2280
ccctcagggg cctcccacac cttctccagc gaggaggtyt cagtcccagc ctcgggaggg 2340
cacctccttt tctgtgcttt cttccctgag gcattcttcc tcatccctag ggtgttgtgt 2400
agaactcttt ttaaactcta tgctccgagt agagttcatc tttatattaa acttcccctg 2460
ttcaaataaa aaaaaaaaaa a 2481
<210> 16
<211> 475
<212> PRT
<213> Homo sapiens
<400> 16
Met Ala Ala Lys Ser Gln Pro Asn Ile Pro Lys Ala Lys Ser Leu Asp
1 5 10 15
Gly Val Thr Asn Asp Arg Thr Ala Ser Gln Gly Gln Trp Gly Arg Ala
20 25 30
Trp Glu Val Asp Trp Phe Ser Leu Ala Ser Val Ile Phe Leu Leu Leu
35 40 45
Phe Ala Pro Phe Ile Val Tyr Tyr Phe Ile Met Ala Cys Asp Gln Tyr
50 55 60
CA 02318303 2000-07-13
WO 99/37674 PGT/US99/01404
Ser Cys Ala Leu Thr Gly Pro Val Val Asp Ile Val Thr Gly His Ala
65 70 75 80
Arg Leu Ser Asp Ile Trp Ala Lys Thr Pro Pro Ile Thr Arg Lys Ala
85 90 95
Ala Gln Leu Tyr Thr Leu Trp Val Thr Phe Gln Val Leu Leu Tyr Thr
100 105 110
Ser Leu Pro Asp Phe Cys His Lys Phe Leu Pro Gly Tyr Val Gly Gly
115 120 125
Ile Gln Glu Gly Ala Val Thr Pro Ala Gly Val Val Asn Lys..Tyr Gln
130 135 140
Ile Asn Gly Leu Gln Ala Trp Leu Leu Thr His Leu Leu Trp Phe Ala
145 150 155 16Q
Asn Ala His Leu Leu Ser Trp Phe Ser Pro Thr Ile Ile Phe Asp Asn
165 170 175
Trp Ile Pro Leu Leu Trp Cys Ala Asn Ile Leu Gly Tyr Ala Val Ser
180 185 190
Thr Phe Ala Met Val Lys Gly Tyr Phe Phe Pro Thr Ser Ala Arg Asp
195 200 205
Cys Lys Phe Thr Gly Asn Phe Phe Tyr Asn Tyr Met Met Gly Ile Glu
210 215 220
Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu Phe Phe Asn
225 230 235 240
Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu Ser Phe Ala
245 250 255
Ala Lys Gln Arg Glu Leu His Ser His Val Thr Asn Ala Met Val Leu
260 265 270
Val Asn Val Leu Gln Ala Ile Tyr Val Ile Asp Phe Phe Trp Asn Glu
275 280 285
Thr Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His Phe Gly Trp
290 295 300
Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Tyr Leu Tyr Thr Leu
305 310 315 320
Gln Gly Leu Tyr Leu Val Tyr His Pro Val Gln Leu Ser Thr Pro His
325 330 335
Ala Val Gly Val Leu Leu Leu Gly Leu Val Gly Tyr Tyr Ile Phe Arg
340 345 350
Val Ala Asn His Gln Lys Asp Leu Phe Arg Arg Thr Asp Gly Arg Cys
355 360 365
Leu Ile Trp Gly Arg Lys Pro Lys Val Ile Glu Cys Ser Tyr Thr Ser
370 375 380
16
CA 02318303 2000-07-13
VVO 99/37674 PCTNS99/01404
Ala Asp Gly Gln Arg His His Ser Lys Leu Leu Val Ser Gly Phe Trp
385 390 395 400
Gly Val Ala Arg His Phe Asn Tyr Val Gly Asp Leu Met Gly Ser Leu
405 410 415
Ala Tyr Cys Leu Ala Cys Gly Gly Gly His Leu Leu Pro Tyr Phe Tyr
420 425 430
Ile Ile Tyr Met Ala Ile Leu Leu Thr His Arg Cys Leu Arg Asp Glu
435 440 445
His Arg Cys Ala Ser Lys Tyr Gly Arg Asp Trp Glu Arg Tyr Thr Ala
450 455 460
Ala Val Pro Tyr Arg Leu Leu Pro Gly Ile Phe
465 470 475
<210> 17
<211> 1518
<212> DNA
<213> Homo sapiens
<400> 17
cttccccact ggctcttggt ttatgagttc cccttttaag gatctgttgt gacttaccta 60
tctgggctag tgacctcaga tgtctcagac tgagcatctt accactgttt ctggttgatc 120
ccttcactca tggtcttaac acatttgcac ttcctctcat ctcagagagt acagtcacgg 180
ggcagagctt gcatagggat ccaggtgtta ctagtcttac tctggagctg gtccaactca 240
gtttcatggc acagaactag attaggtctc cactgcgcag tctgttttac tgcttaggga 300
aagccagctt ttctacccac acacgtttag tttgaagagt atctattttt ggagggttct 360
ttgggaggtt gggcaggctt ctttggatcc cagatacatt tagagctttt tgcattaagt 420
gtgaggaaaa taacttctct ttgatgatgt tgatacacca tgtkggcacc ytggggcaca 480
gcggtttagc tggggagatt ccatgagaat gaacecaaac tactcttctt tgctagggtc 540
ctttacccac acagaggtga gcctttcagg ttcttcattt tgcttagttt cttcccttgt 600
ccttggcatt taagaggcat ccatgtgtta gccagccaaa gccccctgaa ggagctggct 660
gctttaaagg atttacttgg gaggatgtca satggctttg ccttctgcag acttcattta 720
ttttaatctt tttatggctc ctttctcttg ctttaaaaca ggattataag cacacagcag 780
gtactgacac ctgaagtctt actaaattcc tgtcctcagg ccatcctttt tctcctgaaa 840
cctggactcc aattttcaat gacgtttttg tttttctctt tcaagcctaa ctatgggaca 900
gctttacgag aaggaaaaag atgaagatgg attcttatat gtggcctaca gcggagagaa 960
cacttttggc ttctgagggc cattgctggg ctaggtgcac cgtaactgct tgtgtatctt 1020
gtaaatagcc asccattttc agttattawa ccagaacctc ttmacataga cctattagtg 1080
catttgtaac tggatttatt tcttaatata tkggaaggtt ttgtttcctt agactagtaa 1140
attatcatac agagttttat tttgagtttt tctttttgtg cattgtcctc atgcctgtat 1200
tctccaggaa acttgtcctt ctggaaatca tatkgaatga tatttctata tcgaagtgag 1260
gtaggtgcgg tattaaagtg aaagggaagg tgatgcattt attctgggtt atgcttgaag 1320
tgttagatgg ctaagtatta aaattatcca aattaaatcc ttagcagtca gaacacttgc 1380
ttcactagaa tatgccaact gccaatcatg ttggactgag ctaatttgtt cctctttctg 1440
aaactattaa ggtaaataat taacaataaa aattctctta taaaggcaaa aaaaaaaaaa 1500
aaaaaaaaaa aaaaaasa 1518
<210> 18
<211> 55
<212> PRT
<213> Homo sapiens
<400> 18
Met Val Leu Thr His Leu His Phe Leu Ser Ser Gln Arg Val Gln Ser
17
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
1 5 10 15
Arg Gly Arg Ala Cys Ile Gly Ile Gln Val Leu Leu Val Leu Leu Trp
20 25 30
Ser Trp Ser Asn Ser Val Ser Trp His Arg Thr Arg Leu Gly Leu His
35 40 45
Cys Ala Val Cys Phe Thr Ala
50 55
<210> 19
<211> 2097
<212> DNA
<213> Homo sapiens
<400> 19
ctcttgagta cctggggctt gcagatgcat gccaccacac ccggctaatt tttttttttt 60
ttaaatagag atggggtctt gttctgttgc ccargctggt ctggaactcc tggcttcaat 120
cagtcctccc acctcagctt cccaaagctc tgggsttata ggcatgagcc actgtacctg 180
tccacctgag aaattttcta agcctggatt cagtcttatg aaatataata ctttgaaatg 240
cacaataact ttgaaaatga aactcattgc ttttcatttc accaggagtt actaactata 300
ataagcttta gagcaaattc tccttagata tgatttttgt tattattaga aacacatact 360
atcttgataa ctaaattttg ccaatcattc ttcttgacta gtggtcttta tatatacata 420
catatatata tatatataca tatatatata tatgaggaat tttccataag tgacttgaaa 480
aatacagaat gcactccatg gtaggtctgt tcagtgttat caggaatact gtttctcatc 540
ttcctttctt ggtgtccctt tgcaggggtt gtgtttgcac attatggtcc cgtctggaga 600
caacaaagga agttctctca ttcaactctt cgtcattttg ggttgggaaa acttagcttg 660
gagcccaaga ttattgagga gttcaaatat gtgaaagcag aaatgcaaaa gcacggagaa 720
gaccccttct gccctttctc catcatcagc aatgccgtct ctaacatcat ttgctccttg 780
tgctttggcc agcgctttga ttacactaat a9tgagttca agaaaatgct tggttttatg 840
tcacgaggcc tagaaatctg tctgaacagt caagtcctcc tggtcaacat atgcccttgg 900
ctttattacc ttccctttgg accatttaag gaattaagac aaattgaaaa ggatataacc 960
agtttcctta aaaaaatcat caaagaccat caagagtctc tggatagaga gaaccctcag 1020
gacttcatag acatgtacct tctccacatg gaagaggaga ggaaaaataa tagtaacagc 1080
agttttgatg aagagtactt attttatatc attggggatc tctttattgc tgggactgat 1140
accacaacta actctttgct ctggtgcctg ctgtatatgt cgctgaaccc cgatgtacaa 1200
gaaaaggttc atgaagaaat tgaaagagtc attggcgcca accgagctcc ttccctcaca 1260
gacaaggccc agatgcccta cacagaagcc accatcatgg aagtgcagag gctaactgtg 1320
gtggtgccgc ttgccattcc tcatatgacc tcagagaaca cagtgctcca agggtatacc 1380
attcctaaag gcacattgat cttacccaac ctgtggtcag tacatagaga cccagccatt 1440
tgggagaaac cggaggattt ctaccctaat cgatttctgg atgaccaagg acaactaatt 1500
aaaaaagaaa cctttattcc ttttgggata gggaagcggg tgtgtatggg agaacaactg 1560
gcaaagatgg aattattcct aatgtttgtg agcctaatgc agagtttcgc atttgcttta 1620
cctgaggatt ctaagaagcc cctcctgast ggaagatttg gtctaacttt agccccacat 1680
ccattta8ta taactatttc aaggagatga agagcatctc caagaagaga tggtaaaaag 1740
atatatsaat acatatcctt ctaagcagat tcttcctact gcaaaggaca gtgaatccag 1800
caactcagtg gatccaagct gggctcagag gtcggaagga gggtagagca cactgggagg 1860
tttcatcttg gaggattcct cagcaggata cttcagccat tttagtaatg caggtctgtg 1920
atttggggga tagaaaacaa agtacctatg aaacgggata tctggatttt acttgcagtg 1980
gcttccaccg atgggccaat cttctcattt cttagtgcct cagacatccc atatgtasaa 2040
tgagagtaat aaaacttggc ttctctctac ctctcagcac taaaaaaaaa aaaaaaa 2097
<210> 20
<211> 398
<212> PRT
<213> Homo sapiens
<220>
1$
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
<221> UNSURE
<222> (379)
<400> 20 -
Val Leu Ser Gly Ile Leu Phe Leu Ile Phe Leu Ser Trp Cys Pro Phe
1 5 10 15
Ala Gly Val Val Phe Ala His Tyr Gly Pro Val Trp Arg Gln Gln Arg
20 25 30
Lys Phe Ser His Ser Thr Leu Arg His Phe Gly Leu Gly Lys Leu Ser
35 40 45
Leu Glu Pro Lys Ile Ile Glu Glu Phe Lys Tyr Val Lys Ala Glu Met
50 55 60
Gln Lys His Gly Glu Asp Pro Phe Cys Pro Phe Ser Ile Ile Ser Asn
65 70 75 80
Ala Val Ser Asn Ile Ile Cys Ser Leu Cys Phe Gly Gln Arg Phe Asp
85 90 95
Tyr Thr Asn Ser Glu Phe Lys Lys Met Leu Gly Phe Met Ser Arg Gly
100 105 110
Leu Glu Ile Cys Leu Asn Ser Gln Val Leu Leu Val Asn Ile Cys Pro
115 120 125
Trp Leu Tyr Tyr Leu Pro Phe Gly Pro Phe Lys Glu Leu Arg Gln Ile
130 135 140
Glu Lys Asp Ile Thr Ser Phe Leu Lys Lys Ile Ile Lys Asp His Gln
145 150 155 160
Glu Ser Leu Asp Arg Glu Asn Pro Gln Asp Phe Ile Asp Met Tyr Leu
165 170 175
Leu His Met Glu Glu Glu Arg Lys Asn Asn Ser Asn Ser Ser Phe Asp
180 185 190
Glu Glu Tyr Leu Phe Tyr Ile Ile Gly Asp Leu Phe Ile Ala Gly Thr
195 200 205
Asp Thr Thr Thr Asn Ser Leu Leu Trp Cys Leu Leu Tyr Met Ser Leu
210 215 220
Asn Pro Asp Val Gln Glu Lys Val His Glu Glu Ile Glu Arg Val Ile
225 230 235 240
Gly Ala Asn Arg Ala Pro Ser Leu Thr Asp Lys Ala Gln Met Pro Tyr
245 250 255
Thr Glu Ala Thr Ile Met Glu Val Gln Arg Leu Thr Val Val Val Pro
260 265 270
Leu Ala Ile Pro His Met Thr Sex Glu Asn Thr Val Leu Gln Gly Tyr
275 280 285
Thr Ile Pro Lys Gly Thr Leu Ile Leu Pro Asn Leu Trp Ser Val His
290 295 300
19
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
Arg Asp Pro Ala Ile Trp Glu Lys Pro Glu Asp Phe Tyr Pro Asn Arg
305 310 315 320
Phe Leu Asp Asp Gln Gly Gln Leu Ile Lys Lys Glu Thr Phe Ile Pro
325 330 335
Phe Gly Ile Gly Lys Arg Val Cys Met Gly Glu Gln Leu Ala Lys Met
340 345 350
Glu Leu Phe Leu Met Phe Vai Ser Leu Met Gln Ser Phe Ala Phe Ala
355 360 365
Leu Pro Glu Asp Ser Lys Lys Pro Leu Leu Xaa Gly Arg Phe Gly Leu
370 375 380
Thr Leu Ala Pro His Pro Phe Asn Ile Thr Ile Ser Arg Arg
385 390 395
<210> 21
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 21
cnaagttcta ttgggagatg gagtttgtg 29
<210> 22
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 22
cnatccatgg tacatggtca gaagctcat 29
<210> 23
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
CA 02318303 2000-07-13
WO 99/37674 PGT/US99/01404
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramiditeresidue
<400> 23
tngagcaggt caggatacac tggaaaaga 29
<210> 24
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramiditeresidue
<400> 24
cnactgcctt tgttgctttc cagtagtga 29
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramiditeresidue
<400> 25
tnaatatcca catccccaaa tcctacacg 29
<210> 26
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramiditeresidue
<400> 26
cncttgcagc gggaaggcag agaagtttc 29
<210> 27
<211> 29
<212> DNA
<213> Artificial Sequence
21
CA 02318303 2000-07-13
VSO 99/376'74 PCT/US99/01404
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 27
cntgagccac aatagacaga attcctacc 29
<210> 28
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 28
cngtcagggc gcagctgtat tggtcacaa 29
<210> 29
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 29
acccacacag aagtgagcc 19
<210> 30
<211> 147
<212> PRT
<213> Homo sapiens
<400> 30
Met Ala Ser Pro Ser Gly Leu Cys Val Leu Val Arg Leu Pro Lys Leu
1 5 10 15
Ile Cys Gly Gly Lys Thr Leu Pro Arg Thr Leu Leu Asp Ile Leu Ala
20 25 30
Asp Gly Thr Ile Leu Lys Val Gly Val Gly Cys Ser Glu Asp Ala Ser
35 40 45
Lys Leu Leu Gln Asp Tyr Gly Leu Val Val Arg Gly Cys Leu Asp Leu
50 55 60
Arg Tyr Leu Ala Met Arg Gln Arg Asn Asn Leu Leu Cys Asn Gly Leu
65 70 75 80
22
CA 02318303 2000-07-13
WO 99/37674 PCT/US99/01404
Ser Leu Lys Ser Leu Ala Glu Thr Val Leu Asn Phe Pro Leu Asp Lys
85 90 95
Ser Leu Leu Leu Arg Cys Ser Asn Trp Asp Ala Glu Thr Leu Thr Glu
100 105 110
' Asp Gln Val Ile Tyr Ala Ala Arg Asp Ala Gln Ile Ser Val Ala Leu
115 120 125
Phe Leu His Leu Leu Gly Tyr Pro Phe Ser Arg Asn Ser Pro Gly Glu
130 135 140
Lys Lys Arg
145
<210> 31
<211> 176
<212> PRT
<213> Homo sapiens
<400> 31
Met Thr Asp Cys Leu Val Ile Lys His Phe Leu Arg Lys Ile Ile Met
1 5 10 15
Val His Pro Lys Val Arg Phe His Phe Ser Val Lys Val Asn Gly Ile
20 25 30
Leu Ser Thr Glu Ile Phe Gly Val Glu Asn Glu Pro Thr Leu Asn Leu
35 40 45
Gly Asn Gly Ile Ala Leu Leu Val Asp Ser Gln His Tyr Val Ser Arg
50 55 60
Pro Asn Phe Gly Thr Ile Glu Ser His Cys Ser Arg Ile His Pro Val
65 70 75 BO
Leu Gly His Pro Val Met Leu Phe Ile Pro Glu Asp Val Ala Gly Met
85 90 95
Asp Leu Leu Gly Glu Leu Ile Leu Thr Pro Ala Ala Ala Leu Cys Pro
100 105 110
Ser Pro Lys Val Ser Ser Asn Gln Leu Asn Arg Ile Ser Ser Val Ser
115 120 125
Ile Phe Leu Tyr Gly Pro Leu Gly Leu Pro Leu Ile Leu Ser Thr Trp
130 135 140
Glu Gln Pro Met Thr Thr Phe Phe Lys Asp Thr Ser Ser Leu Val Asp
145 150 155 160
Trp Lys Ile Pro Phe Val Tyr Asp Thr Gln Phe Gly Ser Gln Phe Gly
165 170 175
<210> 32
<211> 89
<212> PRT
<213> Homo Sapiens
23
CA 02318303 2000-07-13
VIrO 99/37674 PCT/US99/OI404
<400> 32
Met Gly Ser Leu Ser Thr Ala Asn Val Glu Phe Cys Leu Asp Val Phe
1 5 10 15
Lys Glu Leu Asn Ser Asn Asn Ile Gly Asp Asn Ile Phe Phe Ser Ser
20 25 30
Leu Ser Leu Leu Tyr Ala Leu Ser Met Val Leu Leu Gly Ala Arg Gly
35 40 45
Glu Thr Ala Glu Gln Leu Glu Lys Val Leu His Phe Ser His Thr Val
50 55 60
Asp Ser Leu Lys Pro Gly Phe Lys Asp Ser Pro Lys Cys Ser Gln Ala
65 70 75 80
Gly Arg Ile His Ser Glu Phe Gly Val
24
