Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/093,045
(converted to a provisional application from non-provisional application Ser:
No.
08/929,007), filed September 8, 1997, 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
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 0 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
2 5 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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S1,JMMARY 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 69 to nucleotide 908;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 270 to nucleotide 908;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bn97_1 deposited under accession
number
ATCC 98535;
(e) a poIynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bn97_1 deposited under accession number ATCC 98535;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone bn97 1 deposited under accession number ATCC
98535;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone bn97_1 deposited under accession number ATCC 98535;
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
(1) 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 69 to nucleotide 908; the nucleotide sequence of SEQ ID
NO:I from
nucleotide 270 to nucleotide 908; the nucleotide sequence of the full-length
protein coding
sequence of clone bn97_1 deposited under accession number ATCC 98535; or the
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nucleotide sequence of a mature protein coding sequence of clone bn97_1
deposited under
accession number ATCC 98535. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone bn97_1
deposited under accession number ATCC 98535. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:2 from amino acid 1 to amino acid 83. 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 135
to amino acid 144 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
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 N0:2;
2 0 (b) the amino acid sequence of SEQ ID N0:2 from amino acid 1 to
amino acid 83;
(c) fragments of the amino acid sequence of SEQ ID N0:2 comprising
eight consecutive amino acids of SEQ ID N0:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 5 bn97_1 deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 1 to amino acid 83. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
3 0 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 protein comprising a fragment of the amino acid sequence of SEQ ID
N0:2
having biological activity, the fragment comprising the amino arid sequence
from amino
acid 135 to amino acid 144 of SEQ ID N0:2.
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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 562 to nucleotide 777;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 236 to nucleotide 673;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bn268_ll deposited under accession
number ATCC 98535;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bn268_11 deposited under accession number ATCC 98535;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone bn268_11 deposited under accession number
ATCC 98535;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone bn268_l l deposited under accession number ATCC 98535;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:4;
(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
2 5 (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).
3 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:3 from nucleotide 562 to nucleotide 777; the nucleotide sequence of SEQ ID
N0:3
from nucleotide 236 to nucleotide 673; the nucleotide sequence of the full-
length protein
coding sequence of clone bn268_ll deposited under accession number ATCC 98535;
or
the nucleotide sequence of a mature protein coding sequence of clone bn268_11
deposited
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under accession number ATCC 98535. In other preferred embodiments, the
polynucleotfde encodes the full-length or a mature protein encoded by the cDNA
insert
of clone bn268_11 deposited under accession number ATCC 98535. 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 1 to amino
acid 37.
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 31 to amino acid 40 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
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 N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 1 to
2 0 amino acid 37;
(c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
eight consecutive amino acids of SEQ ID N0:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone
bn268_11 deposited under accession number ATCC 98535;
2 5 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 1 to amino acid 37. 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
3 0 eight (more preferably twenty, most preferably thirty) consecutive amino
acids 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 31 to amino acid 40 of SEQ ID N0:4.
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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 286 to nucleotide 1686;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 544 to nucleotide 1686;
{d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 365 to nucleotide 1260;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cb96_10 deposited under accession
number ATCC 98535;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cb96_10 deposited under accession number ATCC 98535;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cb96_10 deposited under accession number
ATCC 98535;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone cb96_10 deposited under accession number ATCC 98535;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
2 5 comprising eight consecutive amino acids of SEQ ID N0:6;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
3 0 (m) a polynucleoHde that hybridizes under stringent conditions to any
one of the polynucleotides specified in (ar(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 286 to nucleotide 1686; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 544 to nucleotide 1686; the nucleotide sequence of SEQ ID N0:5
from
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nucleotide 365 to nucleotide 1160; the nucleotide sequence of the full-length
protein
coding sequence of clone cb96_10 deposited under accession number ATCC 98535;
or the
nucleotide sequence of a mature protein coding sequence of clone cb96_10
deposited
under accession number ATCC 98535. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cb96_10 deposited under accession number ATCC 98535. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:6 from amino acid 28 to amino
acid
395. 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 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 228 to amino acid 237 of SEQ ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
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 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ ID N0:6 from amino acid 28 to
amino acid 395;
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
2 5 eight consecutive amino acids of SEQ ID N0:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone
cb96_10 deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6 or the amino acid
sequence
3 0 of SEQ ID N0:6 from amino acid 28 to amino acid 395. 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 acids
of SEQ ID
N0:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:6
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having biological activity, the fragment comprising the amino acid sequence
from amino
acid 228 to amino acid 237 of SEQ ID NO:b.
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
N0:7;
{b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 99 to nucleotide 1049;
(c) a poiynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 222 to nucleotide 1049;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 632 to nucleotide 998;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cb213_ll deposited under accession
number ATCC 98535;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cb213_11 deposited under accession number ATCC 98535;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cb213_11 deposited under accession number
2 0 ATCC 98535;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone cb213_11 deposited under accession number ATCC 98535;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
2 5 (j) 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;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
3 0 (1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in {a)-(j).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 99 to nucleotide 1049; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 222 to nucleotide 1049; the nucleotide sequence of SEQ ID N0:7 from
nucleotide 632 to nucleotide 998; the nucleotide sequence of the full-length
protein coding
sequence of clone cb213_ll deposited under accession number ATCC 98535; or the
nucleotide sequence of a mature protein coding sequence of clone cb213_11
deposited
under accession number ATCC 98535. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cb213_l l deposited under accession number ATCC 98535. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:8 from amino acid 187 to amino
acid
300. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
NO:B
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 NO:B having biological activity, the fragment comprising the amino acid
sequence
from amino acid 153 to amino acid 162 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 0 ID N0:7.
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;
2 5 (b) the amino acid sequence of SEQ ID NO:B from amino acid 187 to
amino acid 300;
(c) fragments of the amino acid sequence of SEQ ID N0:8 comprising
eight.consecutive amino acids of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone
3 0 cb213_ll deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:8 or the amino acid
sequence
of SEQ ID N0:8 from amino acid 187 to amino acid 300. 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 N0:8 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:8, or a 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 acid 153 to amino acid 162 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
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 3003 to nucleotide 3137;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 3072 to nucleotide 3137;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 2713 to nucleotide 3114;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cj457 4 deposited under accession
number ATCC 98535;
(f) a polynucleotide encoding the full-length protein encoded by the
2 0 cDNA insert of clone cj457_4 deposited under accession number ATCC 98535;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cj457_4 deposited under accession number
ATCC 98535;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 5 insert of clone cj457_4 deposited under accession number ATCC 98535;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity, the fragment
3 0 comprising eight consecutive amino acids of SEQ ID N0:10;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
*rB
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(m) a polynucleotide~that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 3003 to nucleotide 3137; the nucleotide sequence of SEQ
ID N0:9
from nucleotide 3072 to nucleotide 3137; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 2713 to nucleotide 3114; the nucleotide sequence of the full-length
protein
coding sequence of clone cj457 4 deposited under accession number ATCC 98535;
or the
nucleotide sequence of a mature protein coding sequence of clone cj457_4
deposited under
accession number ATCC 98535. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone cj457_4
deposited under accession number ATCC 98535. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 37. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:10 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:10, or a
polynucleotide encoding
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 17
2 0 to amino acid 2b of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
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 NO:20;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to
amino acid 37;
(c) fragments of the amino acid sequence of SEQ ID N0:10 comprising
3 0 eight consecutive amino acids of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
cj457 4 deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid
sequence
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of SEQ ID N0:10 from amino acid 1 to amino acid 37. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
sequence of SEQ ID N0:10 having biological activity, the fragment preferably
comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
N0:10, 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 17 to amino acid 26 of SEQ ID N0:10.
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
N0:11 from nucleotide 284 to nucleotide 1357;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide b03 to nucleotide 1233;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cz653_ll deposited under accession
number ATCC 98535;
(e) a polynucleotide encoding the full-length protein encoded by the
2 0 cDNA insert of clone cz653_11 deposited under accession number ATCC 98535;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cz653_11 deposited under accession number
ATCC 98535;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
2 5 insert of clone cz653_ll deposited under accession number ATCC 98535;
{h) a polynucleotide 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
3 0 comprising eight consecutive amino acids of SEQ ID N0:12;
(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
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(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 284 to nucleotide 1357; the nucleotide sequence of SEQ
ID N0:11
from nucleotide 603 to nucleotide 1233; the nucleotide sequence of the full-
length protein
coding sequence of clone cz653_11 deposited under accession number ATCC 98535;
or the
nucleotide sequence of a mature protein coding sequence of clone cz653_11
deposited
under accession number ATCC 98535. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cz653_l l deposited under accession number ATCC 98535. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:12 from amino acid 147 to
amino acid
358. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:12
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 fragment comprising the amino
acid
sequence from amino acid 174 to amino acid 183 of SEQ ID N0:12.
2 0 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID NO:11.
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:
2 5 (a) the amino acid sequence of SEQ ID N0:12;
(b) the amino acid sequence of SEQ ID N0:12 from amino acid 147 to
amino acid 358;
(c) fragments of the amino acid sequence of SEQ ID N0:12 comprising
eight consecutive amino acids of SEQ ID N0:12; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
cz653_11 deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12 or the amino acid
sequence
of SEQ ID N0:12 from amino acid 147 to amino acid 358. In further preferred
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embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID NO: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
sequence from amino acid 174 to amino acid 183 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;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 621 to nucleotide 1763;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13 from nucleotide 1461 to nucleotide 1763;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone dx138 4 deposited under accession
number ATCC 98535;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone dx138 4 deposited under accession number ATCC 98535;
2 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone dx138_4 deposited under accession number
ATCC 98535;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone dx138_4 deposited under accession number ATCC 98535;
2 5 (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;
3 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
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(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 621 to nucleotide 1763; the nucleotide sequence of SEQ
ID N0:13
from nucleotide 1461 to nucleotide 1763; the nucleotide sequence of the full-
length
protein coding sequence of clone dx138 4 deposited under accession number ATCC
98535; or the nucleotide sequence of a mature protein coding sequence of clone
dx138 4
deposited under accession number ATCC 98535. In other preferred embodiments,
the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone dx138 4 deposited under accession number ATCC 98535. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:14 from amino acid 83 to amino
acid
229. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:14
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 biological activity, the fragment comprising the amino
acid
sequence from amino acid 185 to amino acid 194 of SEQ ID N0:14.
2 Q Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:13.
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:
2 5 (a) the amino acid sequence of SEQ ID N0:14;
(b) the amino acid sequence of SEQ ID N0:14 from amino acid 83 to
amino acid 229;
(c) fragments of the amino acid sequence of SEQ ID N0:14 comprising
eight consecutive amino acids of SEQ ID N0:14; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
dx138_4 deposited under accession number ATCC 98535;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:14 or the amino acid
sequence
of SEQ ID N0:14 from amino acid 83 to amino acid 229. In further preferred
CA 02302705 2000-03-06
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embodiments, the present invention provides 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 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 185 to amino acid 194 of SEQ ID N0:14.
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;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 119 to nucleotide 295;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 191 to nucleotide 295;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ij167 5 deposited under accession
number
ATCC 98535;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ij167 5 deposited under accession number ATCC 98535;
2 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ij167 5 deposited under accession number ATCC
98535;
(g) a poiynucleotide encoding a mature protein encoded by the cDNA
insert of clone ij167 5 deposited under accession number ATCC 98535;
2 5 (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;
3 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
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(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
N0:15 from nucleotide 119 to nucleotide 295; the nucleotide sequence of SEQ ID
N0:15
from nucleotide 191 to nucleotide 295; the nucleotide sequence of the full-
length protein
coding sequence of clone ij167 5 deposited under accession number ATCC 98535;
or the
nucleotide sequence of a mature protein coding sequence of clone ij167_5
deposited under
accession number ATCC 98535. In other preferred embodiments, the
polynucleotide
encodes the frill-length or a mature protein encoded by the cDNA insert of
clone ij167_5
deposited under accession number ATCC 98535. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:16 from amino acid 1 to amino acid 26. 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 24
to amino acid 33 of SEQ ID NO:Ib.
2 0 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:15.
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:
2 5 (a) the amino acid sequence of SEQ ID N0:16;
(b) the amino acid sequence of SEQ ID N0:16 from amino acid 1 to
amino acid 26;
(c) fragments of the amino acid sequence of SEQ ID N0:16 comprising
eight consecutive amino acids of SEQ ID N0:16; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
ij167_5 deposited under accession number ATCC 98535;
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 1 to amino acid 26. In further preferred
embodiments,
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the present invention provides 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 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 24 to amino acid 33 of SEQ ID N0:16.
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.
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
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
2 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
effective amount of a composition comprising a protein of the present
invention and a
2 5 pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A 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
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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.
As used herein a "secreted" 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
which are transported across the membrane of the endoplasmic reticulum.
Clone "bn97 1"
A polynucleotide of the present invention has been identified as clone
"bn97_1".
bn97_1 was isolated from a human adult placenta cDNA library 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. bn97 1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"bn97_1 protein").
The nucleotide sequence of bn97_1 as presently determined is reported in SEQ
ID
N0:1. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the bn97_1 protein corresponding to the foregoing
nucleotide
2 5 sequence is reported in SEQ ID N0:2. Amino acids 55 to 67 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 68, or
are a transrnembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bn97_1 should be approximately 1700 bp.
3 0 The nucleotide sequence disclosed herein for bn97 1 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bn97_1 demonstrated at least some identity with
sequences
identified as AA046689 (zk72h06.s1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 488411 3'), D30934 (Human fetal-lung cDNA 5'-end sequence), 878820
(yi90b03.r1
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Homo sapiens cDNA clone 146477 5'), and 891687 (yq10h09.s1 Homo sapiens cDNA
clone 196577 3'). The predicted amino acid sequence disclosed herein for
bn97_1 was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted bn97 1 protein demonstrated at least
some
identity with sequences identified as A 10431 (Hepatitis-B virus surface
antigen P31). The
bn97_1 protein also shows some identity (30% identity, 50% conserved amino
acids) to
both bovine and human lectin-like receptor for oxidized LDL (low-density
lipoprotein).
While this homology is weak, it gets stronger (44% identity and 62% conserved
amino
acids) in the lectin-like domain. Further, the 3' untranslated region of the
bovine receptor
has seven mRNA unstabilising sequences (ATTTA) and bn97_1 has four in its 3'
untranslated region. This lectin-like receptor for oxidized LDL (designated
LOX-1,
Sawamura et al., 1997, Nature 386: 73-77) is an integral membrane protein
which binds
oxidized low-density lipoproteins, internalizes them into the endothelial
cells and
destroys them, thus playing a crucial role in the pathogenesis of
atherosderosis. Based
upon identity, bn97_1 proteins and each identical protein or peptide may share
at least
some activity.
Clone "bn268 11"
A polynudeotide of the present invention has been identified as clone
"bn268_11".
2 0 bn268_l l was isolated from a human adult placenta cDNA library was
identified as
encoding a secreted or transmembrane protein on the basis of computer analysis
of the
amino acid sequence of the encoded protein. bn268_11 is a full-length done,
including the
entire coding sequence of a secreted protein (also referred to herein as
"bn268_ll
protein').
2 5 The nucleotide sequence of bn268_l l as presently determined is reported
in SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the bn268_11 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
3 0 bn268_I1 should be approximately 1050 bp.
The nucleotide sequence disclosed herein for bn268_11 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bn268_11 demonstrated at least some identity with
sequences
CA 02302705 2000-03-06
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identified as D62832 (Human aorta cDNA 5'-end GEN-330C09) and U20159 (Mus
musculus 76 kDa tyrosine phosphoprotein SLP-76 mRNA, complete cds). The
predicted
amino acid sequence disclosed herein for bn268_l l was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted bn268_11 protein demonstrated at least some identity with sequences
identified
as D83171 (GDP-GTP exchange protein for Rholp [Saccharomyces cerevisiae]).
Based
upon identity, bn268_11 proteins and each identical protein or peptide may
share at least
some activity. The TopPredII computer program predicts a potential
transmembrane
domain within the bn268_11 protein sequence centered around amino acid 33 of
SEQ ID
N0:4; this region may also function as a signal sequence.
Clone "cb96 10"
A polynucleotide of the present invention has been identified as clone
"cb96_10".
cb96_10 was isolated from a human fetal brain cDNA library was identified as
encoding
a secreted or transmembrane protein on the basis of computer analysis of the
amino acid
sequence of the encoded protein. cb96_10 is a full-length clone, including the
entire
coding sequence of a secreted protein {also referred to herein as "cb96_10
protein').
The nucleotide sequence of cb96_10 as presently determined is reported in SEQ
ID NO:S. What applicants presently believe to be the proper reading frame and
the
2 0 predicted amino acid sequence of the cb96_10 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6. Amino acids 74 to 86 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 87, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 cb96_10 should be approximately 2100 bp.
The nucleotide sequence disclosed herein for cb96_10 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cb96_10 demonstrated at least some identity with
sequences
identified as AA459012, AA459236, AA256744 (zs31h1l.rl Soares NbHTGBC Homo
3 0 Sapiens cDNA clone 686853 5'), N54489 (yv40f07.s1 Soares fetal liver
spleen 1NFLS
Homo sapiens cDNA clone 245221 3'), and N57339 (yw81h07.r1 Homo sapiens cDNA
clone 258685 5'). The predicted amino acid sequence disclosed herein for
cb96_10 was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
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BLASTX search protocol. The predicted cb96_10 protein demonstrated at least
some
identity with sequences identified as X80036 (ascorbate peroxidase
[Arabidopsis
thaliana]). Based upon identity, cb96_10 proteins and each identical protein
or peptide
may share at least some activity. The TopPredII computer program predicts
seven
potential transmembrane domains within the cb96_10 protein sequence, centered
around
amino acid residues 25, 80,125, 225, 300, 350, and 440 of SEQ ID N0:6.
Therefore, cb96_10
is likely to be an integral membrane protein with multiple helices in the
membrane; it also
contains the sequence motif of the actinin-type actin-binding domains that are
believed
to anchor actin to the cell membrane.
Clone "cb213 11"
A polynucleotide of the present invention has been identified as clone
"cb213_11".
cb213_11 was isolated from a human fetal brain cDNA library was identified as
encoding
a secreted or transmembrane protein on the basis of computer analysis of the
amino acid
sequence of the encoded protein. cb213_ll is a full-length clone, including
the entire
coding sequence of a secreted protein (also referred to herein as "cb213_11
protein"}.
The nucleotide sequence of cb213_ll as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the cb213_I1 protein corresponding to the
foregoing
2 0 nucleotide sequence is reported in SEQ ID N0:8. Amino acids 29 to 41 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 42, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cb213_11 should be approximately 2400 bp.
2 5 The nucleotide sequence disclosed herein for cb213_11 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cb213_12 demonstrated at least some identity with
sequences
identified as AA332165 (EST36344 Embryo, 8 week I Homo sapiens cDNA 5' end)
and
834507 (g58a03.r1 Homo sapiens cDNA clone 36801 5'). The predicted amino acid
3 0 sequence disclosed herein for cb213 11 was searched against the GenPept
and GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
cb213_11 protein demonstrated at least some identity with sequences identified
as U39847
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WO 99113066 PCT/US98118724
(A013 ankyrin [Caenorhabditis elegans]}. Based upon identity, cb213_ll
proteins and
each identical protein or peptide may share at least some activity.
Clone "c~'457 4"
A polynucleotide of the present invention has been identified as clone
"cj457_4".
cj457 4 was isolated from a human fetal brain cDNA library was identified as
encoding
a secreted or transmembrane protein on the basis of computer analysis of the
amino acid
sequence of the encoded protein. cj457 4 is a full-length clone, including the
entire coding
sequence of a secreted protein {also referred to herein as "cj457 4 protein'}.
The nucleotide sequence of cj457_4 as presently determined is reported in SEQ
ID
N0:9. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the cj457_4 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID NO:10. Amino acids 11 to 23 are a predicted
leader/ signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 24, or
are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cj457 4 should be approximately 3350 bp.
The nucleotide sequence disclosed herein for cj457 4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. cj457 4 demonstrated at least some identity with
sequences
identified as T92881 (ye22a10.s1 Homo sapiens cDNA clone 118458 3') and T92488
(ye21g09.r1 Homo sapiens cDNA clone 118432 5'}. Based upon identity, cj457_4
proteins
and each identical protein or peptide may share at least some activity. The
TopPredII
computer program predicts a potential transmembrane domain within the cj457_4
protein
2 5 sequence, centered around amino acid 17 of SEQ ID NO:10; this region may
also function
as a signal sequence.
Clone"cz653 11"
A polynucleotide of the present invention has been identified as clone
"cz653_11".
3 0 cz653_11 was isolated from a human adult testes cDNA library was
identified as encoding
a secreted or transmembrane protein on the basis of computer analysis of the
amino acid
sequence of the encoded protein. cz653_ll is a full-length clone, including
the entire
coding sequence of a secreted protein (also referred to herein as "cz653_ll
protein').
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The nucleotide sequence of cz653_11 as presently determined is reported in SEQ
ID NO:11. . What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the cz653_11 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cz653_11 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for cz653_11 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cz653_ll demonstrated at least some identity with
sequences
identified as AA024740 (ze76c09.s1 Soares fetal heart NbHHI9W Homo sapiens
cDNA
clone 364912 3'), AA203204 (zx57b04.r1 Soares fetal liver spleen 1NF'LS S 1
Homo
sapiens cDNA clone 446575 5' similar to contains element MSR1 repetitive
element),
W72894 (zd59e06.s1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone 344962
3'), and W76099 (zd59e06.r1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone
344962 5'). The predicted cz653_11 demonstrated similarity to various WD-40
repeat
containing proteins such as beta transducin-like protein (L28125) and
coatomer,
beta-prime subunit (AJ006523). The homology appears to be due to the presence
of the
Beta-transducin family Trp-Asp repeats signature (WD-40) beginning at residue
262 of
SEQ ID N0:12. The WD-40 repeat has been thought to be a protein-protein
interaction
2 0 domain. Based upon identity, cz653_11 proteins and each identical protein
or peptide
may share at least some activity. The TopPredII computer program predicts a
potential
transmembrane domain within the cz653_11 protein sequence centered around
amino
acid 200 of SEQ ID N0:12.
2 5 Clone "dx138 4"
A polynucleotide of the present invention has been identified as clone "dx138
4".
dx138 4 was isolated from a human adult testes cDNA library was identified as
encoding
a secreted or transmembrane protein on the basis of computer analysis of the
amino acid
sequence of the encoded protein. dx138 4 is a full-length clone, including the
entire
3 0 coding sequence of a secreted protein (also referred to herein as "dx138_4
protein').
The nucleotide sequence of dx138_4 as presently determined is reported in SEQ
ID N0:13. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the dx138 4 protein corresponding to the
foregoing
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WO 99!13066 PCTNS98118724
nucleotide sequence is reported in SEQ ID N0:14. Amino acids 268 to 280 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 281, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
dx138 4 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for dx138 4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. dx138 4 demonstrated at least some identity with
sequences
identified as AA108970 (m163a06.r1 Stratagene mouse testis (#937308) Mus
musculus
cDNA clone 516658 5'), AA280976 (zs97fOl.rl Soares NbHTGBC Homo Sapiens cDNA
clone 711577 5' similar to contains Alu repetitive element), H99316
(yx23a03.s1 Homo
Sapiens cDNA clone 262540 3'), T36050 (EST96120 Homo sapiens cDNA 5'), X85637
(H.sapiens mRNA for expressed sequence tag, clone CAM tEST417 (A)), and 222280
(H.sapiens DNA sequence). Based upon identity, dx138_4 proteins and each
identical
protein or peptide may share at least some activity.
Clone "ij_167 5"
A polynucleotide of the present invention has been identified as clone "ij167
5".
ij 167 5 was isolated from a human adult blood (peripheral blood mononuclear
cells treated
2 0 in vivo with G-CSF) cDNA library was identified as encoding a secreted or
transmembrane
protein on the basis of computer analysis of the amino acid sequence of the
encoded
protein. ij167 5 is a full-length clone, including the entire coding sequence
of a secreted
protein (also referred to herein as "ij167 5 protein").
The nucleotide sequence of ij167 5 as presently determined is reported in SEQ
ID
2 5 N0:15. What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the ij167 5 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:16. Amino acids 12 to 24 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 25, or are a transmembrane domain.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ij167 5 should be approximately 1050 bp.
The nucleotide sequence disclosed herein for ij167 5 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
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WO 99/13066 PCT/US98/18724
FASTA search protocols. ij167_5 demonstrated at least some identity with
sequences
identified as N71115 (za87h10.s1 Homo sapiens cDNA clone 299587 3'), T85491
(yd78bOl.r1 Homo sapiens cDNA clone 114313 5'), W04374 (za43f06.r1 Soares
fetal liver
spleen 1NFLS Homo Sapiens cDNA clone 295331 S'), W05476 (za87h10.r1 Soares
fetal
lung NbHLI9W Homo Sapiens cDNA clone 299587 5'), and W40146 (zb74d09.r1 Soares
fetal lung NbHLI9W Homo sapiens cDNA clone 309329 5'). The predicted amino
acid
sequence disclosed herein for ij167 5 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
ij167 5
protein demonstrated at least some identity with sequences identified as
M96653
(adenylyl cyclase, type 6 [Mus musculus]). Based upon identity, ij167_5
proteins and
each identical protein or peptide may share at least some activity. The
TopPredII
computer program predicts a potential transmembrane domain within the ij167_5
protein
sequence, centered around amino acid 40 of SEQ ID N0:16.
Deposit of Clones
Clones bn97_l, bn268_ll, cb96_10, cb213_ll, cj457_4, cz653_ll, dx138 4, and
ij167 5 were deposited on September 4,1997 with the American Type Culture
Collection
(10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an
original deposit
under the Budapest Treaty and were given the accession number ATCC 98535, from
2 0 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.
Each clone has been transfected into separate bacterial cells (E. coIi) in
this
2 5 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
pED6 or
pNOTs vector depicted in Figures 1A and 1B, respectively. The pED6dpc2 vector
("pED6") was derived from pED6dpc1 by insertion of a new polylinker to
facilitate
3 0 cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 4485-4490); the
pNOTs vector
was derived from pMT2 (Kaufman et al.,1989, Mol. CeII. 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 ClaI site. In some instances, the deposited clone can
become "flipped"
26
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
(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
oligonucleotide 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
bn97_1 SEQ ID N0:17
bn268_11 SEQ ID N0:18
cb96_10 SEQ ID N0:19
cb213_11 SEQ ID N0:20
cj457_4 SEQ ID N0:21
2 0 cz653_l l SEQ ID N0:22
dx138 4 SEQ ID N0:23
ij 167 5 SEQ ID N0:24
In the sequences listed above which include an N at position 2, that position
is occupied
2 5 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)).
The design of the oligonucleotide probe should preferably follow these
3 0 parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ('N's"), if any;
(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).
27
CA 02302705 2000-03-06
WO 99113066 PCT/US98/18724
The oligonucleotide should preferably be labeled with g-3zP ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Uruncorporated
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 ul 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
colonies to nitrocellulose filters and lyse, 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 lzg/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
2 5 500 mL of ZX 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 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
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
3 0 be employed.
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.
28
*rB
CA 02302705 2000-03-06
WO 99/13066 PCTIUS98/18724
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 0 773-77$ (1992) and in
R.S.
McDowell, et al., J. Amer. Chem. Soc.114. 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to earner 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
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.
2 0 The present invention also provides genes corresponding to the
polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
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
2 5 to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification. and/or amplification of genes in
appropriate
3 0 genomic libraries or other sources of genomic materials. An "isolated
gene" is a gene that
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
29
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
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 "UruGene 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 antisense
polynucleotides or ribozymes that bind and/or cleave the mRlVA transcribed
from the
gene (Albert and Morris,1994, Trends Pharmacol. Sci.15(7): 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
2 0 multiple copies of the genes) corresponding to the polynucleotide
sequences disclosed
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
2 5 also provided (see European Patent No. 0 649 464 Bl, incorporated by
reference herein).
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
3 0 can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et aL,1994, Proc. Natl. Acad. Sci. USA
91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negaHve genetic selection strategies (Mansour
et al.,1988,
CA 02302705 2000-03-06
WO 99/13066 PCTIUS98/18724
Naticre 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 invention 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.
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
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
2 0 identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(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.
2 5 In particular, sequence identity may be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
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
3 0 Molecular Biology 215: 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
31
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
reference herein). WU-BLAST version 2.0 executable programs for several UNIX
platforms can be downloaded from ftp://blast.wustl.edulblast/executabies. 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
turned off in alI 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
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
2 5 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
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.
2 0 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
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
2 5 polynucleotide, but with significant sequence similarity to the given
protein or
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 polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
3 0 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
32
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
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 hamudryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus,
Rattzts noruegicus,
Cricetulus grisetts, Felis catus, Mustela vison, Canis familiaris,
Oryctolagtts cuniculus, Bos taunts,
Ovis aries, Sus scrofa, and Equus caballats, 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; O'Brien et aL, 1993,
Nature
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., 2997, 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
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
2 0 60% sequence identity (more preferably, at least 75% identity; most
preferably at least 90%
identity) with the given polynucleotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
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
2 5 screening a suitable nucleic acid source from individuals of the
appropriate species.
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
3 0 stringent conditions, to polynucleotldes described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
33
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybrid Hybridization TemperatureWash
ConditionHybrid Length and Temperature
(bp)$ Buffer' and Buffers
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; ixSSC, 50%
formamide
B DNA:DNA <50 TB*; IxSSC TB*; lxSSC
C DNA:RNA 2 50 67C; IxSSC -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, 50%
formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50%
formamide
H DNA:DNA <50 TH*; 4xSSC TH*; 4xSSC
I DNA:RNA 2 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50%
formamide
] DNA:RNA c50 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 2 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50%
formamide
2 P DNA:RNA <50 TP*; 6xSSC T,,*; 6xSSC
0
Q RNA:RNA Z SO 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50%
formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
x: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing poiynucleotides. When
2 5 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
hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
3 0 (IxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and
wash buffers; washes are
performed for 15 minutes after hybridization is complete.
"T8- 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 according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
34
CA 02302705 2000-03-06
WO 99113066 PCT/US98/18724
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log~p[Na']) + 0.41(%G+C) -
(b00/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
hybridization buffer ([Na'] for lxSSC = 0.165 M).
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
2 0 Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to
produce the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 1~5, 537-566 (1990). As defined herein
"operably
linked" means that the isolated polynucleotide of the invention and an
expression control
2 5 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
protein. Mammalian host cells include, for example, monkey COS cells, Chinese
Hamster
3 0 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 in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
CA 02302705 2000-03-06
WO 99/13066 PCTIUS98/18724
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, Bacilla~s subtilis, Salmonella fyphimurizcm,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
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 Agricultural
Experiment
Station Bulletin No. 1555 (1987),, incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
2 0 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 rnay then be purified from such culture (i.e., frorn 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 5 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.
3 0 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
36
CA 02302705 2000-03-06
WO 99/130b6 PCT/US98/18724
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).
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
1 5 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 structural and/or
conformational
2 0 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.
The proteins provided herein also include proteins characterized by amino acid
2 5 sequences similar to those of purified proteins but into which
modification are naturally
provided or deliberately engineered. For example, modifications in the peptide
or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
3 0 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,
insertion or deletion retains the desired activity of the protein.
37
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WO 99/13066 PCT/US98/18724
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein 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
given the disclosures herein. Such modifications are believed to be
encompassed by the
presentinvention.
USES AND BIOLOGICAL ACTIVITY
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
2 0 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
(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
2 5 probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
3 0 immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
described in Gyuris et al.,1993, Celt 75: 791-803 and in Rossi et al.,1997,
Proc. Natl. Acad.
38
CA 02302705 2000-03-06
WO 99/i3066 PCTlUS98/18724
Sci. USA 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.
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 all 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
2 0 the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel
eds.,1987.
2 5 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
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
3 0 be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
can be added to the medium in or on which the microorganism is cultured.
39
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WO 99/13066 PCT/US98/18724
Cytokine and Cell Proliferation/Differentiation ActivitT
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
induce production of other cytokines in certain cell populations. 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
2 0 133:327-341, 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
cells or thymocytes include, without limitation, those described imc
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immt~noIog~. J.E.e.a.
2 5 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.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without (imitation, those described in: Measurement of Human
and Murine
3 0 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky,
P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., j. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
Immicnology. 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
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. Coligan 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 Suppress, in~t Activity
2 0 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
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
2 5 as effecting the cytolytic activity of NK cells and other cell
populations. These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
3 0 herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various
fungal infections
such as candidiasis. Of 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
treatment of cancer.
41
CA 02302705 2000-03-06
WO 99113066 PCTNS98118724
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be 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 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
2 0 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
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
2 5 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
3 0 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-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
42
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
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
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, 89:21102-11105 (1992). In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
2 0 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
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 5 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
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
3 0 the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice ar NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
43
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
murine 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.
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
2 0 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,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 5 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can 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 B7-1-like activity and/or B7-3-like
activity. The
3 0 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
lymphocyte antigens) on the surface of the tumor cell provides the necessary
44
*rB
CA 02302705 2000-03-06
WO 99113066 PCT/US98/18724
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
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
(3z
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 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
2 0 limitation, those described in: Current Protocols in Immunology, Ed by J.
E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974,1982; Handa
et al.,
2 5 J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-
3500,1986; Takai et al.,
J. Imrnunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
3 0 Cellular Immunology 133:327-341,1991; Brown et al., J. Irnmunol.153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell
function: In vitro
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immicnology.
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,
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.I-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
3500,1986; Takai
et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., j. Immunol. 149:3778-
3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,1995;
1 S 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,
2 0 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,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 5 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
84:111-1I7, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hernat,Qpoiesis Re~ulatin,~~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
marginal biological activity in support of colony forming cells or of factor-
dependent cell
46
CA 02302705 2000-03-06
WO 99113066 PCTIUS98/18724
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional 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 thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
2 0 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.
Assays for embryonic stem cell differentiation {which will identify, among
others,
2 5 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.
Assays for stem cell survival and differentiation (which will identify, among
3 0 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cetts. R.I. 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
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
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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 aI. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of strornal 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 Activit;~
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
2 0 well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 5 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
3 0 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
invention, which induces tendon/ligament-like tissue or other tissue formation
in
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circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
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
2 0 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
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 5 Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
3 0 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.
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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
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic 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. W091 /07491 (skin, endothelium ).
2 0 Assays for wound healing activity include, without limitation, those
described in:
Winter, E,lsidermal 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).
Activin/Inhibin Activit3r
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
3 0 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-
CA 02302705 2000-03-06
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(3 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.
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; Vaie 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 Activity
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
2 0 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 5 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
3 0 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
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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 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146,1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et
al. J. of
Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
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
2 0 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.
Recentor/Ligand Activity
2 5 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,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
3 0 cellular adhesion molecules (such as selectins, integrins and their
ligands) and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
development of cellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
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WO 99/13066 PCT/US98/18724
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:
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
2 0 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
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
2 5 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.
3 0 ~'adherin/Tumor Invasion Suppressor Activity
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
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
53
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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
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
2 0 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
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
2 5 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
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
3 0 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
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
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WO 99/13066 PCT/US98/18724
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.
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.
2 0 A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
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 inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
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
3 0 agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
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);
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 iineages 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 AND DOSING
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.
2 0 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
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
2 5 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
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
3 0 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
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
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WO 99/13066 PCT/US98118724
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
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 MHC 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.
2 0 The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
2 5 monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. 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.
3 0 As used herein, the term "therapeutically effective amount" means the
total
amount of each active component of the pharmaceutical composition or method
that is
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
57
CA 02302705 2000-03-06
WO 99113066 PCT/US98118724
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
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 cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(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
2 0 cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection.
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
2 5 composition of the invention may additionally contain a solid carrier such
as a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
3 0 may be added. The liquid form of the pharmaceutical composition may
further contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
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WO 99113066 PCT/US98/18724
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
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, isotorucity, 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 Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
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
2 0 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
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 pg
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 ug to
about 1 mg)
2 5 of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
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
3 0 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
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
59
CA 02302705 2000-03-06
WO 99113066 PCT/US98118724
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
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.
211, 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
2 0 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
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
2 5 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
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.
3 0 The choice of matrix material is based on biocompatibility,
biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered 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
2 0 acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
2 5 are prevented from infiltrating the matrix, thereby providing the protein
the opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins 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
3 0 (EGF), platelet derived growth factor (PDGF), transforming growth factors
(TGF-a and
TGF-p), and insulin-like growth factor (IGF).
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.
61
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WO 99/1306b PCT/US98118724
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
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.
2 0 Treated cells can then be introduced in uivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
62
CA 02302705 2000-03-06
WO 99113066 PCT/US98i18724
SEQUENCE LISTING
<110> Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Evans, Cheryl
Merberg, David
Treacy, Maurice
Agostino, Michael J.
Spaulding, Vikki
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> 6052A
<140>
<141>
<160> 24
<170> PatentIn Ver. 2.0
<210> 1
<211> 1776
<212> DNA
<213> Homo sapiens
<400> 1
agctcacagt agcccggcgg cccagggcaa tccgaccaca tttcactctc accgctgtag 60
gaatccagat gcaggccaag tacagcagca cgagggacat gctggatgat gatggggaca 120
ccaccatgag cctgcattct caagcctctg ccacaactcg gcatccagag ccccggcgca 180
cagagcacag ggctccctct tcaacgtggc gaccagtggc cctgaccctg ctgactttgt 240
gcttggtgct gctgataggg ctggcagccc tggggctttt gttttttcag tactaccagc 300
tctccaatac tggtcaagac accatttctc aaatggaaga aagattagga aatacgtccc 360
aagagttgca atctcttcaa gtccagaata taaagcttgc aggaagtctg cagcatgtgg 420
ctgaaaaact ctgtcgtgag ctgtataaca aagctggagc acacaggtgc agcccttgta 480
cagaacaatg gaaatggcat ggagacaatt gctaccagtt ctataaagac agcaaaagtt 540
gggaggactg taaatatttc tgccttagtg asaactctac catgctgaag ataaacaaac 600
aagaagacct ggaatttgcc gcgtctcaga gctactctga gtttttctac tcttattgga 660
cagggctttt gcgccctgac agtggcaagg cctggctgtg gatggatgga acccctttca 720
cttctgaact gttccatatt ataatagatg tcaccagccc aagaagcaga gactgtgtgg 780
ccatccttaa tgggatgatc ttctcaaagg actgcaaaga attgaagcgt tgtgtctgtg 840
agagaagggc aggaatggtg aagccagaga gcctccatgt cccccctgaa acattaggcg 900
aaggtgactg attcgccctc tgcaactaca aatagcagag tgagccaggc ggtgccaaag 960
caagggctag ttgagacatt gggaaatgga acataatcag gaaagactat ctctctgact 1020
agtacaaaat gggttctcgt gtttcctgtt caggatcacc agcatttctg agcttgggtt 1080
tatgcacgta tttaacagtc acaagaagtc ttatttacat gccaccaacc aacctcagaa 1140
acccataatg tcatctgcct tcttggctta gagataactt ttagctctct ttcttctcaa 1200
tgtctaatat cacctccctg ttttcatgtc ttccttacac ttggtggaat aagaaacttt 1260
ttgaagtaga ggaaatacat tgaggtaaca tccttttctc tgacagtcaa gtagtccatc 1320
agaaattggc agtcacttcc cagattgtac cagcaaatac acaaggaatt ctttttgttt 1380
gtttcagttc atactagtcc cttcccaatc catcagtaaa gaccccatct gccttgtcca 1440
tgccgtttcc caacagggat gtcacttgat atgagaatct caaatctcaa tgccttataa 1500
gcattccttc ctgtgtccat taagactctg ataattgtct cccctccata ggaatttctc 1560
ccaggaaaga aatatatccc catctccgtt tcatatcaga actaccgtcc ccgatattcc 1620
cttcagagag attaaagacc agaaaaaagt gagcctcttc atctgcacct gtaatagttt 1680
cagttcctat tttcttccat tgacccatat ttataccttt caggtactga agatttaata 1740
ataataaatg taaatactgt gaaaaaaaaa aaaaaa 1776
1
CA 02302705 2000-03-06
WO 99113066 PCT/US98I18724
<210> 2
<211> 280
<212> PRT
<213> Homo sapiens
<400> 2
Met Gln Ala Lys Tyr Ser Ser Thr Arg Asp Met Leu Asp Asp Asp Gly
1 5 10 15
Asp Thr Thr Met Ser Leu His Ser Gln Ala Ser Ala Thr Thr Arg His
20 25 30
Pro Glu Pro Arg Arg Thr Glu His Arg Ala Pro Ser Ser Thr Trp Arg
35 40 45
Pro Val Ala Leu Thr Leu Leu Thr Leu Cys Leu Val Leu Leu Ile Gly
50 55 60
Leu Ala Ala Leu Gly Leu Leu Phe Phe Gln Tyr Tyr Gln Leu Ser Asn
65 70 75 80
Thr Gly Gln Asp Thr Ile Ser Gln Met Glu Glu Arg Leu Gly Asn Thr
85 90 95
Ser Gln Glu Leu Gln Ser Leu Gln Val Gln Asn Ile Lys Leu Ala Gly
100 105 110
Ser Leu Gln His Val Ala Glu Lys Leu Cys Arg Glu Leu Tyr Asn Lys
115 120 125
Ala Gly Ala His Arg Cys Ser Pro Cys Thr Glu Gln Trp Lys Trp His
130 135 140
Gly Asp Asn Cys Tyr Gln Phe Tyr Lys Asp Ser Lys Ser Trp Glu Asp
145 150 155 160
Cys Lys Tyr Phe Cys Leu Ser Glu Asn Ser Thr Met Leu Lys Ile Asn
165 170 175
Lys Gln Glu Asp Leu Glu Phe Ala Ala Ser Gln Ser Tyr Ser Glu Phe
180 185 190
Phe Tyr Ser Tyr Trp Thr Gly Leu Leu Arg Pro Asp Ser Gly Lys Ala
195 200 205
Trp Leu Trp Met Asp Gly Thr Pro Phe Thr Ser Glu Leu Phe His Ile
210 215 220
Ile Ile Asp Val Thr Ser Pro Arg Ser Arg Asp Cys Val Ala Ile Leu
225 230 235 240
Asn Gly Met Ile Phe Ser Lys Asp Cys Lys Glu Leu Lys Arg Cys Val
245 250 255
Cys Glu Arg Arg Ala Gly Met Val Lys Pro Glu Ser Leu His Val Pro
260 265 270
Pro Glu Thr Leu Gly Glu Gly Asp
275 280
2
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WO 99/13066 PCTIUS98118724
<210> 3
<211> 947
<212> DNA
<213> Homo Sapiens
<400> 3
caactatccc ataatttatt tattcttctt caatgtttgt aaagtgcatg agtcatgttc 60
acacttgaag tctagtagtg cactgtaata attcattttt taaaagatta tttaatgccc 120
atttcaaaat acagtagttt acacagctac agaaacaatt tggggcaagt tttaaaacac 180
tgaaacagta atagttattg gtgtcacata aaactgattt gttttttaca gccaaacctc 240
tgtcagtcag aggcattcat tagttttata catgtaattt gaaaatcact aaacctcgtt 300
ttctcagcag caataattta agaggcttca aaaatataat ttcactctta tttagtattt 360
tttcctgggg ggatttttac gtaatttttt tatgaaaaga caaatgcatg ttgagataac 420
ttctgggatt aaaatagtct tttgctttac ttttttggtt tcctaaaaca actttattga 480
cttttagtcc atactgttat atttttgtct taaagaaaat ttaaactaca aataccaaaa 540
gaaaacattt taaatttagg gatgagactt tggtgtatcg tgggtctagg tttaatgaac 600
acatctgggg ttaagttggc atttcttcac atctccacac ccacaccaac catcacagcc 660
ccccaccaac cttctcccaa ccccaaaagc attgtccagg gatatagatt ttaccaaagg 720
cttcctggga agacgaggga gcaacacttt agattaaatg tgatcagact ttcctattag 780
atatggctct tctgtctctt gttatccccc tgacagctct gccataaagt cccttctcct B40
catccttccc aaacaggctg tataagtgct ttgaggtaat taaactcttt cctccagttt 900
acaaatatca cttaacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 947
<210> 4
<211> 72
<212> PRT
<213> Homo Sapiens
<400> 4
Met Arg Leu Trp Cys Ile Val Gly Leu Gly Leu Met Asn Thr Ser Gly
1 5 10 15
Val Lys Leu Ala Phe Leu His Ile Ser Thr Pro Thr Pro Thr Ile Thr
20 25 30
Ala Pro His Gln Pro Ser Pro Asn Pro Lys Ser Ile Val Gln Gly Tyr
35 40 45
Arg Phe Tyr Gln Arg Leu Pro Gly Lys Thr Arg Glu Gln His Phe Arg
50 55 60
Leu Asn Val Ile Arg Leu Ser Tyr
65 70
<210> 5
<211> 2120
<212> DNA
<213> Homo Sapiens
<400> 5
cgctgacttg ggcaatgggg ccggtggggt ttgggggcgg aagagaccct cggggttgag 60
aagtatgtgg tggcctttcg tcccctgtaa aacattgtca cacggtgtgg ggcggcagcg 120
ctggatcttt gcaaggctat tttggcattc tgctggatat atgttcgtaa ataccaaagt 180
cggcgggaaa gtgaagttgt ctccaccata acagcaattt tttctctagc aattgcactt 240
atcacatcag cacttctacc agtggatata tttttggttt cttacatgaa aaatcaaaat 300
ggtacattta aggactgggc taatgctaat gtcagcagac agattgagga cactgtatta 360
tacggttact atactttata ttctgttata ttgttctgtg tgttcttctg gatccctttt 420
gtctacttct attatgaaga aaaggatgat gatgatacta gtaaatgtac tcaaattaaa 480
3
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acggcactca agtatacttt gggatttgtt gtgatttgtg cactgcttct tttagttggt 540
gcctttgttc cattgaatgt tcccaataac aaaaattcta cagagtggga aaaagtgaag 600
tccctatttg aagaacttgg aagtagtcat ggtttagctg cattgtcatt ttctatcagt 660
tctctgacct tgattggaat gttggcagct ataacttaca cagcctatgg catgtctgcg 720
ttacctttaa atctgataaa aggcactaga agcgctgctt atgaacgttt ggaaaacact 7$0
gaagacattg aagaagtaga acaacacatt caaacgatta aatcaaaaag caaagatggt 840
cgacctttgc cagcaaggga taaacgcgcc ttaaaacaat ttgaagaaag gttacgaaca 900
cttaagaaga gagagaggca tttagaattc attgaaaaca gctggtggac aaaattttgt 960
ggcgctctgc gtcccctgaa gatcgtctgg ggaatatttt tcatcttagt tgcattgctg 1020
tttgtaattt ctcttttctt gtcaaattta gataaagctc ttcattcagc tggaatagat 1080
tctggtttca taatttttgg agctaacctg agtaatccac tgaatatgct tttgccttta 1140
ctacaaacag ttttccctct tgattatatt cttataacaa ttattattat gtactttatt 1200
tttacttcaa tggcaggaat tcgaaatatt ggcatatggt tcttttggat tagattatat 1260
aaaatcagaa gaggtagaac caggccccaa gcactccttt ttctctgcat gatacttctg 1320
cttattgtcc ttcacactag ctacatgatt tatagtcttg ctccccaata tgttatgtat 1380
ggaagccaaa attacttaat agagactaat ataacttctg ataatcataa aggcaattca 1440
accctttctg tgccaaagag atgtgatgca gatgctcctg aagatcagtg tactgttacc 1500
cggacatacc tattccttca caagttctgg ttcttcagtg ctgcttacta ttttggtaac 1560
tgggcctttc ttggggtatt tttgattgga ttaattgtat cctgttgtaa agggaagaaa 1620
tcggttattg aaggagtaga tgaagattca gacataagtg atgatgagcc ctctgtctat 1680
tctgcttgac agccttctgt cttaaaggtt ttataatgct gactgaatat ctgttatgca 1740
tttttaaagt attaaactaa cattaggatt tgctaactag ctttcatcaa aaatgggagc 1800
atggctataa gacaactata ttttattata tgttttctga agtaacattg tatcatagat 1860
taacatttta aattaccata atcatgctat gtaaatataa gactactggc tttgtgaggg 1920
aatgtttgtg caaaattttt tcctctaatg tataatagtg ttaaattgat taaaaatctt 1980
ccagaattaa tattcccttt tgtcactttt tgaaaacata ataaatcatt tgtatctgtg 2040
ccttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100
aaaaaaaaaa aaaaaaaaaa 2120
<210> 6
<211> 467
<212> PRT
<213> Homo sapiens
<400> 6
Met Lys Asn Gln Asn Gly Thr Phe Lys Asp Trp Ala Asn Ala Asn Val
1 5 10 15
Ser Arg Gln Ile Glu Asp Thr Val Leu Tyr Gly Tyr Tyr Thr Leu Tyr
20 25 30
Ser Val Ile Leu Phe Cys Val Phe Phe Trp Ile Pro Phe Val Tyr Phe
35 40 45
Tyr Tyr Glu Glu Lys Asp Asp Asp Asp Thr Ser Lys Cys Thr Gln Ile
50 55 60
Lys Thr Ala Leu Lys Tyr Thr Leu Gly Phe Val Val Ile Cys Ala Leu
65 70 75 80
Leu Leu Leu Val Gly Ala Phe Val Pro Leu Asn Val Pro Asn Asn Lys
85 90 95
Asn Ser Thr Glu Trp Glu Lys Val Lys Ser Leu Phe Glu Glu Leu Gly
100 105 110
Ser Ser His Gly Leu Ala Ala Leu Ser Phe Ser Ile Ser Ser Leu Thr
lI5 120 125
Leu Ile Gly Met Leu Ala Ala Ile Thr Tyr Thr Ala Tyr Gly Met Ser
4
*rB
CA 02302705 2000-03-06
WO 99113066 PCTIUS98118724
130 135 140
Ala Leu Pro Leu Asn Leu Ile Lys Gly Thr Arg Ser Ala Ala Tyr Glu
145 150 155 160
Arg Leu Glu Asn Thr Glu Asp Ile Glu Glu Val Glu Gln His Ile Gln
165 170 175
Thr Ile Lys Ser Lys Ser Lys Asp Gly Arg Pro Leu Pro Ala Arg Asp
180 185 190
Lys Arg Ala Leu Lys Gln Phe Glu Glu Arg Leu Arg Thr Leu Lys Lys
195 200 205
Arg Glu Arg His Leu Glu Phe Ile Glu Asn Ser Trp Trp Thr Lys Phe
210 215 220
Cys Gly Ala Leu Arg Pro Leu Lys Ile Val Trp Gly Ile Phe Phe Ile
225 230 235 240
Leu Val Ala Leu Leu Phe Val Ile Ser Leu Phe Leu Ser Asn Leu Asp
245 250 255
Lys Ala Leu His Ser Ala Gly Ile Asp Ser Gly Phe Ile Ile Phe Gly
260 265 270
Ala Asn Leu Ser Asn Pro Leu Asn Met Leu Leu Pro Leu Leu Gln Thr
275 280 285
Val Phe Pro Leu Asp Tyr Ile Leu Ile Thr Ile Ile Ile Met Tyr Phe
290 295 300
Ile Phe Thr Ser Met Ala Gly Ile Arg Asn Ile Gly Ile Trp Phe Phe
305 310 315 320
Trp Ile Arg Leu Tyr Lys Ile Arg Arg Gly Arg Thr Arg Pro Gln Ala
325 330 335
Leu Leu Phe Leu Cys Met Ile Leu Leu Leu Ile Val Leu His Thr Ser
340 345 350
Tyr Met Ile Tyr Ser Leu Ala Pro Gln Tyr Val Met Tyr Gly Ser Gln
355 360 365
Asn Tyr Leu Ile Glu Thr Asn Ile Thr Ser Asp Asn His Lys Gly Asn
370 375 380
Ser Thr Leu Ser Val Pro Lys Arg Cys Asp Ala Asp Ala Pro Glu Asp
385 390 395 400
Gln Cys Thr Val Thr Arg Thr Tyr Leu Phe Leu His Lys Phe Trp Phe
405 410 415
Phe Ser Ala Ala Tyr Tyr Phe Gly Asn Trp Ala Phe Leu Gly Val Phe
420 425 430
Leu Ile Gly Leu Ile Val Ser Cys Cys Lys Gly Lys Lys Ser Val Ile
435 440 445
Glu Gly Val Asp Glu Asp Ser Asp Ile Ser Asp Asp Glu Pro Ser Val
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
450 455 460
Tyr Ser Ala
465
<210> 7
<211> 2487
<212> DNA
<213> Homo Sapiens
<400> 7
gttccgaaat aaaagatttt gcaaaccact ttcctacgta cgtccactgt agtttttgca 60
gatacaacac tagctgtagc aaagcctatg taaatcatat gatgagcttt catagtaacc 120
gtccaagcaa aaggttttgt atttttaaga agcattcaga aaatctccgg ggcattactc 180
tagtgtgcct taattgtgat ttcctaagtg atgtttctgg cttagataat atggctacac 240
acttaagtca acataaaact catacttgcc aagttgtaat gcagaaagtt tctgtttgta 300
tcccaacttc tgagcacctt tctgaattaa aaaaagaagc tcccgcaaag gaacaagaac 360
ctgtgtctaa ggaaattgca agacctaaca tggctgaaag agaaacagaa acatcaaatt 420
ctgaaagtaa acaagataaa gctgcttctt caaaagaaaa aaatggatgt aatgcaaatt 480
catttgaagg ctcatcaaca acaaaaagtg aagaaagcat aacagtttca gataaggaaa 540
atgaaacctg tcttgcagac caggaaactg gctcaaaaaa catcgtcagt tgtgattcaa 600
atattggtgc agataaagtg gaaaagaaaa aacaaataca acacgtttgt caggaaatgg 660
agttgaagat gtgccaaagt tcagaaaaca taatcttatc tgatcagatt aaagatcaca 720
actccagtga agccagattt tcttcaaaga atattaagga tttgcgatta gcatcagata 780
atgtaagcat tgatcagttt ttgagaaaaa gacatgaacc tgaatctgtt agttctgatg 840
ttagcgagca aggcagtatt catttggaac ctctgactcc atccgaggta cttgagtatg 900
aagccacaga gattcttcag aaaggtagtg gtgatccttc agccaagact gatgaagtag 960
tgtctgatca aacagatgac attcctggag gaaataaccc tagcacaaca gaggcaacag 1020
tagacctgga agatgaaaaa gaaagaagtt gaaattagtc attttaagtt tcagtgtacc 1080
aacgataagg gcatttggaa cagtgctatc aggtgagctc agtggtgctg ttgtaggttc 1140
agaaatggaa atatgtaagg gaggtcacac atacacttta cctgtatgtt caacctatgt 1200
tatcaaacaa atcaattcac caataatagc atgattagta gggattccca aaaagttttt 1260
aaaaacacga acaggatttt aatgataatt aaatttgcag tggaaaggtc tcatttaatg 1320
gttttcaagg aaatgggatt tggttgctga catgaattga tgatattagt aatatttata 1380
aagcctttca aacttccatc aatcctaagc taaaaatctt tattacctgt atatcctttt 1440
cagttaactg agaggaaggg atttggaaac catgtacttt tggggagtaa ttgattaaaa 1500
acaatggctg attggcattg ttaatgaagg ctttatttgt gaggatgatg ctggtaaatg 1560
gagcatgctt agagtactaa attgatctaa tgagaatttg gatgaacata aacttaattt 1620
tggatttaat ataacattcc agtcagacgc atgtaaacag aatatttgaa tctttgtacc 1680
tccatacaag tgttagcctg ccaggctgta agcttacctt aattaaactt tcagtgaaag 1740
tggaattatt aagatataaa tttatatttg tgctttttgt cagtgtgtaa gctgtgtaga 1800
aattctttga tgtattagtt gtattaatgt aaagtagaaa cccattgttg aaactcctgt 1860
agctattatg cttttaatat tgttttaatg atcttcctta gaaataggcc cataaaaatg 1920
gtctggaagc caaaccaaag tatggtataa tgtagatatt gtaaagcagt aaactgaaaa 1980
catgtcctgg catgtattca gccatgttta agtgactttt ctgtaattgt aaaataaaaa 2040
cttcaaatgg gacctaaaac agtgatgtaa aagaactggt tttggaaatt tagcctaatt 2100
tatctataag atggctgcta aattgatttt tcagttcttt ttatcatcta gaatataata 2160
gatatagaaa tgaataatat gaagaacagt agtttgcttt gaaatactaa taaactttta 2220
tttaaaatgc ttcattttta cttcttaaaa tgtgctttgg attcttaaat tttgtttcac 2280
tgaatgttca atgttttaaa tggcgattaa aatactctgc tgtatatagt agtttttgag 2340
taaatatttg caataaaaat ctgcccccga ataaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460
aaaaaaaaaa aaaaaaaaaa aaaaaaa 2487
<210> 8
<211> 317
<212> PRT
<213> Homo Sapiens
6
CA 02302705 2000-03-06
WO 99113066 PCT/US98/18724
<400> 8
Met Met Ser Phe His Ser Asn Arg Pro Ser Lys Arg Phe Cys Ile Phe
2 5 10 15
Lys Lys His Ser Glu Asn Leu Arg Gly Ile Thr Leu Val Cys Leu Asn
20 25 30
Cys Asp Phe Leu Ser Asp Val Ser Gly Leu Asp Asn Met Ala Thr His
35 40 45
Leu Ser Gln His Lys Thr His Thr Cys Gln Val Val Met Gln Lys Val
50 55 60
Ser Val Cys Ile Pro Thr Ser Glu His Leu Ser Glu Leu Lys Lys Glu
65 70 75 80
Ala Pro Ala Lys Glu Gln Glu Pro Val Ser Lys Glu Ile Ala Arg Pro
85 90 95
Asn Met Ala Glu Arg Glu Thr Glu Thr Ser Asn Ser Glu Ser Lys Gln
100 105 110
Asp Lys Ala Ala Ser Ser Lys Glu Lys Asn Gly Cys Asn Ala Asn Ser
115 120 125
Phe Glu Gly Ser Ser Thr Thr Lys Ser Glu Glu Ser Ile Thr Val Ser
130 135 140
Asp Lys Glu Asn Glu Thr Cys Leu Ala Asp Gln Glu Thr Gly Ser Lys
145 150 155 160
Asn Ile Val Ser Cys Asp Ser Asn Ile Gly Ala Asp Lys Val Glu Lys
165 170 175
Lys Lys Gln Ile Gln His Val Cys Gln Glu Met Glu Leu Lys Met Cys
180 185 190
Gln Ser Ser Glu Asn Ile Ile Leu Ser Asp Gln Ile Lys Asp His Asn
195 200 205
Ser Ser Glu Ala Arg Phe Ser Ser Lys Asn Ile Lys Asp Leu Arg Leu
210 215 220
Ala Ser Asp Asn Val Ser Ile Asp Gln Phe Leu Arg Lys Arg His Glu
225 230 235 240
Pro Glu Ser Val Ser Ser Asp Val Ser Glu Gln Gly Ser Ile His Leu
245 250 255
Glu Pro Leu Thr Pro Ser Glu Val Leu Glu Tyr Glu Ala Thr Glu Ile
260 265 270
Leu Gln Lys Gly Ser Gly Asp Pro Ser Ala Lys Thr Asp Glu Val Val
275 280 285
Ser Asp Gln Thr Asp Asp Ile Pro Gly Gly Asn Asn Pro Ser Thr Thr
290 295 300
Glu Ala Thr Val Asp Leu Glu Asp Glu Lys Glu Arg Ser
305 310 315
7
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
<210> 9
<211> 3495
<222> DNA
<213> Homo Sapiens
<400> 9
tttatttttc aaatcataat tttaaaatga tagataccat tttgtgataa caacaattca 60
gaaaacaatt ttctatcctc ttagttgaaa gaatgtaggt acagtttgga tacttgtact 120
ttaattttag agtaaacatc tgcattatac tcttatagat aatagaatta tttagttaag 180
aaattcttta cagtaaatga gataatgtgt gaaaaagtat tttgtaaatg ctgaggattc 240
tacaaatgat agttgttatt ttcatgtgta tttgtaagat catgtccatt tcatgaatat 300
aggacttcac ataaaaaaag actttctcaa gacaacttta tattctagta tttttctgtt 360
gtaaaaagta ttaactattt acttttattt tgttatacat ttattttaat atccatgtgt 420
ttattatagt aaatttgaaa tgaaatcctg aaaaacagaa tttttttaaa cacagacctc 480
acaccaatat taattttttc tctacataat ttaaaactac ataaattaag tacttaaaat 540
ttatattgaa ggccaccaag aacttaggtt gaatcttaga aaatttaaat aactattttt 600
aaagttaccc aacttaatat tttaattttt taatatttat cttcctttac taattcttga 660 ,
taaataatag cattagactt gataaaataa aaaagaattt tagagtagaa ttaatatatc 720
aaaaggggta tatcaaccaa attggtgtca gattgtattc attctctcat cacataaaga 780
tttttctttt gataggtgat gctcatatga acctttggtt tagaatctat atatgtacat 840
gtgtatgtat gtagatagta tggttgtata cacacatata taccaaacac catgaatttt 900
agcagtctgt gatgatcagc aaaaaagcac ataaagtaaa attagttgac catgctaaat 960
tcaattctgg aatttttttt tatttgggca tttctagaac tttttacatt tgaaagtaca 1020
tgatgagtat tagtaacgat gacttatgta taatcagaat ctttatgaca atttagtttt 1080
acaaggtcag aagagatgag tttgctaaac ccagctgtga tacctcagtt ggaaagggaa 1140
ttcaaaggta tgctttgtag aacagaaaag tatagttttt ttttcatgaa ctttaatcat 1200
tttctgtttt tcctctatgt gagtcagcta caaaagtggt ctaattttta caacagtaga 1260
acttcctcct tttctactgt aatcttccca ctgactttac tgcacaggta tgaaatacta 1320
gtgtattgga tcttcagtaa cctttttatt tcctagatga ttgaaatata ggtatttact 1380
ccatttaaac caggtgataa gatgatgtaa atactcaggg agggtattaa cttgttactt 1440
ttgctcgttt ggggtgtaaa gtgccatgac tgaataatct tcaattcatg attctagagt 1500
aagtttaatt tggaaaaagg ggcttcacac atggtggtgg ttgaacattg attcttttat 1560
acttaaaaag atgaaaatgt tttgtggact gatacatttt atcttactga atatgaattg 1620
tttatgtatc tctactgtca aatagccttt ttgaaactca ggaaagacaa aggttcaatt 1680
acaccacttt tgtcaataag caaaccaggt attttttttt tctcctgttg tctggatatg 1740
gcaatagatt ttttaaattg ctgtgagaac ccatatatga aaagagagga gttgaattgt 1800
gtgtgccttt tatgtcttga gatttatatg tggaaaagac gacatctact tcaaactgta 1860
tttttttcgt tttttttttt tttttgggga aggggggaga acggggtctt gctctgtcgc 1920
ccaggctgga gtgcagtggc gcgatctcag ctgactgcaa cctccacctc ccgggttcaa 1980
gggattctgc ctcagcctcc cgagtagctg agaccacagg tgcgtgccac cacacccggc 2040
taattttttt gtatttttag tagagacggg gtttagtaga gacggatcac tcctgaccac 2100
gtgatccgcc cacctcggcc tcccaaagtg ctgggattac aggcgtgagc caccaccccc 2160
ggcctgtatt ttcagagagg agagcttggt gtttttgtgg tgccaagtgg taagataatg 2220
tctctttgag gcttcctatg gactgccttt attttagtaa actcaagaca ccagttaacc 2280
tcaacagagt tttggcctta ttagaatttg ttgtgcatct tattgaaagc caggtttaca 2340
tcacctcacc ccattattct ttttagttaa ataaatttac catgccaagt aaccagaatg 2400
gagcaaattg gttgatcttt aaggcagtag gtttgactag ctagctatca ttattgtcac 2460
atctaatgct aggcaccaga aaccatttga gccaggagtg tgaatgaata attcccagag 2520
acactttaga cattttttaa tgttttatat gacattttac atttgtgtga ttgccttaga 2580
tattaaattt tcctagtgct gataaaaaca gcaacattca taacttattt tatatattgt 2640
tccaaagaaa agaatttgtt ttaatggttt caaaataact gcacctgaat ttgtttatgt 2700
gccttaagtt ctctagtgct atttcaactt ttttttcaat ctaaatgaag cttaccttag 2760
ataaggttca tatttgtttc ctatagagta aataaacttc cccttcttaa attgtgtaat 2820
aagcaccaac gtgtggttgc ttggcagaat gagaatgtta agggagattg ttggatgttt 2880
ggagtttcat tatatttttt gtttttattt tttgatacct aggtgctttt taaaatattc 2940
agacaaatat ctatcttaca ttgattaaac ccgtgtaaat tcatttgcag tatctacatc 3000
gaatgtcaaa aaagtatact tatttttgtt ccatacttat gtacaatttt ttccctcttc 3060
aggctttttc atttaccttt ttgaaaaagc acttactctc cccttcccta tcacccctcc 3120
8
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
cccaaggttt ctttatttaa atttttattg agagttgttg gagctctaag acaatacaaa 3180
tttagagttg aacaaaagta taatctgctt tacaactagt atagacctaa ggtcatttgc 3240
tttcaattag aggctccaga gtcttcatag tggaaagaat gctttgtatt taattgttct 3300
tagttaagtt gtagcacgtg aatacttact tacatgtttt gtttaaatat acttcttgca 3360
tagtttaatt ttttaaaagt tgtatctaat aaaatgtctt ttaaccatta ttacttgact 3420
atatggttgt attaaatttt gtttacgaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480
aaaaaaaaaa aaaaa 3495
<210> 10
<211> 45
<212> PRT
<213> Homo Sapiens
<400> 10
Met Ser Lys Lys Tyr Thr Tyr Phe Cys Ser Ile Leu Met Tyr Asn Phe
1 5 10 15
Phe Pro Leu Gln Ala Phe Ser Phe Thr Phe Leu Lys Lys His Leu Leu
20 25 30
Ser Pro Ser Leu Ser Pro Leu Pro Gln Gly Phe Phe Ile
35 40 45
<210> 11
<211> 1398
<212> DNA
<213> Homo Sapiens
<400> 11
gttgatccat ctgagaaagg gatcatgaac tagacagaat gaacagcctt agaggcacag 60
actcttgaac gggacggtgg tggtatgact agtgcagagt gtttagagat cactcagttt 120
ttaaagactg gcctttatcg tgtctcagtg cagccgaggc agagcctttg aaggatgcga 180
tgttgtcatt cttactaatc tagtccagcc gctgaggtga ctttcaacgg cagaccgtct 240
cctgagcgcc ccaggtagaa tttcaaaagt ctccgggacc attatggcag tcaagtggac 300
gggtgggcat tcttctcctg tcctctgcct gaatgcaagt aaagaagggc tgctggcttc 360
tggagcagag ggcggagatc tcacggcttg gggtgaagat ggaactccat taggacacac 420
gcggttccaa ggggctgatg atgttaccag tgtcttattt tctccctcct gtcccaccaa 480
gctctatgcc tcacatggag aaaccattag tgtactggat gtcaggtccc tcaaagattc 540
cttggaccat tttcatgtga atgaagaaga aatcaattgt ctttcattga atcaaacgga 600
aaacctgctg gcttctgctg acgactctgg ggcaatcaaa atcctagact tggaaaacaa 660
gaaagttatc agatccttga agagacattc caatatctgc tcctcagtgg cttttcggcc 720
tcagaggcct cagagcctgg tgtcatgtgg actggatatg caggtgatgc tgtggagtct 780
tcaaaaagcc cgaccactct ggattacaaa tttacaggag gatgaaacag aagaaatgga 840
aggcccacag tcacctggtc agctcttaaa ccctgcccta gcccattcta tctctgtggc 900
ttcgtgtggt aatattttta gttgtggtgc agaagatggt aaggttcgaa tctttcgggt 960
gatgggagtt aagtgtgaac aggaactggg atttaagggc cacacttcag gggtatccca 1020
ggtctgcttt ctcccagaat cctatttgct gcttactgga gggaatgatg ggaagatcac 1080
gttgtgggat gcaaacagtg aagttgagaa aaaacagaag agtcccacaa aacgtaccca 1140
caggaagaaa cctaaaagag gaacttgcac caagcagggt ggaaatacta acgcttcagt 1200
aacagatgag gaagaacatg gcaacatttt accgaagcta aatattgaac atggagaaaa 1260
agtgaactgg ctcttgggta caaaaataaa gggacaccaa aatatattag tagctgatca 1320
aactagttgt atatctgtat accccttaaa tgaattttaa atccaataaa aacatttgaa 1380
gaaaaaaaaa aaaaaaaa 1398
<210> 12
<211> 358
<212> PRT
<213> Homo Sapiens
9
CA 02302705 2000-03-06
WO 99/13066 PCT/US98118724
<400> 12
Met Ala Val Lys Trp Thr Gly Gly His Ser Ser Pro Val Leu Cys Leu
1 5 10 15
Asn Ala Ser Lys Glu Gly Leu Leu Ala Ser Gly Ala Glu Gly Gly Asp
20 25 30
Leu Thr Ala Trp Gly Glu Asp Gly Thr Pro Leu Gly His Thr Arg Phe
35 40 45
Gln Gly Ala Asp Asp Val Thr Ser Val Leu Phe Ser Pro Ser Cys Pro
50 55 60
Thr Lys Leu Tyr Ala Ser His Gly Glu Thr Ile Ser Val Leu Asp Val
65 70 75 80
Arg Ser Leu Lys Asp Ser Leu Asp His Phe His Val Asn Glu Glu Glu
85 90 95
Ile Asn Cys Leu Ser Leu Asn Gln Thr Glu Asn Leu Leu Ala Ser Ala
100 105 110
Asp Asp Ser Gly Ala Ile Lys Ile Leu Asp Leu Glu Asn Lys Lys Val
115 120 125
Ile Arg Ser Leu Lys Arg His Ser Asn Ile Cys Ser Ser Val Ala Phe
130 135 140
Arg Pro Gln Arg Pro Gln Ser Leu Val Ser Cys Gly Leu Asp Met Gln
145 150 155 160
Val Met Leu Trp Ser Leu Gln Lys Ala Arg Pro Leu Trp Ile Thr Asn
165 170 175
Leu Gln Glu Asp Glu Thr Glu Glu Met Glu Gly Pro Gln Ser Pro Gly
180 185 190
Gln Leu Leu Asn Pro Ala Leu Ala His Ser Ile Ser Val Ala Ser Cys
195 200 205
Gly Asn Ile Phe Ser Cys Gly Ala Glu Asp Gly Lys Val Arg Ile Phe
210 215 220
Arg Val Met Gly Val Lys Cys Glu Gln Glu Leu Gly Phe Lys Gly His
225 230 235 240
Thr Ser Gly Val Ser Gln Val Cys Phe Leu Pro Glu Ser Tyr Leu Leu
245 250 255
Leu Thr Gly Gly Asn Asp Gly Lys Ile Thr Leu Trp Asp Ala Asn Ser
260 265 270
Glu Val Glu Lys Lys Gln Lys Ser Pro Thr Lys Arg Thr His Arg Lys
275 280 285
Lys Pro Lys Arg Gly Thr Cys Thr Lys Gln Gly Gly Asn Thr Asn Ala
290 295 300
Ser Val Thr Asp Glu Glu Glu His Gly Asn Ile Leu Pro Lys Leu Asn
305 310 315 320
1~
CA 02302705 2000-03-06
WO 99113066 PCTIUS98/I8724
Ile Glu His Gly Glu Lys Val Asn Trp Leu Leu Gly Thr Lys Ile Lys
325 330 335
Gly His Gln Asn Ile Leu Val Ala Asp Gln Thr Ser Cys Ile Ser Val
340 345 350
Tyr Pro Leu Asn Glu Phe
355
<210> 13
<211> 2132
<212> DNA
<213> Homo Sapiens
<400> 13
ccggaggtag ctaccacggc ctgtgtcaac gactaaagct ccagtacagc ggcgccctca 60
gacagctggg agggtggctc tggccgggag cggcggccgg tgagctaccg cgaggaggag 120
cggcggaggc gacctcggcc cggccctgca ctggccgccc ggcaggcgcg acatgagcct 180
ggtctggcat ccgcgggatg ctccttaagc cccttctccg gctgttaacc tccggggaac 240
ggttgtgacc acaccgacac gtattttaca gataaatcat tcttgcggcg gcgggtcgaa 300
cacgtttatt tattttttat tttctcaaca agcttttacc cagcacctgt ccagtgaaac 360
aacttgataa tcgtttcgag gggcgtccgc cgggttagga agccactgcc tggcagcttg 420
tggaagcctc atttgcaaag ccacccctca gatgttttga agatcgtgac gtcttgtaac 480
tagcagtgtg tgcacagaat cctactcaag gaacgtcttg gcccagcgat gcaaagaact 540
gaagtttcaa gctggaagag cctgtattgt cctcacaata gtatagaaga attcaagaga 600
ggagagagag acagcaccga atgaagactg taaaagaaaa gaaggaatgc cagagattga 660
gaaaatctgc caagactagg agggtaaccc agaggaaacc gtcttcaggg cctgtttgct 720
ggctatgcct tcgagaacct ggggatcccg aaaaattagg ggaatttctt cagaaagaca 780
atatcagcgt gcattatttc tgtcttatct tatctagtaa gctgcctcag aggggccagt 840
ccaacagagg tttccatgga tttctgcctg aagacatcaa aaaggaggca gcccgggctt 900
ctaggaagat ctgctttgtg tgcaagaaaa agggagctgc tatcaactgc cagaaggatc 960
agtgcctcag aaacttccat ctgccttgtg gccaagaaag gggttgcctt tcacaatttt 1020
ttggagagta caaatcattt tgtgacaaac atcgcccaac acagaacatc caacatgggc 1080
atgtggggga ggaaagctgc atcttatgtt gtgaagactt atcccaacag agtgttgaga 1140
acatccagag cccgtgttgt agtcaagcca tctaccaccg caagtgcata cagaaatatg 1200
cccacacatc agcaaagcat ttcttcaaat gtccacagtg taacaatcga aaagagtttc 1260
ctcaagaaat gctgagaatg ggaattcata ttccagacag agatgctgcc tgggaactcg 1320
agccaggggc tttctcagac ttatatcagc gctatcagca ctgtgatgcc cccatctgtc 2380
cgtatgaaca aggcagagac agctttgagg atgaagggag gtggtgcctc attctgtgtg 1440
ctacatgcgg atcccacgga acccacaggg actgctcctc tcttagattt aacagtaaga 1500
aatgggagtg tgaggagtgt tcacctgctg cagccacaga ctacatacct gaaaactcag 1560
gggacatccc ttgctgcagc agcaccttcc accctgagga acatttctgc agagacaaca 1620
ccttggaaga gaatccgggc ctttcttgga ctgattggcc agaaccttcc ttattagaaa 1680
agccagagtc ctctcgtggc aggaggagct actcctggag gtccaagggt gtcagaatca 1740
ctaacagctg caaaaaatcc aagtaacacc ttctgagtag ctgctgtccc acacaatagg 1800
gtatgaagct gcgctcctcc atcgggtttg gggagggagc actctgggac tgtgagacaa 1860
ggaagcaggg ccagcagtga gactatgagc caagcaaaga gaagtctcag tggagcatga 1920
ggagggagca gtccagatgc caacaaggaa atgcgtttat ggctacaaga gtgcctctgc 1980
tttctcctcc tctcctccca ccaaggattc ttccacctta atcttgtttt catatgcctc 2040
ttcttacttc acccatgttt gttgttatgc aaataaaggt tttctctccc aaaaaaaaaa 2100
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa as 2132
<210> 14
<211> 381
<212> PRT
<213> Homo Sapiens
<400> 14
11
CA 02302705 2000-03-06
WO 99/13066 PCT/US98/18724
Met Lys Thr Val Lys Glu Lys Lys Glu Cys Gln Arg Leu Arg Lys Sex
1 5 10 15
Ala Lys Thr Arg Arg Val Thr Gln Arg Lys Pro Ser Ser Gly Pro Val
20 25 30
Cys Trp Leu Cys Leu Arg Glu Pro Gly Asp Pro Glu Lys Leu Gly Glu
35 40 45
Phe Leu Gln Lys Asp Asn Ile Ser VaI His Tyr Phe Cys Leu Ile Leu
50 55 60
Ser Ser Lys Leu Pro Gln Arg Gly Gln Ser Asn Arg Gly Phe His Gly
65 70 75 80
Phe Leu Pro Glu Asp Ile Lys Lys Glu Ala Ala Arg Ala Ser Arg Lys
85 90 95
Ile Cys Phe Val Cys Lys Lys Lys Gly Ala Ala Ile Asn Cys Gln Lys
100 105 110
Asp Gln Cys Leu Arg Asn Phe His Leu Pro Cys Gly Gln Glu Arg Gly
115 120 125
Cys Leu Ser Gln Phe Phe Gly Glu Tyr Lys Ser Phe Cys Asp Lys His
130 135 140
Arg Pro Thr Gln Asn Ile Gln His Gly His Val Gly Glu Glu Ser Cys
145 150 155 160
Ile Leu Cys Cys Glu Asp Leu Ser Gln Gln Ser Val Glu Asn Ile Gln
165 170 175
Ser Pro Cys Cys Ser Gln Ala Ile Tyr His Arg Lys Cys Ile Gln Lys
180 185 190
Tyr Ala His Thr Ser Ala Lys His Phe Phe Lys Cys Pro Gln Cys Asn
195 200 205
Asn Arg Lys Glu Phe Pro Gln Glu Met Leu Arg Met Gly Ile His Ile
210 215 220
Pro Asp Arg Asp Ala Ala Trp Glu Leu Glu Pro Gly Ala Phe Ser Asp
225 230 235 240
Leu Tyr Gln Arg Tyr Gln His Cys Asp Ala Pro Ile Cys Pro Tyr Glu
245 250 255
Gln Gly Arg Asp Ser Phe Glu Asp Glu Gly Arg Trp Cys Leu Ile Leu
260 265 270
Cys Ala Thr Cys Gly Ser His Gly Thr His Arg Asp Cys Ser Ser Leu
275 280 285
Arg Phe Asn Ser Lys Lys Trp Glu Cys Glu Glu Cys Ser Pro Ala Ala
290 295 300
Ala Thr Asp Tyr Ile Pro Glu Asn Ser Gly Asp Ile Pro Cys Cys Ser
305 310 . 315 320
12
CA 02302705 2000-03-06
WO 99/13066 PCT/IJS98/18724
Ser Thr Phe His Pro Glu Glu His Phe Cys Axg Asp Asn Thr Leu Glu
325 330 335
Glu Asn Pro Gly Leu Ser Trp Thr Asp Trp Pro Glu Pro Ser Leu Leu
340 345 350
Glu Lys Pro Glu Ser Ser Arg Gly Arg Arg Ser Tyr Ser Trp Arg Ser
355 360 365
Lys Gly Val Arg Ile Thr Asn Ser Cys Lys Lys Ser Lys
370 375 380
<210> 15
<211> 984
<212> DNA
<213> Homo sapiens
<400> 15
gtcacgtgga acctcttaat ctcagcatcc ggagctccag gaagggaaaa tttcaagtca 60
gatagaattc tatatatacc atttctttgg aaccttcagc cctcaagatt ccaacatcat 120
gacctcagtt tcaacacagt tgtccttagt cctcatgtca ctgcttttgg tgctgcctgt 180
tgtggaagca gtagaagccg gtgatgcaat cgcccttttg ttaggtgtgg ttctcagcat 240
tacaggcatt tgtgcctgct tgggggtata tgcacgaaaa agaaatggac agatgtgact 300
ttgaaaggcc tactgagtca aacctcaccc tgaaaacctt tgcgctttag aggctaaacc 360
tgagatttgg tgtgtgaaag gttccaagaa tcagtaaata agggagtttc acatttttca 420
ttgtttccat gaaatggcaa caaacataca tttataaatt gaaaaaaaaa tgttttcttt 480
acaacaaata atgcacagaa aaatgcagcc tataatttgc tagttaggta gtcaaagaag 540
taagatggct gaaatttaca taagtaatat ttcataatct tagaattctc tcaaagcatg 600
tgaaatagga agaaggaagt tcttgcccag aatcttagga aatcaccact gttcggttat 660
aatcactgcc tcctgaatcg ttgaggagtc ttttaaatta gatttttgtt ttgttgtctc 720
ccaagttaat attatattta gatatcagag agtcaggcaa aaaggaaaac ttttatctct 780
agggaaaaaa catttagaaa aatgtattca gtgtatctaa tactgaaatg cggaaaaaaa 840
tttaatgtta aaaaaaaact atagacattg acatggaaaa gagatttaat gttttgaaaa 900
aaaaacttta tattaactga gtaacatcct cctgatgaga agtactatat taaatataaa 960
cccattatgt tataaaaaaa aaaa 984
<210> 16
<211> 59
<212> PRT
<213> Homo sapiens
<400> 16
Met Thr Ser Val Ser Thr Gln Leu Ser Leu Val Leu Met Ser Leu Leu
1 5 10 15
Leu Val Leu Pro Val Val Glu Ala Val Glu Ala Gly Asp Ala Ile Ala
20 25 30
Leu Leu Leu Gly Val Val Leu Ser Ile Thr Gly Ile Cys Ala Cys Leu
35 40 45
Gly Val Tyr Ala Arg Lys Arg Asn Gly Gln Met
50 55
<210> 1?
<211> 28
<212> DNA
<213> Homo sapiens
13
CA 02302705 2000-03-06
WO 99113066 PCTIUS98118724
<400>
17
gttcctacagcggtgagagtgaaatgtg 28
<210>
18
<211>
28
<212>
DNA
<213> Sapiens
Homo
<400>
18
ccgatacaccaaagtctcatccctaaat 28
<210>
19
<211>
28
<212>
DNA
<213> sapiens
Homo
<400>
19
gagctagtgtgaaggacaataagcagaa 28
<210>
20
<211>
28
<212>
DNA
<213> Sapiens
Homo
<400>
20
gtcgctaacatcagaactaacagattca 28
<210>
21
<211>
28
<212>
DNA
<213> Sapiens
Homo
<400>
21
gagatactgcaaatgaatttacacgggt 28
<210>
22
<211>
28
<212>
DNA
<213> Sapiens
Homo
<400>
22
gtcacacttaactcccatcacccgaaag 28
<210>
23
<211>
28
<2I2>
DNA
<213> sapiens
Homo
<400>
23
tctgaggcactgatccttctggcagttg 28
<210>
24
<211>
28
<212>
DNA
<213> Sapiens
Homo
<400>
24
tgagggctgaaggttccaaagaaatggt 28
14
