Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,XXX
(converted to a provisional application from non-provisional application 08 /
873,488), filed
June 12,1997, 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
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 2178 to nucleotide 2513;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 2364 to nucleotide 2513;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1980 to nucleotide 2311;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone c1181 3 deposited under accession
number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone c1181_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone c1181 3 deposited under accession number
2 0 ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone c1181 3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
2 5 (j) 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;
(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
N0:1 from nucleotide 2178 to nucleotide 2513; the nucleotide sequence of SEQ
ID N0:1
from nucleotide 2364 to nucleotide 2513; the nucleotide sequence of SEQ ID
N0:1 from
nucleotide 1980 to nucleotide 2311; the nucleotide sequence of the full-length
protein
coding sequence of clone c1181_3 deposited under accession number ATCC 98456;
or the
nucleotide sequence of a mature protein coding sequence of clone c1181 3
deposited
under accession number ATCC 98456. In other preferred embodiments, -the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone c1181 3 deposited under accession number ATCC 98456. 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 67.
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 51 to amino acid 60 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 0 ID N0:1.
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 5 (b) the amino acid sequence of SEQ ID N0:2 from amino acid 1 to
amino acid 67;
(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
3 0 c1181_3 deposited under accession number ATCC 98456;
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 67. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
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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 acid sequence
from amino
acid 51 to amino acid 60 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 207 to nucleotide 893;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1 to nucleotide 527;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cr1044_1 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cr1044_1 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 0 protein coding sequence of clone cr1044_1 deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone cr1044_1 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid
2 5 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
3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 207 to nucleotide 893; the nucleotide sequence of SEQ ID
N0:3
from nucleotide 1 to nucleotide 527; the nucleotide sequence of the full-
length protein
coding sequence of clone cr1044_1 deposited under accession number ATCC 98456;
or the
nucleotide sequence of a mature protein coding sequence of clone cr1044_1
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cr1044_1 deposited under accession number ATCC 98456. 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 107.
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 109 to amino acid 118 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
2 0 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 5 amino acid 107;
(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
cr1044_1 deposited under accession number ATCC 98456;
3 0 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 107. 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
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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
N0:4having biological activity, the fragment comprising the amino acid
sequence from
amino acid 109 to amino acid 118 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) , a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 77 to nucleotide 400;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 118 to nucleotide 392;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cz251 1 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cz251 1 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cz251 1 deposited under accession number
2 0 ATCC 98456;
(g) a polynudeotide encoding a mature protein encoded by the cDNA
insert of clone cz251 1 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 77 to nucleotide 400; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 118 to nucleotide 392; the nucleotide sequence of the full-length
protein coding
sequence of clone cz251_1 deposited under accession number ATCC 98456; or the
nucleotide sequence of a mature protein coding sequence of clone cz251_1
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cz251_1 deposited under accession number ATCC 98456. 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 15 to amino
acid
105. 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
polynucleoiide 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 49 to amino acid 58 of SEQ ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
2 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ ID N0:6 from amino acid 15 to
2 5 amino acid 105;
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
eight consecutive amino acids of SEQ ID N0:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone
cz251 1 deposited under accession number ATCC 98456;
3 0 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
of SEQ ID N0:6 from amino acid 15 to amino acid 105. 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
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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
N0:6having biological activity, the fragment comprising the amino acid
sequence from
amino acid 49 to amino acid 58 of SEQ ID N0:6.
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 13 to nucleotide 501;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1 to nucleotide 506;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ddl2_7 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ddl2_7 deposited under accession number
2 0 ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ddl2 7 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
2 5 (i) 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;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 13 to nucleotide 501; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 1 to nucleotide 506; the nucleotide sequence of the full-length
protein coding
sequence of clone ddl2_7 deposited under accession number ATCC 98456; or the
nucleotide sequence of a mature protein coding ,sequence of clone ddl2_7
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ddl2_7 deposited under accession number ATCC 98456. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:8 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:8, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 76
to amino acid 85 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
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
2 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8 comprising
eight consecutive amino acids of SEQ ID N0:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone
2 5 ddl2_7 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:8. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
preferably
3 0 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 N0:8having biological activity, the fragment comprising the amino acid
sequence
from amino acid 76 to amino acid 85 of SEQ ID N0:8.
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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
N0:9;
(b) a polyrtucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 778 to nucleotide 1083;
{c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 931 to nucleotide 1083;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 802 to nucleotide 1056;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fn191 3 deposited under accession
number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fn191 3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fn191 3 deposited under accession number
ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone fn191_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:10 having biological activity, the fragment
2 5 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
3 0 (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 778 to nucleotide 1083; the nucleotide sequence of SEQ ID
N0:9
from nucleotide 931 to nucleotide 1083; the nucleotide sequence of SEQ ID N0:9
from
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nucleotide 802 to nucleotide 1056; the nucleotide sequence of the full-length
protein
coding sequence of done fn191 3 deposited under accession number ATCC 98456;
or the
nucleotide sequence of a mature protein coding sequence of clone fn191 3
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone fn191 3 deposited under accession number ATCC 98456. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:10 from amino acid 1 to amino
acid
93. 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 NO:10, or a
polynudeotide encoding a protein comprising a fragment of the amino acid
sequence of
SEQ ID NO:10 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 46 to amino acid 55 of SEQ ID N0:10.
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 0 consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID N0:10 from amino acid 1 to
amino acid 93;
{c) fragments of the amino acid sequence of SEQ ID N0:10 comprising
2 5 eight consecutive amino acids of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
fn191 3 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10 or the amino acid
sequence
3 0 of SEQ ID N0:10 from amino acid 1 to amino acid 93. 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
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N0:10having biological activity, the fragment comprising the amino acid
sequence from
amino acid 46 to amino acid 55 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 390 to nucleotide 1355;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 1384 to nucleotide 1736;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone gm196 4 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone gm196 4 deposited under accession number ATCC 98456;
{f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone gm196 4 deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone gm196 4 deposited under accession number ATCC 98456;
(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
2 5 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
3 0 (1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:11 from nucleotide 390 to nucleotide 1355; the nucleotide sequence of SEQ
ID N0:11
from nucleotide 1384 to nucleotide 1736; the nucleotide sequence of the full-
length protein
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coding sequence of clone gm196 4 deposited under accession number ATCC 98456;
or the
nucleotide sequence of a mature protein coding sequence of clone gm196 4
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone gm196_4 deposited under accession number ATCC 98456. 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 156
to amino acid 165 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:11.
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:
(a) the amino acid sequence of SEQ ID N0:12;
(b) fragments of the amino acid sequence of SEQ ID N0:12 comprising
2 0 eight consecutive amino acids of SEQ ID N0:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
gm196_4 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12. In further
preferred
2 5 embodiments, the present invention provides a protein comprising a
fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:12, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:12 having biological activity, the fragment comprising the amino
acid
3 0 sequence from amino acid 156 to amino acid 165 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;
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 879 to nucleotide 1391;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 519 to nucleotide 1074;
(d) a polyriucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone gn114_1 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone gn114_1 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone gn114_1 deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gn114_1 deposited under accession number ATCC 98456;
{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;
2 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-{g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
{l) a polynucleotide that hybridizes under stringent conditions to any
2 5 one of the polynucleotides specified in (a~(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 879 to nucleotide 1391; the nucleotide sequence of SEQ
ID N0:13
from nucleotide 519 to nucleotide 1074; the nucleotide sequence of the full-
length protein
coding sequence of clone gn114_1 deposited under accession number ATCC 98456;
or the
3 0 nucleotide sequence of a mature protein coding sequence of clone gn114_1
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone gn114_1 deposited under accession number ATCC 98456. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
I4
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comprising the amino acid sequence of SEQ ID N0:14 from amino acid 1 to amino
acid
65. 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 80 to amino acid 89 of SEQ ID N0:14.
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:
(a) the amino acid sequence of SEQ ID N0:14;
(b) the amino acid sequence of SEQ ID N0:14 from amino acid 1 to
amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID N0:14 comprising
eight consecutive amino acids of SEQ ID N0:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 0 gn114_1 deposited under accession number ATCC 98456;
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 1 to amino acid 65. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
2 5 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
N0:14having biological activity, the fragment comprising the amino acid
sequence from
amino acid 80 to amino acid 89 of SEQ ID N0:14.
3 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 225 to nucleotide 1508;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 252 to nucleotide 1508;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 1 to nucleotide 302;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone hj968_2 deposited under accession
number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone hj968_2 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone hj968_2 deposited under accession number
ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone hj968_2 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:16;
(j) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID N0:16 having biological activity, the
fragment
comprising eight consecutive amino acids of SEQ ID N0:16;
(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
2 5 of (i) or (j) above ; and
(m) a polynucleotide 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
N0:15 from nucleotide 225 to nucleotide 1508; the nucleotide sequence of SEQ
ID N0:15
3 0 from nucleotide 252 to nucleotide 1508; the nucleotide sequence of SEQ ID
N0:15 from
nucleotide 1 to nucleotide 302; the nucleotide sequence of the full-length
protein coding
sequence of clone hj968_2 deposited under accession number ATCC 98456; or the
nucleotide sequence of a mature protein coding sequence of clone hj968_2
deposited
under accession number ATCC 98456. In other preferred embodiments, the
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polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone hj968 2 deposited under accession number ATCC 98456. 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 209 to amino acid 218 of SEQ ID N0:16.
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:
(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;
2 0 (c) fragments of the amino acid sequence of SEQ ID N0:16 comprising
eight consecutive amino acids of SEQ ID N0:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone
hj968_2 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 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,
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
3 0 N0:16, or a protein comprising a fragment of the amino acid sequence of
SEQ ID
N0:16having biological activity, the fragment comprising the amino acid
sequence from
amino acid 209 to amino acid 218 of SEQ ID N0:16.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
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(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 1113 to nucleotide 1274;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 1233 to nucleotide 1274;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 894 to nucleotide 1309;
(e) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone hkl0_3 deposited under accession
number
ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone hkl0 3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone hkl0 3 deposited under accession number ATCC
98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone hkl0_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:18;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:18 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:18;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
2 5 (a)-(h) above;
(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).
3 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:17 from nucleotide 1113 to nucleotide 1274; the nucleotide sequence of SEQ
ID N0:17
from nucleotide 1233 to nucleotide 1274; the nucleotide sequence of SEQ ID
N0:17 from
nucleotide 894 to nucleotide 1309; the nucleotide sequence of the full-length
protein
coding sequence of clone hkl0 3 deposited under accession number ATCC 98456;
or the
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nucleotide sequence of a mature protein coding sequence of clone hkl0_3
deposited under
accession number ATCC 98456. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone hkl0_3
deposited under accession number ATCC 98456. In further preferred embodiments,
the
present invention provides a polynucleotide encoding a protein comprising a
fragment
of the amino acid sequence of SEQ ID N0:18 having biological activity, the
fragment
preferably comprising eight (more preferably twenty, most preferably thirty)
consecutive
amino acids of SEQ ID N0:18, or a polynucleotide encoding a protein comprising
a
fragment of the amino acid sequence of SEQ ID N0:18 having biological
activity, the
fragment comprising the amino acid sequence from amino acid 22 to amino acid
31 of SEQ
ID N0:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
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 N0:18;
(b) fragments of the amino acid sequence of SEQ ID N0:18 comprising
eight consecutive amino acids of SEQ ID N0:18; and
2 0 (c) the amino acid sequence encoded by the cDNA insert of clone
hkl0_3 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:18. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
2 5 amino acid sequence of SEQ ID N0:18 having biological activity, the
fragment preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:18, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:18 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 22 to amino acid 31 of SEQ ID N0:18.
3 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19;
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 96 to nucleotide 1145;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 109 to nucleotide 539;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone hm236_1 deposited under accession
number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone hm236_1 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone hm236_1 deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone hm236_1 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:20;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:20 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:20;
2 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
2 5 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:19 from nucleotide 96 to nucleotide 1145; the nucleotide sequence of SEQ ID
N0:19
from nucleotide 109 to nucleotide 539; the nucleotide sequence of the full-
length protein
coding sequence of clone hm236_1 deposited under accession number ATCC 98456;
or the
3 0 nucleotide sequence of a mature protein coding sequence of clone hm236_1
deposited
under accession number ATCC 98456. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone hm236_1 deposited under accession number ATCC 98456. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
CA 02293825 1999-12-10
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comprising the amino acid sequence of SEQ ID N0:20 from amino acid 6 to amino
acid
148. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:20
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:20, or a
polynucleotide encoding a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:20 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 170 to amino acid 179 of SEQ ID N0:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:20;
(b) the amino acid sequence of SEQ ID N0:20 from amino acid 6 to
amino acid 148;
(c) fragments of the amino acid sequence of SEQ ID N0:20 comprising
eight consecutive amino acids of SEQ ID N0:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 0 hm236_1 deposited under accession number ATCC 98456;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:20 or the amino acid
sequence
of SEQ ID N0:20 from amino acid 6 to amino acid 148. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
2 5 sequence of SEQ B7 N0:20 having biological activity, the fragment
preferably comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
N0:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:20having biological activity, the fragment comprising the amino acid
sequence from
amino acid 170 to amino acid 179 of SEQ ID N0:20.
3 0 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
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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
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
pharmaceutically acceptable Garner.
BRIEF DESCRIPTION OF THE DRAWI1~TGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
2 5 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
3 0 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
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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 "c1181 3"
A polynucleotide of the present invention has been identified as clone "c1181
3".
c1181 3 was isolated from a human adult retina cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. c1181 3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"c1181 3 protein').
The nucleotide sequence of c1181 3 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 c1181 3 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:2. Amino acids 50 to 62 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 63, or
are a transmembrane domain.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
c1181 3 should be approximately 2800 bp.
The nucleotide sequence disclosed herein for 4181 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. c1181 3 demonstrated at least some similarity with
sequences
identified as C16190 (Human aorta cDNA 5'-end GEN-241B04) and L36900
(Saccharomyces cerevisiae mitochondrion transfer RNA-Serl (tRNA-Ser) gene and
varI
gene, complete cds). Based upon sequence similarity, c1181 3 proteins and each
similar
protein or peptide may share at least some activity. The TopPredII computer
program
predicts a potential transmembrane domain within the c1181 3 protein sequence
centered
3 0 around amino acid 77 of SEQ ID N0:2.
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Clone"cr1044 1"
A polynucleotide of the present invention has been identified as clone
"cr1044_1".
cr1044_1 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. cr1044_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "cr1044_1 protein').
The nucleotide sequence of cr1044_1 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 cr1044_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4. Amino acids 158 to 170 of SEQ
ID N0:4
are a predicted leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 171 of SEQ ID N0:4, or are a transmembrane domain.
Base pairs
175 to 237 of SEQ ID N0:3 are a possible intron; if this sequence were removed
from SEQ
ID N0:3, another potential cr1044_1 reading frame and predicted amino acid
sequence
that could be encoded by basepairs 45 to 830 of SEQ ID N0:3 is reported in SEQ
ID
N0:31. Amino acids 7 to 19 of SEQ ID N0:31 are a predicted leader/signal
sequence,
with the predicted mature amino acid sequence beginning at amino acid 20 of
SEQ ID
2 0 N0:31, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cr1044_1 should be approximately 3200 bp.
The nucleotide sequence disclosed herein for cr1044_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. cr1044_1 demonstrated at least some similarity
with sequences
identified as N99156 (zb81g04.s1 Soares senescent fibroblasts NbHSF Homo
Sapiens
cDNA clone 310038 3'), Q46852 (clone of recombinant human kappa casein gene
fragment), and T20727 (Human gene signature HUMGS01945). The predicted amino
acid
sequence disclosed herein for cr1044_1 was searched against the GenPept and
GeneSeq
3 0 amino acid sequence databases using the BLASTX search protocol. The
predicted
cr1044_1 protein demonstrated at least some similarity to sequences identified
~s M 16279
(antigen [Homo sapiens]) and U82164 (human CD99 type II). Based upon sequence
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similarity, cr1044_1 proteins and each similar protein or peptide may share at
least some
activity.
Clone "cz251 1"
A polynucleotide of the present invention has been identified as clone "cz251
1".
cz251 1 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. cz251 1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"cz251 1 protein").
The nucleotide sequence of cz251 1 as presently determined is reported in SEQ
ID
N0:5. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the cz251 1 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:6.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cz251 1 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for cz251 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. cz251 1 demonstrated at least some similarity with
sequences
identified as 855084 (yg87a06.r1 Homo sapiens cDNA clone 40244 5') and U00930
(Human clone C4E 1.63 (CAC)n/(GTG)n repeat-containing mRNA). The predicted
amino
acid sequence disclosed herein for cz251 1 was searched against the GenPept
and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
2 5 predicted cz251_1 protein demonstrated at least some similarity to the
sequence identified
as 268751 (FOlG4.1 [Caenorhabditis elegans]). Based upon sequence similarity,
cz251 1
proteins and each similar protein or peptide may share at least some activity.
The
nucleotide sequence of cz251 1 may contain CAAA-like repeats.
3 0 Clone "ddl2 7"
A polynucleotide of the present invention has been identified as clone
"ddl2_7".
ddl2 7 was isolated from a human adult testes cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
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identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ddl2_7 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ddl2_7 protein").
The nucleotide sequence of ddl2 7 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 ddl2_7 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ddl2_7 should be approximately 1550 bp.
The nucleotide sequence disclosed herein for ddl2_7 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ddl2_7 demonstrated at least some similarity with
sequences
identified as AA257999 (zs34a02.s1 Soares NbHTGBC Homo Sapiens cDNA clone
687050 3'). Based upon sequence similarity, ddl2_7 proteins and each similar
protein or
peptide may share at least some activity.
Clone "fn191 3"
A polynucleotide of the present invention has been identified as clone "fn191
3".
2 0 fn191 3 was isolated from a human fetal brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. fn191 3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
2 5 "fn191 3 protein")
The nucleotide sequence of fn191_3 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 fn191 3 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID NO:10. Amino acids 39 to 51 are a predicted
leader/signal
3 0 sequence, with the predicted mature amino acid sequence beginning at amino
acid 52, or
are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fn191 3 should be approximately 3000 bp.
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The nucleotide sequence disclosed herein for fn191 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fn191 3 demonstrated at least some similarity with
sequences
identified as AA046787 (zk72fn7.r1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 488389 5' similar to gb:L07077 ENOYIrCOA HYDRATASE (HUMAN);contains
Alu repetitive element), 613132 (human chromosome 7 STS sWSS3349; single
read),
T06013 (EST03902 Homo sapiens cDNA clone HFBDL25), T08594 (EST06486 Homo
sapiens cDNA clone HIBBG72 5' end), and W28342 {45g9 Human retina cDNA
randomly
primed sublibrary Homo sapiens cDNA). Based upon sequence similarity, fn191 3
proteins and each similar protein or peptide may share at least some activity.
The
nucleotide sequence in the 5' untranslated region of fn191_3 may contain some
repetitive
elements.
Clone "gm1964"
A polynucleotide of the present invention has been identified as clone
"gm196_4".
gm196 4 was isolated from a human adult uterus cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. gm196 4 is a full-
length
2 0 clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "gm196_4 protein').
The nucleotide sequence of gm196_4 as presently determined is reported in SEQ
ID N0:11. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the gm196 4 protein corresponding to the
foregoing
2 5 nucleotide sequence is reported in SEQ ID N0:12. Another potential gm196 4
reading
frame and predicted amino acid sequence is encoded by basepairs 1364 to 2809
of SEQ ID
N0:11 and is reported in SEQ ID N0:32. A frameshift in the nucleotide sequence
of SEQ
ID N0:11 could join the reading frames of SEQ ID N0:12 and SEQ ID N0:32. The
TopPredII computer program predicts two potential transmembrane domains within
the
3 0 amino acid sequence of SEQ ID N0:32. Preferred fragments of the amino acid
sequence
of SEQ ID N0:32 comprise amino acids 1 to 163 or amino acids 236 to 245.of SEQ
ID
N0:32.
27
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WO 98/56805 PCTNS98/12076
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
gm196 4 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for gm196_4 was searched against the
GenBank .and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. gm196_4 demonstrated at least some similarity with
sequences
identified as AA233552 (zr43a10.s1 Soares NhHMPu S1 Homo Sapiens cDNA clone
666138 3'), AB005666 (Homo Sapiens mRNA for GTPase-activating protein,
complete
cds), F12887 (H. sapiens partial cDNA sequence; clone c-3fh09), T25078 (Human
gene
signature HUMGS07218), and T75264 (yc88g09.r1 Homo Sapiens cDNA clone 23008
5').
The predicted amino acid sequence disclosed herein for gm196_4 was searched
against
the GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol. The predicted gm196 4 protein demonstrated at least some similarity
to the
sequence identified as M64788 (GTPase activating protein [Homo sapiens]).
Based upon
sequence similarity, gm196 4 proteins and each similar protein or peptide may
share at
least some activity.
Clone"gn114 1"
A polynucleotide -of the present invention has been identified as clone
"gn114_1".
gn114_1 was isolated from a human adult blood (peripheral blood mononuclear
cells
2 0 treated with granulocyte-colony stimulating factor in vivo) cDNA library
using methods
which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or
was identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. gn114_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
2 5 "gn114_1 protein").
The nucleotide sequence of gn114_1 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 gn114_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:14. Amino acids 26 to 38 of SEQ
ID N0:14
3 0 are a predicted leader/signal sequence, with the predicted mature amino
acid sequence
beginning at amino acid 39 of SEQ ID N0:14, or are a transmembrane domaiu~.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
gn114_1 should be approximately 1500 bp.
28
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
The nucleotide sequence disclosed herein for gn114_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. gn114_1 demonstrated at least some similarity with
sequences
identified as AA370547 (EST82206 Prostate gland I Homo sapiens cDNA S' end),
C04732
(Human Heart cDNA, clone 3NHC3910), and C05361 (Human Heart cDNA, clone
3NHC2451 ). Based upon sequence similarity, gn114_1 proteins and each similar
protein
or peptide may share at least some activity. The TopPredII computer program
predicts
an additional potential transmembrane domain within the gn114_1 protein
sequence
centered around amino acid 90 of SEQ ID N0:14.
Clone "hj968 2"
A polynucleotide _of the present invention has been identified as clone "hj968
2".
hj968 2 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. hj968 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"hj968_2 protein").
The nucleotide sequence of hj968_2 as presently determined is reported in SEQ
ID
2 0 N0:15. What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the hj968 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:16. Amino acids 1 to 9 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 10, or are a transmembrane domain.
2 5 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
hj968_2 should be approximately 1800 bp.
The nucleotide sequence disclosed herein for hj968_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. hj968 2 demonstrated at least some similarity with
sequences
3 0 identified as AA071746 (mf16h07.r1 Life Tech mouse brain Mus musculus cDNA
clone
405277 5') and AA325286 (EST28500 Cerebellum Ii Homo sapiens cDNA 5' end). The
predicted amino acid sequence disclosed herein for hj968 2 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
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WO 98/56805 PCT/iJS98/12076
The predicted hj968 2 protein demonstrated at least some similarity to the
sequence
identified as U23528 (translated cosmid B0034.[Caenorhabditis elegans]). Based
upon
sequence similarity, hj968_2 proteins and each similar protein or peptide may
share at
least some activity.
Clone "hkl0 3"
A polynucleotide of the present invention has been identified as clone "hkl0
3".
hkl0 3 was isolated from a human fetal brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. hkl0 3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"hkl0_3 protein").
The nucleotide sequence of hkl0_3 as presently determined is reported in SEQ
ID
N0:17. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the hkl0_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:18. Amino acids 28 to 40 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 41, or are a transmembrane domain.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
hkl0_3 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for hkl0_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. hkl0_3 demonstrated at least some similarity with
sequences
2 5 identified as AA305975 (EST 176966 Jurkat T-cells VI Homo sapiens cDNA 5'
end similar
to S. cerevisiae hypothetical protein FAA3-BET1), F18016 (H.sapiens EST
sequence
016-T), N36880 (yy37e07.s1 Homo sapiens cDNA clone 273444 3'), and 887757
(yo45a08.s1 Homo sapiens cDNA clone 180854 3'). The predicted amino acid
sequence
disclosed herein for hkl0 3 was searched against the GenPept and GeneSeq amino
acid
3 0 sequence databases using the BLASTX search protocol. The predicted hkl0 3
protein
demonstrated at least some similarity to the sequence identified as 238113
.(CAI 0.21
[Saccharomyces cerevisiae]). Based upon sequence similarity, hkl0 3 proteins
and each
similar protein or peptide may share at least some activity.
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
Clone "hm236 1"
A polynucleotide of the present invention has been identified as clone
"hm236_1".
hm236_1 was isolated from a human adult testes (teratocarcinoma NCCIT) cDNA
library
using methods which are selective for cDNAs encoding secreted proteins (see
U.S. Pat.
No. 5,536,637), or was identified as encoding a secreted or transmembrane
protein on the
basis of computer analysis of the amino acid sequence of the encoded protein.
hm236_1
is a full-length clone, including the entire coding sequence of a secreted
protein (also
referred to herein as "hm236_1 protein').
The nucleotide sequence of hm236_1 as presently determined is reported in SEQ
ID N0:19. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the hm236_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:20.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
hrn236_1 should be approximately 1800 bp.
The nucleotide sequence disclosed herein for hm236_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. hm236_1 demonstrated at least some similarity with
sequences
identified as AA026169 (zkO1b03.r1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 469229 5'), AA046211 {zk77e04.s1 Soares pregnant uterus NbHPU Homo
Sapiens
2 0 cDNA clone 488862 3'), AA223088 (PMY0368 KGla Lambda Zap Express cDNA
Library
Homo sapiens cDNA 5'), AB002368 and AC003010 (Human mRNA for KIAA0370 gene,
partial cds), AC002399 (Human chromosome 16p11.2 BAC clone CTT987SK-A-481B3;
HTGS phase 1,18 unordered pieces, sequencing in progress), 886676 (ym86f03.r1
Homo
sapiens cDNA clone), and T21501 (Human gene signature HUMGS02874). The
predicted
2 5 amino acid sequence disclosed herein for hm236_1 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted hm236_1 protein demonstrated at least some similarity to sequences
identified
as AB002368 (KIAA0370 [Homo sapiens]). Based upon sequence similarity, hm236_1
proteins and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones c1181 3, cr1044_1, cz251 1, ddl2_7, fn191 3, gm196_4, gn114_l, hj968_2,
hkl0_3, and hm236_1 were deposited on June 12,1997 with the American Type
Culture
Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.)
as an
31
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
original deposit under the Budapest Treaty and were given the accession number
ATCC
98456, from which each clone comprising a particular polynucleotide is
obtainable. All
restrictions on the availability to the public of the deposited material will
be irrevocably
removed upon the granting of the patent, except for the requirements specified
in 37
S 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. coli} in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector
("pED6") was derived from pED6dpc1 by insertion of a new polylinker to
facilitate
cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 4485-4490); the pNOTs
vector
was derived from pMT2 (Kaufman et aL,1989, Mol. Cell. Biol. 9: 946-958) by
deletion of
the DHFR sequences, insertion of a new polylinker, and insertion of the M13
origin of
replication in the CIaI site. In some instances, the deposited clone can
become "flipped"
(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
2 0 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
2 5 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
3 0 c1181 3 SEQ ID N0:21
cr1044_1 SEQ ID N0:22
cz251 1 SEQ ID N0:23
ddl2_7 SEQ ID N0:24
fn191 3 SEQ ID N0:25
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WO 98/56805 PCT/US98/12076
gm196 4 SEQ ID N0:26
gn114_1 SEQ ID N0:27
hj968 2 SEQ ID N0:28
hkl0 3 SEQ ID N0:29
hm236_1 SEQ ID N0:30
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-3zP ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
2 0 labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
2 5 The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 Ill of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 Izg/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
3 0 dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ilg/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.
33
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in,
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
10 by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
2 0 protein may be in linear form or they may be cyclized using known methods,
for example,
as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc.1~ 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to Garner molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
2 5 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.
3 0 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
34
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
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 mRNA transcribed from
the
2 0 gene (Albert and Morns,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
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
2 5 stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 B1, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
3 0 polynucleotide sequences disclosed herein have been partially or
completely inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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 positlve/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human
models~for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present 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
2 0 identity (more preferably, at least 75% identity; most preferably at least
90% or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
2 5 (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.
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
3 0 polynucleotide with a different species of origin from that of a given
protein or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
36
*rB
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues may be isolated and identified by making suitable probes or primers
from
the sequences provided herein and screening a suitable nucleic acid source
from the
desired species. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaezcs, Hylobates
concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus
norvegicus,
Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus
cunicuha, Bos taums,
Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been
created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
(O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; 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; OBrien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
2 0 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 aiternaHve 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 5 60% sequence identity (more preferably, at least 75% identity; most
preferably at least 90%
identity) with the given polynucieotide, 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
3 0 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.
37
CA 02293825 1999-12-10
WO 98/56805 PCTIUS98/12076
The present invention also includes polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash
and
ConditionHybrid LengthBuffer' Temperature
(bp)= and Buffer'
A DNA:DNA Z 50 65C; lxSSC-or- 65C; 0.3xSSC
42C; lxSSC, 50%
formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA x 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50%
formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 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 T"*; 4xSSC T~*; 4xSSC
I DNA:RNA 2 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50%
formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
2 K RNA:RNA Z 50 70C; 4xSSC -or- 67C; lxSSC
0
50C; 4xSSC, 50%
formamide
L RNA:RNA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA 2 50 50C; 4xSSC -or- 50C; ZxSSC
40C; 6xSSC, 50%
formamide
N DNA:DNA <50 TN*; 6xSSC T~,*; 6xSSC
O DNA:RNA 2 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50%
formamide
2 P DNA:RNA <50 T,.*; 6xSSC TP*; 6xSSC
5
Q RNA:RNA Z 50 60C; 4xSSC -or- 60'C; 2xSSC
45C; 6xSSC, 50%
formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
#: The hybrid length is that anti«pated for the hybridized regions) of the
hybridizing polynu«eotides. When
3 0 hybridizing a polynu«eotide to a target polynucleotide of unknown
sequence, the hybrid length is assumed
38
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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 mmplementarity.
t: SSPE (lxSSPE is 0.15M NaCI, lOmM NaI-IZPO" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and ISmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
"TB - 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 +
C bases). For hybrids between 18 and 49 base pairs in length,
T°,(°C) = 81.5 + 16.6(log,a[Na*]} + 0.41 (%G+C) -
(600/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
2 0 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
2 5 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
Kaufman et aL, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce
the protein
3 0 recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein
"operably
linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
3 5 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
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
39
*rB
CA 02293825 1999-12-10
WO 98!56805 PCT/US98/12076
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.
Alternatively, it may be possible to produce the protein in lower eukaryotes
such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subfilis, Salmonella typhimurium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
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., lnvitrogen, 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
2 0 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."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
2 5 expressed protein may then be purified from such culture (i.e., from
culture medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
3 0 Sepharose~; one or more steps involving hydrophobic interaction
chromatography using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
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
CA 02293825 1999-12-10
WO 98/S680S PCT/US98/12076
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 BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
InVitrogen, respectively. The protein can also be tagged with an epitope and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (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 transgenic
animals, e.g., as a component of the milk of transgeruc cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
2 0 known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
2 5 compounds and in immunological processes for the development of
antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberately engineered. For example, modifications in the peptide
or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
3 0 of interest in the protein sequences may include the alteration,
substitution, replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
41
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
(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.
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
2 0 recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
2 5 with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
3 0 examination of expression patterns; to raise anti-protein antibodies using
DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
42
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
described in Gyuris et al.,1993, Cell 75: 791-803 and in Rossi et al.,1997,
Proc. Natl. Acad.
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.
5 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
10 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
15 inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agorusts 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.
2 0 Methods for performing the uses listed above are well known to those
skilled in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Bergen S.L. and A.R. Kimmel
eds.,1987.
25
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
3 0 source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
can be added to the medium in or on which the microorganism is cultured.
43
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
Cytokine and Cell Proliferation/Differentiation Activity
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, Imxnunologic 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 in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
2 5 Coligan eds. Vol I 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 limitation, 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. 173:1205-1212, 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
44
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
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 Stimulatin og r Suppressing 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.
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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
46
CA 02293825 1999-12-10
WO 98/56805 PCTNS98/12076
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, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
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 marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
47
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
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
48
CA 02293825 1999-12-10
WO 98/56805 PCTNS98/12076
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
~iZ
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 78488-2492,1981; 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. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
3 0 Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol.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 Th1/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. ImmunoL 144:3028-3033,1990; and Assays for B cell
function: In vitro
49
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antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
ICruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Tmmunol.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;
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., Cytometty 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
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Regulati ~ Activity
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
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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 Cells. 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 al. eds. Vol pp.1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.139-
162, Wiley-Liss,
Inc., New York, NY.1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
2 0 well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogeruc agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
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, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/InhibinActivity
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-
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CA 02293825 1999-12-10
WO 98/56805 PGTNS98/12076
~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; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et
al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chemokinetic 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
CA 02293825 1999-12-10
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include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. ICruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:13'l0-
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.152:58b0-5867,1994; Johnston et al. J. of Immunol.153: 1762-1768,1994.
Hemostatic and Thrombolytic 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.
Receptor/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 iigands 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
56
CA 02293825 1999-12-10
WO 98156805 PCT/US98/12076
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 arid
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)), ischeinia-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 l:L-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
3 0 Cadherin/Tumor Invasion Su~pressor 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
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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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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;
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CA 02293825 1999-12-10
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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 lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION 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.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
2 0 salts, buffers, stabilizers, solubilizers, and other materials well known
in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
2 5 such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-
8, IIr9, IL-10, IL-11,
IL-12, ILrl3, IL-14, IL-15,1FN, 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
in treatment. Such additional factors and/or agents may be included in the
3 0 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
CA 02293825 1999-12-10
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of the cytokine, lyrnphokine, 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 iriay 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 Garners, 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
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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 earner 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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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, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringei s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
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 ug
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 pg to
about i 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
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CA 02293825 1999-12-10
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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. 85, 2149-2154 (1963); J.L. Krstenansky, et
aL, FEBS Lett.
211, 10 (198. 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
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CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
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 autoiogous 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 osteogeruc 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-Vii), 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.
CA 02293825 1999-12-10
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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 vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
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SEQUENCE LISTING
(1)
GENERAL
INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
Howes, Steven H.
Fechtel, Kim
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
2 (D) STATE; MA
5
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
3 (A) MEDIUM TYPE: Floppy disk
0
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version
#1.30
3 (vi) CURRENT APPLICATION DATA:
5
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
4 (viii) ATTORNEY/AGENT INFORMATION:
O
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
45 (A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2)
INFORMATION
FOR
SEQ
ID
N0:1:
50
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2696 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
55 (D) TOPOLOGY: linear
67
CA 02293825 1999-12-10
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: I:
GTTGTACCAT TCTTGCCAAC TTCTGGGCTG GCAGTATGGA GTCATCTCCC60
TATCTTTCAT
1 TGCCTGTGTG AAATCTACTT TCTGAATTCT GCCATTTCCC TCTTCACACT120
O GTCTCCTGGG
TTATCTTTGC TTCCTCACAT CCCTATCTCT CTTCCTATAA ACTGGCTCCC180
GTCACTTCCA
TGATCCCTTC AGTGGCfiTCT GAGCTGGTCT CCCTGACCCC AAAGCCTCAG240
CCTTCCAGTC
TCCCTACAAA ATCTCAGCAA GTTCATTTTA GGTTAAAATT TGGACATATT300
TTAAATACGG
CTCACCACTT CATGTGAAAA TGATGGCACC CTACCAAGCA GTTTGCAGAG360
TTACGGTAAC
2 TGTTTCATGC TAATGATGTT ATTCATCCAG TTACAATTTT CTCAAAACTC420
O CTTTGGGCAC
TCTTTATTTT TAATCAAATT TTAAAGCCAA TATTTCATTT TGAGAATATG480
AATTAAATTG
GGAAATTCAT CCTTGTGGTA CAGTTTACAG ATTTTTAATG TTTACCCATT540
TATCCTGTTT
TTTGATATAT TAATTTCCCA TATAGCTCCA GAGTTATGTG ATATTATTTC600
TTTGCCAGTA
TATTAGAAAA TGATTAATTT CTCATGACCA ACTTCTGAAA AGAAAGACCC660
AATGCAAAAT
3 GCAATCTATT ACAATTATTT TTTTGAATAA AAAAGAATAT ATTATAGTTC720
O TTTAACATTT
GATATTTTAA ATTTGACATA TTCTTGATAT TTGTAAGAAA TTTCCACTGA780
ATGAATTTTA
CACAATTCAG ATACTACCAA TTAACTAATT CTAGCCTAAA CAAATAACAT840
TATTTTTAAA
TAACAAAATC TTTAAAAATA ATTTTCTATT TTGAACTTTT AGCCATAATG900
TAAGAAAATA
AAATTTTCTA GCAGAATAAT CAAAGAGTGA AACAAAGTTC CAACATGTTT960
TTTCTTTGCA
4 ATTAAACATG GCACTTTTAC AGTTATTTAT TATTCATATC AGTGCACTTA1020
O CCGACTTCAT
ATTTTCAAAT CAAAATACAG TGTTTTTCTC CAGTGAAATC CTTATTCTCA1080
TGACTGATAG
AAAACATTGC CAATTTTGAT ATTTCCAGAG TTAATGTTAA ATTATTTGAA1140
AGAAAATTAT
TTAAAATAAT AAAAATAGAC ATTTCAAGAC TATTTCTTAT CACATAATTC1200
AAAAAGTACT
TGGATCAAAT CCTACAGAGT TTCTCCACTA AAATTCTACT TGTGCAGAGG1260
GCATTGAAAC
5 GCATGAAAAT CAACAGCAGC TTAGTTAGGT TAATTAATTC GGTTAATTAA1320
O GCACCTACTA
CATGCTCAGC TCTATGCTAG GTGTCATGAG GAATTAAAAG GACATGTAAT1380
GCACATTTTC ~
TGATTTCAAG GAGCTTTAAA TATTATTGTG TAGAAAAAGT TAACATCTAT1440
GAAAATAGAA
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GTGGGGCAAT TTTGTGCTTA ATTCCATGGT CCAGATACAT CAAAAAATCA ATGTGGGCTG 1500
TCAAAGAAGG TTTCTTGATA GTCATGAGTC AGCCTGATTC TTGAAAGGAT ATGTGGAATA 1560
TAAAATTTTA TTTATATTCC TTTTGAGAAA ATACTGAGAA AACCATCTTC1620
CCTGGAAAAG
AGAACGTATT GTAAAGAAAG TACATGAAAT TGAAGGTTGA ATATCCAACA1680
TCCCCCACAC
TGCCCCAGTG TCTCTGCTCC CTTACTGAGC CTTACTATTA TTCTTCATAG1740
CCCTATCACT
ACCTAGTCTA GTATTCACTG AACTGTGTCA TCCACTAGAA TATGAGCATA1800
ATGAGAGCAG
AGACTACACC TGTCGGTTCA GTATTCTATC CTCAGCACAT AGAATGGTAC1860
CTGGCACATA
1 5 GCAGATGCTA AAATAAAATT TAAATGAATA AATTAATTCA ATCAACAACT1920
TCAAGGTGTT
ATTATTACCT ACAACTATTG TTTACAAGAG GTATGCACCG TGGAAGATCC1980
TGGAGACACA
ACAATGAATA AAGCCAAGCC AGTTCCTGCC CCCGTGGAGC TTGTAGTCAA2040
GACATTGAAC
AAGTGATCAG AAAGATGTTG ACTGCTGCAG CAGAGGGTTG CAAGCTGCTC2100
ATGAGTATAT
AACAAGTAGC CCTAACCAAA GCATTCTCTC CCTTGGTTTA ATGTCCACCC2160
ATTGAGGTGA
2 5 CTGCTAAATA CTAATCCATG ACTCTATCCC TTGGCATTCA AACTCACACA2220
TCCACTTACC
TGCCTCTCCA ACCTCATCTC CCTCCACTCA CAAGAGCCCA TCATATTATT2280
CATCAAAATG
AAACTGCACC CAGTTCTTCT GAACATATTA CCTTACAAAA CTTTCATTTA2340
TGCCTGGTGA
CTCTCATCAG GCATTCAAAA GCTTTCCCTC AGTGCTTCAG GGCTCTTCCT2400
TTTCTTCCCT
TTATACATAC ACCTTTATGT ATCTTCATAC GTACCCTGCA TAACCTCATA2460
TATCTTAGCA
3 5 TTTACCATAT TCTGTTGAAA AACTGTTTCC ATTTCTCTTT ACTTACTAGA2520
ATGTAAACAG
ATGCACAATG TTGAGAAAAT GAAAAGTGAC AACTTTGTTT ACAAGTTTAG2580
AAATTATCAG
ATTCTCACYT AAGCTCTAGT CTCTGTAAAG TCCACAACTA CTYAATAAAA2640
GTGAAGAAAA
ATGTTAACAG AGAGGGAGGA ATCAAAAACA AAGAACTATT TAAAAAAAAA2696
AAAAAA
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Thr Leu Ser Leu Gly Ile Gln Thr His Thr Ser Thr Tyr Leu Pro
1 5 10 15
Leu Gln Pro His Leu Pro Pro Leu Thr Arg Ala His His Ile Ile His
20 25 30
Gln Asn Glu Thr Ala Pro Ser Ser Ser Glu.His Ile Thr Leu Gln Asn
35 40 45
Phe His Leu Cys Leu Val Thr Leu Ile Arg His Ser Lys Ala Phe Pro
50 55 60
25 Gln Cys Phe Arg Ala Leu Pro Phe Leu Pro Phe Ile His Thr Pro Leu
65 70 75 80
Cys Ile Phe Ile Arg Thr Leu His Asn Leu Ile Tyr Leu Ser Ile Tyr
85 90 95
His Ile Leu Leu Lys Asn Cys Phe His Phe Ser Leu Leu Thr Arg Met
100 105 110
2 5 (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3614 base pairs
(B) TYPE: nucleic acid
3 0 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
4 O CGCGCTAACT GTGCTCCTCC GGGGCCCTCC GCCTGCTCCC AGCCATGGTG GCCTGGCGCT 60
CGGCGTTCCT TGTCTGCCTC GCTTTCTCCT TGGCCACCCT GGTCCAGCGA GGATCTGGGG 120
ACTTTGATGA TTTTAACCTG GAGGATGCAG TGAAAGAAAC TTCCTCAGTA AAGCGATCAC 180
TGTAAGGATG ACATGGGAGG AGCCATGTGA AGCACTCAGC ACAGTCCTTG GAACAAGAGC 240
CATGGGACCA CACCACCACC ACCACAACCA ATAGGCCAGG AACCACCAGA GCTCCGGCAA 300
5 O AACCTCCAGG TAGTGGATTG GACTTGGCTG ATGCTTTGGA TGATCAAGAT GATGGCCGCA 360
GGAAACCGGG TATAGGAGGA AGAGAGAGAT GGAACCATGT AACCACCACG ACCAAGAGGC' 420
CAGTAACCAC CAGAGCTCCA GCAAATACTT TAGGAAATGA TTTTGACTTG GCTGATGCCC 480
70
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TGGATGATCG AAATGATCGA GATGATGGCC GCAGGAAACCGGAGGAGGTT540
AATTGCTGGA
TTTCAGACAA GGATCTTGAA GACATAGTAG GGGGTGGAGAGACAAGGGTA600
ATACAAACCT
AAGGTGATGG CCGGTACGGC AGCAATGACG ACCCTGGATCGCAGAGCCTG660
TGGCATGGTG
GCACCATTGC CGGGGTGGCC AGCGCCCTGG CCATGGCCCTGTCTCCAGCT720
CATCGGTGCC
ACATCTCCTA CCAGCAGAAG AAGTTCTGCT TCAGCATTCAAACGCAGACT780
GCAGGGTCTC
ACGTGAAGGG AGAGAACCTG GAAGCCGTGG TATGTGAGGAAAATACTCCA840
ACCCCAAGTG
CGTTGCACAC GCAGTCTGCA GAGCCGCCGC CGCCGCCCGAATCTGAGGGC900
ACCAGCCCGG
1 CCTGTCCAGC TGCAGGCATG CACAATGGTG CCACCGCTTGCCCCCCACCC960
5 TCACCCGGCT
CTTCATTTGG ACCCGCAGCT GCTGTGCTGC TCTGTGCCGTTTGGTCTGAG1020
CGGCTCCTTG
TTTCCCGGAT GAGCTCTGGG TGTTTGTGAG TTTGGTTTCTCCAAGCGTGC1080
CTGCCCTGCC
TGAGACTTGG TGCCGAAATT CAAGAGCCAG CTCTGATAGACAGCCTCGGG1140
AAGCCAGCAC
AGCTGCTGAG CCACCAACTC CCAAAGCCAG CCTGCCTCCAGCACAGGATG1200
GCTTTACTGA
2 CGGGGGCCAA GATGATGCTG AGGCCTGATG ACATTTATGCAGAGTTTGAA1260
5 TTAGGGGACA
CTCAAGGGAC TGTGACCCCT GCACACTGGA GTGGCTCATTTCTGCCAATA1320
GTGGCAGGTT
GACAGCCCCT GACAGTGGCC TCAAGGAGCT GCAGGTGGGGGCACCCACTT1380
GGCTCAGCCT
GGAGCCCCTG CAAGGAGCGA ACCGGTCAGC ACCAAGTAACCGCAGCACCC1440
ACCACACACA
AGGATGATGG TTTCACTTCA GTCTTCCCCA TCCCAGGTTTGGCTTCCGGA1500
TATGTTGCTG
3 GAGCCGGTCC AAGCGGAGGC TTTCAGTGAT TTAAGTACAACGTGATAGTC1560
5 ACATGCATCT
CTGCCTTGAG AGCTTAGGAA TCTTCCGGAT AAGTATGAAGGCCTGTTTCC1620
CAATTCGTAG
CATCTGATTC CATAGGGGGC TGGGTGTGGC TTCGGGTTGAGTCTTTAGCA1680
CATGAGAAAG
ATCATTTCTG CACCGGAGAT GAGTTTTATC CTGTGTTGGGCACCCTCCAC1740
GAGAGGTGCT
CCTGTGTCCC TGTTTTGGTA GCAAGAGTGA CCGATGTCAACAAAGCCAGA1800
GAACGAGCAT
4 ATCCTGCTTG TTTGCTTAAA AATGTAATTG GGGGCGGCGGGGGAAAGAGA1860
5 GGGAGGAGAG
CATTCGCTTG GTTTAGTGAA ACGCAGGTGA CTTTGTAGCTCCTACTTGTC1920
CTGTGGTCAG
TGCTCTGAGG GAGAGTGCGT GGGGAGCCAT GCTCACCGTGGAACCCCATG1980
GCAAACACAG
ACTCGCCCCT CACCTGGCGT GGAGCTGCCT GGTTTGGGCTTGGTTTCCTG2040
GGAGCAGAGC
GAATGTTCCT TTGGCCCACA TATGGTTCTG TCCCGGTGAGAGAGGCTCAC2100
CTCTGTTGTC
5 GGGACAGAAC CACATGCTAG GGTCTAGGGC CCCTGTCTACTTTGCTGTGT2160
5 TGATAGTCAG
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CAGAAAGCAC TTCTGAAAGC AGATATGAGT CACCAGACAG GCAGGATCTT2220
ACAAAACTCA
CGGGCCTCTT TGGTCTGCAT GATGGCCCCA TGCGTTTCAT AGGCTGTCCA2280
CTGAGCGGGA
TTGTCTGCTG AGTGGGATGA GCCAACTCCA GTTTCTTAAG GAAACCACTG2340
GAATCTGCAG
CCCCCACATG CATCTGTCTA ACGCATGCCT CGTGTTCGTT TTGCAAACAT2400
GCCTGTGGTG
GAGGGTGGTC AGTTGTAGCC CTGTGCGTCT CAAGGCTGCC TTGTGAGGCC2460
ATTCCCAGTG
CGTGCCCTTG AGCTCCTTAC CACCCCTTTT CCTGCTCGGC CCTTTAATCC2520
CTGACAGACC
TGGACTGTGT GGCTGAAGGG GGACCTGCAG CACTGCAGAA ATGCCTCTGC2580
GTGGTGCCAT
1 GAAGGAAAGA AACCTTGGCC TGGTCTCGAG AAGCTTCCCA TGCTTCAGGA2640
5 AGTTAGTAAG
GGTGGGGTGG CTTGCAGGAT TGGCCTGTTT CCAGGGCCTC CCACACTCAT2700
TGGCCAGATT
GTGAACTTTG TCAGGCTTGT CCCTCCCTGA TACCAAGTAT GTCGAGAACC2760
GATGGCCCCA
CCCTCTGGCT GGTGCTGGGC CGGAGGTGGC TATGGAGGAT TTTGGCATGC2820
GTGGCCTGTC
GCCACCTGGA CAGCGTGACC TCAGGGGTTG TCCACTTTAC CTTTATGGTG2880
AGGCCTGTCG
2 GATGGCTAAG TCCTTGAAAC CCTAGAGCTG TGACGTAGAA TATGTGCTGT2940
5 CTGTGAGACC
GTGTTCCCAG GAGCACTGAC TGCAGTTGAG AGAGACCCAT TTTGCTCTCC3000
CTTACCGCCC
CCCGCCCCGG GT~vCTTTCTG CACAAAGCCT AGAGCCTGGC ACTCAAGCCC3060
ACCGGTGGCA
GCTCCTAGTG ACTGGACATG CCTGGAAGAC CCCTCAGCCT TCTGTTTGCA3120
GAACGTTCAT
TTCAGGAGCT TCTCCTTCCC ACAGACATCT TACACTTGCT CGACACTGCC3180
ACCTGCAGAA
3 GCCTGGCGGG CTCTGGTCAC CATGTGTCTA TCTGAAGGTT GCACTGGCCA3240
5 GCATGGGCCT
GTCCCAAGCG AGAGGGGAGA CACAGTGGAC TGAAAGGACT GGTTGAAAGT3300
GGCCAATCTC
TGTCAGCTTA ATTTGGCAGA GAAAATTTGT AACAACTCTG AGCACATGCT3360
GGGTGAAGTC
ACAGCTCAAG GAAAGATAAA GCTGGGCGGA AGGAGGTGTG CGTGGCTTCT3420
GGGGTGGGAC
CCAGAGGGGA GGCTCTGGGA CAGGGGCTGG GGTTCAGTGC CAGGGCCCTG3480
AGGAAGAAAT
4 GGGGACTGAT CTCAAAATTC CAGAATTCCC TGTACATCTG TTCACGTGCT3540
5 TGTGTCCAGG
TGTGACTTGT AAACTGTCTA GTGTTTGCAT TAAATAAAAT GGCACCGAGC3600
AAAAAAAAAA
AAAAP.AAAAA AAAA 3 614
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE
DESCRIPTION:
SEQ
ID
N0:4:
Val LysHisSerAlaGln SerLeuGluGlnGlu ProTrpAspHisThr
1 5 10 15
Thr ThrThrThrThrAsn ArgProGlyThrThr ArgAlaProAlaLys
20 25 30
Pro ProGlySerGlyLeu AspLeuAlaAspAla LeuAspAspGlnAsp
35 40 45
2 Asp GlyArgArgLysPro flyIleGlyGlyArg GluArgTrpAsnHis
0
50 55 60
Val ThrThrThrThrLys ArgProValThrThr ArgAlaProAlaAsn
65 70 75 80
Thr LeuGlyAsnAspPhe AspLeuAlaAspAla LeuAspAspArgAsn
85 90 g5
Asp ArgAspAspGlyArg ArgLysProIleAla GlyGlyGlyGlyPhe
3 100 105 110
0
Ser AspLysAspLeuGlu AspIleValGlyGly GlyGluTyrLysPro
115 120 125
3 Asp LysGlyLysGlyAsp GlyArgTyrGlySer AsnAspAspProGly
5
130 135 140
Ser GlyMetValAlaGlu ProGlyThrIleAla GlyValAlaSerAla
145 150 155 160
40
Leu AlaMetAlaLeuIle GlyAlaValSerSer TyrIleSerTyrGln
165 170 175
Gln LysLysPheCysPhe SerIleGlnGlnGly LeuAsnAlaAspTyr
45 180 185 190
Val LysGlyGluAsnLeu GluAlaValValCys GluGluProGlnVal
195 200 205
5 Lys TyrSerThrLeuHis ThrGlnSerAlaGlu ProProProProPro
0
210 215 220
Glu Pro Ala Arg Ile
225
73
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(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1077 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xij SEQUENCE DESCRIPTION: SEQ ID N0:5:
AATCGGGAGT CCCAACAGCG TTAGGTTTTT TTTTTTGTTT GTTTGTTTGT60
TTTGTTTTTT
TCCAACCCTC TTTCGGATGG ACGGGGGAAA GAGAGAAAGA AAAACGAGGG120
AAAATCAACA
2 AAATGTGCGA TGCAAAGAGT CGATTTTCGC GGGGTTTGTC AACTTCGCCA180
O CTGCCGCACG
CGAATCGACG TCGTCACGTG ACGGTCTGCC TCCGCCCTTA TTAACTCTCA240
GCCCAGCGGC
GGTTTCCAGG ACCTCAGACT TTTTGCCGAG GCGGCAGTCC CTAGACGAAG300
CGAAGGAGGC
GGCGCCTGCC CCGCCCACAA GAGCTGCCGC GCGCGGGTGT TATAGCTCCA360
CCCCATCTGC
AAAGGAAGGG GGAGCGGAAA GAGCGGGATC TAGCGTGGGA TAAAAGTGGG420
ACTACTACAG
3 TGTAACTGGG CATGCGCCCC TCCTAGAAAT GATGGGAATG CAAAAGCCCT480
O TGACTGCTCC
AGGACTCGAG GGATCCTCGG TGCCAGGATG CTGGGTCAAG CGCTCCGCCG540
GGACAGAGGA
CTCATACCAG GGAAATGGAG CCCAGCCTCG TGATAAACTA CGACCCAAGC600
TGGGGGAGGA
ACCTAGTTTT CGAAAGGAAA ATAATATGCG CAAGCTTTAA CTGAGCAGTG660
GGATGGTCTT
AAATACCAAA GGAATGACTT TAAATCTTGC TGGATGGGAC TGCCACTCAC720
CGCTAGAAAT
4 CGGGGATCAA CAGCAAACTC TGGATGACCC TGTAACCACA TCTCCAGTTC780
O AGCCCGGCGA
GGGGCATCCT CACCCACCAG CAAAGTACCA TCCACCTTAT TGATGACAGG840
GATCCGGGTC
TCCAGGTCCA CATCAAGGTG ATTAGGCTCT TCCATGCACT CCACCTCCAG900
CTGCAAACCC
AGAATCCACC CCCATGAGCA CATACTCTTC TTTGGGGGAG GGAGGGAGGG960
GGAGCAGGGC
CAATGGTAGT CATGAAATGA CTCTAGTATT TTCCATTCCC CCAGTCCCAC1020
TGCCTTCATC
5 AATTATTGGG AATAAAAAGA CAATCTAATC GTCAAAAAAA AAAAAAAAAA1077
O AAAAAAA
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 108 amino acids
5
74
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(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
1O (xi) SEQUENCE
DESCRIPTION:
SEQ
ID
N0:6:
Met AspGlyGly LysArgGluArgLys ThrArgGluAsnGln GlnAsn
1 5 10 15
Val ArgCysLys GluSerIlePheAla GlyPheValAsnPhe AlaThr
20 25 30
Ala AlaArgGlu SerThrSerSerArg AspGlyLeuProPro ProLeu
35 40 45
Leu ThrLeuSer ProAlaAlaValSer ArgThrSerAspPhe LeuPro
50 55 60
Arg ArgGlnSer LeuAspGluAlaLys GluAlaAlaProAla ProPro
65 70 75 80
Thr ArgAlaAla AlaArgGlyCysTyr SerSerThrProSer AlaLys
85 90 95
3 Glu GlyGlyAla GluArgAlaGlySer SerValGly
0
100 105
(2) INFORMATION FOR SEQ ID N0:7:
3 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 588 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TAAGAATTAA AAATGTCATC CAAACAAGAA ATAATGAGTG ACCAGCGGTT TAGACGGGTT 60
5 O GCAAAGGACC CGAGATTTTG GGAAATGCCA GAAAAGGATC GAAAAGTCAA AATTGACAAG 120
AGATTTCGAG CCATGTTTCA TGACAAGAAG TTCAAGTTGA ACTATGCCGT GGATAAAAGA~ 180
GGGCGCCCCA TTAGCCATAG CACTACAGAG GATTTGAAGC GTTTTTACGA CCTTTCAGAT 240
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TCTGATTCCA ATCTCTCTGG TGAAGATAGC AAAGCATTGA GTCAAAAGAA AATAAAGAAG 300
AAAAAAACCC AGACTAAAAA AGAAATCGAT TCAAAAAATC TAGTTGAGAA AAAGAAAGAA 360
ACCAAGAAGG CTAATCACAA GGGTTCTGAA AATAAAACTG ATTTAGATAA TTCTATAGGA 420
ATTAAAAAAA TGAAAACCTC ATGTAAATTT AAGATAGATT CAAACATAAG TCCGAAGAAG 480
GATAGCAAAG AATTTACACA Fu~~AAAAAAAA AAAAAAAAAA AAAAAAAAAA F~L~AAAAAAA 540
AAAAAAAAAA AAAAAAiaAAA AAAAP.AAAAA F~~i~AAAAAAA AAAAAAAA 5g8
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
30
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Ser Ser Lys Gln Glu Ile Met Ser Asp Gln Arg Phe Arg Arg Val
1 5 10 15
Ala Lys Asp Pro Arg Phe Trp Glu Met Pro Glu Lys Asp Arg Lys Val
20 25 30
Lys IleAspLys ArgPheArgAlaMet PheHisAspLysLysPhe Lys
35 40 45
Leu AsnTyrAla ValAspLysArgGly ArgProIleSerHisSer Thr
50 55 60
Thr GluAspLeu LysArgPheTyrAsp LeuSerAspSerAspSer Asn
65 70 75 80
Leu SerGlyGlu AspSerLysAlaLeu SerGlnLysLysIleLys Lys
85 90 95
Lys LysThrGln ThrLysLysGluIle AspSerLysAsnLeuVal Glu
100 105 110
Lys LysLysGlu ThrLysLysAlaAsn HisLysGlySerGluAsn Lys
115 120 125
Thr AspLeuAsp AsnSerIleGlyIle LysLysMetLysThrSer ~Cys
130 135 140
5 Lys PheLysIle AspSerAsnIleSer ProLysLysAspSerLys Glu
5
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145 150 155 160
Phe Thr Gln
(2) INFORMATION FOR SEQ ID N0:9:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2773 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GTATATTTTG GTTTACTTAC TCCTCTATTT CAGAAATTGA AAAAGATCCC60
CAAGGATCTG
TTACTACTGC ATTTCCTTCT TGCTCTGTCT ACAGCCTAGG CCAACTAGTC120
AGGGTCTGGA
2 CATGCATCTC CTAAAGGAAG AACTGTGTAG CACCATTGAT CACAATGTAA180
5 CATTTCCATG
CTGCATTAAG GGTGTCTCTC TCTAATCATG ATTGTACCTG TCTCTTCCTG240
GGTAAAGGGA
GATTTTTTTT TTTTAATGTG TAAAGAATTG ATGCSAGCCA GGAACATGTC300
TGTAGTCCCA
GCTACTTGGG CACACGCCTG TAGTCCMSCG CCACTCGAGC ACACACCTGT360
AGTACCAGCT
ACTCTGGAGG CTGAGGCAGG AGGATCACTT GAGCCCAGGA GATTAAGACT420
GTAGTATACT
3 ATGATCGTGC CTGTGGCTAG CCACTGTGCT CCAGCCTGGG CAACACCATC480
5 GTAAAAATAA
ATAAATAAAT AAATAAATTG GGGAGGACAG CCTCACTGGT ATCAGACTTA540
CAGGACCAGA
TAGACAAGAT GGGTATAAGG GGAGCTGAAG TCTGTGTTCA TATGAGGAAG600
AGAAGACCAA
GCCCTGGGAC TTTGGCTGAA TTCCTCCGTG GGGCTGGACG GCAGTGATCT660
CCTGTTCCCT
ATGTGTAAAC AAAGATTCCA GGGCGTGGTT TTGCACTCCT GTTGTACTCT720
TTTAGAGGTG
4 GAAAAGAGGT GGATACTGAG ATCTAAGAGG AAAGGATAGT CATTCACGTT780
5 CTGAGATATG
CGCTCTCTCT ATTGTTCTCG WACACAAAGG GATAGTCTCT TTTCTGGAGC840
TGATGTCCCT
GCTTGGAGGT TAGCCCCAAA ACATGGCTCT TGTATTGTTC TAAGAGAAAA900
GGCTTTCATT
TTGGTTCTTC TGATTGGTGT TACCTACTGC CTAATATGTG TTCATTTTTT960
GACAGAGAGG
CAGACTATTG AAAAAGTCTG TGTGAACAGA GAGCAGTTCA TTAAGCCCAT1020
TGCTTTCAGT
5 AATGTGGCCT TGACCCCTTC TGCTTCCCCC TTCTCCCATG GAGCATGGCA1080
5 GGGCTTGGTT
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ATTTAGAGTC CATACATGCA AGCCATTGAG AGACTTGTTT GCTCAAATGC1140
AAGTTTGCTC
AAAAACAGGT CCTGAAGGCT TGCTTAGGAT TACAGGGATG CTGGGTAAGA1200
ACACCGTTCC
TCTCTCTCGC TGGAGAAATC CCTGTTTCTC TGACTCCCTT TGTGATCCTC1260
ACAGTAATGT
ATTCTGTGCC ACTGTAGGAC ACAAGGCTCT GGGCCAGTAG AACAGGCAGA1320
GAGGTGACAC
TGGGCAGCAA GCTGAGAGCT CTTTCTAAAT GGAGTGAAGG AATTCAGTGG1380
CCTAGTfiTCG
10.
CCATTCTCTA ATGAGAAACC AAGGCCAGGC TGAAAAGTGC AATTAGATGT1440
GGTGGATTGT
GGTAACGGCC TCCAGATAAA GGGGTTATCC CTGTGGAAGT GACTTTTCCC1500
CATTTGATCC
1 CTTTTCAACT CTAAATGGCC AGGCCCAGAG CAGAAGAAGG GTTGGGTCTG1560
5 GAAGGAAGGC
TCCAAAGGAT GAAAGCTTCT CCCTGATCAT AAGGAAGTGC ATCTTTATAG1620
AATTGTTGTG
CATAATGTCA GTAAATCCCT CTCACTTGAC AAGGGACTGG ATTCATCTTG1680
CCTTGAGACG
20
GGCCAGTAGT TATCAGTGAG TCAAAGCAAA GTGAAAGTTT CAGGAGATGG1740
GACCAATGGT
GCAATGCTCG CCATAACAAA ATTCCTTAAA AATAAi~AAAG CTAATGTTAT1800
AGCAACAAAA
2 AAAGACTGAA GCAAAACCAC ACTGAAATGC ATCCCACTCC AGGAGAGGAA1860
5 TTCTTAGCGT
AACACTCTAA ATAAATGGAA GGAATCATCA CCTTCCTTAT TTTACCCCTG1920
CCTTGTTCAC
CAGGCTGCCC AGTGCTTACC ATGCAGAAAG CAGTCAGCTG TACTCTGGAA1980
GTTTCTGTTC
30
TTCTTTCCTG GGGCTTAGGA TATTCTGGGA GCTGTCTGAG CCTTGTGCCT2040
AAGGCTTATC
AGGTGATATA ATCTTCCTGT TCTGGGCTGC TTGCTGGAGG AATAGGAAGT2100
GACATTTATA
3 AGACACAGGC GGTGTGAGCA TCCATGTGTG GTCTTGGTCT AAACCAGCTC2160
5 TTGAACAGGT
TAAAGCAAAC AGCAATAACA AAACAAAAAC TACTGATGCT GAGCGTTTTG2220
ATCCTAGTAA
TATTTCAAAT ATTGTCCTTC TGCATATGTT CTATCCATAT TTGATTCCAA2280
TATACATTAT
40
TAAGCTTTCT TGGGTACTAT TTTGCTGGGG CTCTTGCGTG AAGGTGGTAC2340
CTGTCTCATG
ATCCTTAAAA GAGAGAGGCT TTTTTCATCC AAAGCTGTAG TGTTGGGAAC2400
TGGGGTGGGA
4 GAGGCACTTT TTGGAATTCT GAAAGAATCA TATCTGTGTA TATACATACT2460
5 GAGTGGGGAA
GGATGGGGGT TGGCAGGGGT TGAGGGAGGT GGGAACAAAC AGTGAGTATG2520
GGAACAGGCA
GTCACCTCGA GTGTGGGAGG TCACCTGGGT CCGTCGTCTT CCTTCTGTAT2580
GGTGTTGGGT
50
TTATGTACAC ACTATAACAC TTCCTGTGTG AGTTCATGTA CCTGTCTGTG2640
AGTGCTTTGG
TGTATTGAGC CTCAGTACAC TCCAAGGGCA TTAAAGTCAA GAACTAGAAC2700
CTGGAAAAAA
5 AAAAAAAAAA AAAAAAAAAA A,F~AAAAAAAA AAAAAAAAAA A,P~F~AAAAAAA2
5 AAAAAAAAAA 7
6
0
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1'~~~AAAAAAA AAA
2773
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:10:
Met Arg Ser Leu Tyr Cys ThrGlnArgAspSer LeuPheSer
Ser Arg
1 5 10 15
Gly Ala Asp Val Pro Ala LeuAlaProLysHis GlySerCys
Trp Arg
20 25 30
Ile Val Leu Arg Glu Lys IleLeuValLeuLeu IleGlyVal
Ala Phe
35 40 45
Thr Tyr Cys Leu Ile Cys PheLeuThrGluArg GlnThrIle
Val His
50 55 60
3 Glu Lys Val Cys Val Asn GlnPheIleLysPro IleAlaPhe
0 Arg Glu
65 70 75 80
Ser Asn Val Ala Leu Thr AlaSerProPheSer HisGlyAla
Pro Ser
85 90 95
Trp Gln Gly Leu Val Ile
100
(2) INFORMATION
FOR
SEQ
ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3119 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
4 (D) TOPOLOGY: linear
5
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
GGAAACATTA TGGATCTGTG GAGCTGCTTA TTTCCAGTGA TGCTGATGGA GCCATCCAAA 60
79
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GGGCTGGAAG ATTCAGAGTG GAAAATGGCT CTTCAGATGA GAATGCAACT120
GCCCTGCCTG
GTACTTGGCG AAGAACAGAC GTGCACTTAG AGAACCCAGA ATACCACACC180
AGATGGTATT
TCAAATATTT TTTAGGACAA GTCCATCAGA ACTACATTGG AAACGATGCC240
GAGAAGAGCC
CTTTCTTCTT GTCCGTGACC CTTTCTGACC AAAACAATCA ACGTGTCCCT300
CAATACCGTG
CAATTCTTTG GAGAAAAACA GGTACCCAGA AAATATGCCT TCCCTACAGT360
CCCACAAAAA
20
CTCTTTCTGT GAAGTCCATC TTAAGTGCCA TGAATCTGGA CAAATTTGAG420
AAAGGCCCCA
GGGAAATTTT TCATCCTGAA ATACAAAAGG ACTTGCTGGT TCTTGAAGAA480
CAAGAGGGCT
1 CTGTGAATTT CAAGTTTGGG GTTCTTTTTG CCAAAGATGG GCAGCTCACT540
5 GATGATGAGA
TGTTCAGCAA TGAAATTGGA AGCGAGCCTT TTCAAAAATT TTTAAATCTT600
CTGGGTGACA
CAATCACTCT AAAGGGCTGG ACGGGCTACC GTGGCGGTCT GGATACCAAA660
AATGATACCA
20
CAGGGATACA TTCAGTTTAT ACTGTGTACC AAGGGCATGA GATCATGTTT720
CATGTTTCCA
CCATGTTGCC ATATTCCAAA GAGAACAAAC AGCAGGTGGA AAGGAAACGC780
CACATTGGAA
2 ACGATATCGT CACCATTGTG TTCCAAGAAG GAGAGGAATC TTCTCCTGCC840
5 TTTAAGCCTT
CCATGATCCG CTCCCACTTT ACACATATTT TTGCCTTAGT GAGATACAAT900
CAACAAAATG
ACAATTACAG GCTGAAAATA TTTTCAGAAG AGAGCGTACC ACTCTTTGGC960
CCTCCCTTGC
30
CAACTCCACC AGTGTTTACA GACCACCAGG AATTCAGGGA CTTTTTGCTA1020
GTGAAATTAA
TTAATGGTGA AAAAGCCACT TTGGAAACCC CAACATTTGC CCAGAAACGT1080
CGGCGTACCC
3 TGGATATGTT GATTAGATCT TTACACCAGG ATTTGATGCC AGATTTGCAT1140
5 AAGAACATGC
TTAATAGACG ATCTTTTAGT GATGTCTTAC CAGAGTCACC CAAGTCAGCG1200
CGGAAGAAAG
AGGAGGCCCG CCAGGCGGAG TTTGTTAGAA TAGGGCAGGC ACTAAAACTG1260
AAATCCATTG
40
TGAGAGGGGA TGCTCCATCA AGCTTGGCAG CTTCAGGGAT CTGTAAAAAA1320
GAGATGACCT
TCCATCAGTG CCCGTGTTTG ACAGAACTCT GCCAGTGAAG CAAATGCATG1380
TGCTTGAGAC
4 CCTGGACCTT CTGGTTCTCA GAGCAGACAA AGGAAAAGAT GCTCGCCTCT1440
5 TTGTCTTCAG
GCTAAGTGCT CTGCAAAAGG GCCTTGAGGG GAAGCAGGCT GGGAAGAGCA1500
GGTCTGACTG
CAGAGAAAAC AAGTTGGAGA AAACAAAAGG CTGCCACCTG TATGCTATTA ACACTCACCA 1560
CAGCAGAGAG CTGAGGATTG TGGTTGCAAT TCGGAATAAA CTGCTTCTGA TCACAAGAAA . 1620
ACACAACAAG CCAAGCGGGG TCACCAGCAC CTCATTGTTA TCTCCCCTGT CTGAGTCACC 1680
5 5 TGTTGAAGAA TTCCAGTACA TCAGGGAGAT CTGTCTGTCT GACTCTCCCA TGGTGATGAC 1740
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CTTAGTGGAT GGGCCAGCTG AAGAGAGTGA CAATCTCATC TGTGTGGCTT1800
ATCGACACCA
ATTTGATGTG GTGAATGAGA GCACAGGAGA AGCCTTCAGG CTGCACCACG1860
TGGAGGCCAA
CAGGGTTAAT TTTGTTGCAG CTATTGATGT GTACGAAGAT GGAGAAGCTG1920
GTTTGCTGTT
GTGTTACAAC TACAGTTGCA TCTATAAAAA GGTTTGCCCC TTTAATGGTG1980
GCTCTTTTTT
GGTTCAACCT TCTGCGTCAG ATTTCCAGTT CTGTTGGAAC~CAGGCTCCCT2040
ATGCAATTGT
CTGTGCTTTC CCGTATCTCC TGGCCTTCAC CACCGACTCC ATGGAGATCC2100
GCCTGGTGGT
GAACGGGAAC CTGGTCCACA CTGCAGTCGT GCCGCAGCTG CAGCTGGTGG2160
CCTCCAGGTC
1 GGATATATAC TTCACAGCAA CTGCAGCTGT GAATGAGGTC TCATCTGGAG2220
5 GCAGCTCCAA
GGGGGCCAGT GCCCGAAATT CTCCTCAGAC ACCCCCGGGC CGAGATACTC2280
CAGTATTTCC
TTCTTCCCTG GGGGAAGGTG AAATTCAATC AAAAAATCTG TACAAGATTC2340
CACTTAGAAA
CCTCGTGGGC AGAAGCATCG AACGACCTCT GAAGTCACCC TTAGTCTCCA2400
AGGTCATCAC
CCCACCCACT CCCATCAGTG TGGGCCTTGC TGCCATTCCA GTCACGCACT2460
CCTTGTCCCT
2 GTCTCGCATG GAGATCAAAG AAATAGCAAG CAGGACCCGC AGGGAACTAC2520
5 TGGGCCTCTC
GGATGAAGGT GGACCCAAGT CAGAAGGAGC GCCAAAGGCC AAATCAAAAC2580
CCCGGAAGCG
GTTAGAAGAA AGCCAAGGAG GCCCCAAGCC AGGGGCAGTG AGGTCATCTA2640
GCAGTGACAG
GATCCCATCA GGCTCCTTGG AAAGTGCTTC TACTTCCGAA GCCAACCCTG2700
AGGGGCACTC
AGCCAGCTCT GACCAGGACC CTGTGGCAGA CAGAGAGGGC AGCCCGGTCT2760
CCGGCAGCAG
3 CCCCTTCCAG CTCACGGCTT TCTCCGATGA AGACATTATA GACTTGAAGT2820
5 AACAGAGTTG
AATCTCATTT GCCATCTTTA GTTTTCTTAT GGAGGTTTAT ACTCTTTAAA2880
CAGTTCTGAT
GTAATTTCTC AACAAAATGT GGCTTTTAGC CTGTCAGTGA TCTATTGGAC2940
CAAACCTTCT
GCACACTCGG CCAGTTCCCT CTCCAATGTC CGGTGCCATC TTTCCTGACC3000
TTTGTTTCTT
TCTGTTCAGG AACCATCAGT CCCCTTGTAA TAAAGGTGGT AGATTTCATT3060
GAGGTTTTAG
ATTGAAACTT TGAATAAATC AAAAATACTC ATTCTTAAAA AAAAAAAAAA3119
AAAAAAAAA
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 322 amino acids
0
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
gl
CA 02293825 1999-12-10
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Asn Leu Asp Lys Phe Glu Lys Gly Pro Arg Glu Ile Phe His Pro
1 5 10 15
Glu Ile Gln Lys Asp Leu Leu Val Leu Glu Glu Gln Glu Gly Ser Val
20 25 30
Asn Phe Lys Phe Gly Val Leu Phe Ala Lys Asp Gly Gln Leu Thr Asp
35 40 45
Asp GluMetPhe SerAsnGluIleGlySerGlu ProPheGlnLysPhe
50 55 60
Leu AsnLeuLeu GlyAspThrIleThrLeuLys GlyTrpThrGlyTyr
65 70 75 80
Arg GlyGlyLeu AspThrLysAsnAspThrThr GlyIleHisSerVal
85 90 95
Tyr ThrValTyr GlnGlyHisGluIleMetPhe HisValSerThrMet
100 105 110
Leu ProTyrSer LysGluAsnLysGlnGlnVal GluArgLysArgHis
115 120 125
3 Ile GlyAsnAsp IleValThrIleValPheGln GluGlyGluGluSer
0
130 135 140
Ser ProAlaPhe LysProSerMetIleArgSer HisPheThrHisIle
145 150 155 160
Phe AlaLeuVal ArgTyrAsnGlnGlnAsnAsp AsnTyrArgLeuLys
165 170 175
Ile PheSerGlu GluSerValProLeuPheGly ProProLeuProThr
4 180 185 190
0
Pro ProValPhe ThrAspHisGlnGluPheArg AspPheLeuLeuVal
195 200 205
Lys LeuIleAsn GlyGluLysAlaThrLeuGlu ThrProThrPheAla
210 215 220
Gln Lys Arg Arg Arg Thr Leu Asp Met Leu Ile Arg Ser Leu His Gln
225 230 235 240
Asp Leu Met Pro Asp Leu His Lys Asn Met Leu Asn Arg Arg Ser Phe
245 250 255
Ser Asp Val Leu Pro Glu Ser Pro Lys Ser Ala Arg Lys Lys Glu Glu
260 265 270
82
*rB
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Ala Arg Gln Ala Glu Phe Val Arg Ile Gly Gln Ala Leu Lys Leu Lys
275 280 285
Ser Ile Val Arg Gly Asp Ala Pro Ser Ser Leu Ala Ala Ser Gly Ile
290 295 300
Cys Lys Lys Glu Met Thr Phe His Gln Cys Pro Cys Leu Thr Glu Leu
305 310 315 320
Cys Gln
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1592 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
' (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GACTTTTATA AAAAAGTCAA GCAGTACAAA AGGGTGTAAA GTGAAGTTAC60
TGTCCTTCCC
3 CTCCATAAAC CCCCTGACCT TGGGAAACTG TTGTTAACAG TTACTTGGGT120
O AACTTTTCAG
ATATTTTGTA TGCATGTACA AATGTGAGTA TCTAATGTAA AAAAATCAAA180
CCAAGATAAA
GTGTAAACTG CTATGATGGA ATCCTGCCTT GTTCTGCTAT TAGTCTTCTG240
TTTAATAATC
AGCTTTGGTA TTAGGACAGT GGTAGGAAGA AGCCAGTATG TCCTGCAACA300
TAATTTGTGG
TTCTGGACTG GTCAGGATTT CCTGAATGCA GCCTTTATCT GGAAGCTCTG360
CCCTTCTCCA
4 TCTGGGATAC GCTTTTTCAT CCATCAAAAC TGTCATCTCC CTCTGTGAAG420
O CCTTCCCTGA
CTATTCTCTG TCCCTCTTTC CTCTCTTCCC ACAAACACAA CTGTGTACGC480
GTGTCACCAA
AGAGTTAATC GTGCTTTTCT CTGTGCTACT TTTATACSTA GTATATGGTC540
CATTGTTTTG
CACTTAATAC ACTCTCTTGT AATGATTTGT TTACATGTCA GTCTCCCAGC600
CAGACTGAGA
GCTCACCAAG GGCAGAAGCC GTGTTTTGTT TACTGCTGTA TTCCTGGTAC660
CTGGTACAAT
5 GCTTGGCATA CAGTTGGATG AACGGGAAAG TAATCTGAGC TGCCGGTGCT720
O GTGGCAGTGC
AAAGTGGGCA TATTTGTGCC CTTGGACCAG ATGTAGCCCT TGATGCATTT780
TGCAGGAACA
CGGCTTAGTT ATTGTTTACT TTGAAGCCCT TTTGCCTCTA CTCTCTCCCA840
TATATCTTCT
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CCTGACAGGG TGAAGTCACC TATAGCATTT CCTAGTGTAT GGAAGTATTA 900
ATTTCTTTCT
TTACTGGAAG AGCTACTAGC TTTTCTTCAT ACAGTTTCCT CTGCTCCAGT 960
TTCATAAGTT
TCTTTTTGGC TTGTATCTGT TTAGGATCAG GTGATATGGC TTCATTTCTC1020
ATGACTGAAG
CCCGGCAACA TAACACTGAA ATTCGAATGG CAGTCAGCAA AGTGGCTGAT 1080
AAAATGGATC
ATCTCATGAC TAAGGTTGAA GAGTTACAGA AACATAGTGC~TGGCAATTCC 1140
ATGCTTATTC
CTAGCATGTC AGTTACAATG GAAACAAGCA TGATTATGAG CAACATCCAG 1200
CGAATCATTC
AGGCCAAGGT GACAGAGGAG TTAGCAGCGG CCACTGCACA GKTCTCTCAT 1260
CTGCAGCTGA
AAATGACTTG CTCACCAAAA AAAGGAAACA GAGCTGCAGA TGCAGCTGAC1320
AGAAAGCCTG
AAGGAGACAG ATCTTCTCAG GGGCCAGCTC ACCAAAGTGC AGGCAAAGCT 1380
CTCAGAGCTC
CAAGAAACYT CTGAGCAAGC ACAGTCCAAA TTCAAAAGTG AAAAGCAGAA CCGGAAACAA 1440
CTGGAACTCA AGGTGACATC CCTGGAGGAG GAACTGACTG ACCTTCGAGT TGAGAAGGAG 1500
TCCTTGGAAA AGGTAAGCTC TACAACCCAG TTTGCCAGAA TTAGCTGTTT AATAAACATT 1560
2 5 TTTATTTTCC TTTTACAAAA AAAAAAAAAA AA 1592
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
3 0 (A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
3 5 (ii) MOLECULE TYPE: protein
4 O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Glu Val Leu Ile Ser Phe Phe Thr Gly Arg Ala Thr Ser Phe Ser
1 5 10 15
45 Ser Tyr Ser Phe Leu Cys Ser Ser Phe Ile Ser Phe Phe Leu Ala Cys
20 25 30
Ile Cys Leu Gly Ser Gly Asp Met Ala Ser Phe Leu Met Thr Glu Ala
35 40 45
Arg Gln His Asn Thr Glu Ile Arg Met Ala Val Ser Lys Val Ala Asp
50 55 60
Lys Met-Asp His Leu Met Thr Lys Val Glu Glu Leu Gln Lys His Ser
65 70 75 80
84
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Ala Gly Asn Ser Met Leu Ile Pro Ser Met Ser Val Thr Met Glu Thr
85 90 95
Ser Met Ile Met Ser Asn Ile Gln Arg Ile Ile Gln Ala Lys Val Thr
100 105 110
Glu Glu Leu Ala Ala Ala Thr Ala Gln Xaa Ser His Leu Gln Leu Lys
115 120 I25
Met Thr Cys Ser Pro Lys Lys Gly Asn Arg Ala Ala Asp Ala Ala Asp
130 135 140
Arg Lys Pro Glu Gly Asp Arg Ser Ser Gln Gly Pro Ala His Gln Ser
145 150 155 160
Ala Gly Lys Ala Leu Arg Ala Pro Arg Asn Xaa
165 170
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1694 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
2 5 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GGGAAACGGG AAGCCGCTGC AAGTCCACCG CCTCAGCTACCCAGATTGGGATCTGCCCAG60
GCCCGCTTTA TGGACTAGTG TGGGCGGCAG GCTCCTTTCCGTCCCTGCCCTGCTGTACCC120
CGCTCCTTGG AGACCCCCTG TATCCCTCCC GCAAGGTGGAATCCGCAGGCTGGAGGCTCC180
4 O CAGGGGAGGC AAACGCCTGG CCCTGCCCTG CACCATGACCCTCCTGCTGC240
CCCCACGCCG
TGCCCCTTCT GCTGGCCTCT CTGCTCGCGT CCTGCTCCTG TAACAAAGCC AACAAGCACA 300
AGCCATGGAT TGAGGCAGAG TACCAGGGCA TCGTCATGGA GAATGACAAC 360
ACGGTCCTAC
TGAATCCACC ACTCTTTGCC TTGGACAAGG ATGCCCCGCT GCGCTATGCA 420
GGTGAGATCT
GCGGCTTCCG GCTCCATGGG TCTGGGGTGC CCTTTGAGGC TGTGATCCTT 480
GACAAGGCGA
5 O CAGGAGAGGG GCTGATCCGG GCCAAGGAGC CTGTGGACTG CGAGGCCCAG540
AAGGAACACA
CCTTCACCAT CCAGGCCTAT GACTGTGGCG AGGGCCCCGA CGGGGCCAAC 600
ACCAAGAAGT~
CCCACAAGGC CACTGTGCAT GTGCGGGTCA ACGATGTGAA CGAGTTTGCC 660
CCAGTGTTTG
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TGGAACGGCT GTATCGTGCG GCTGTGACAG AGGGGAAGCT GTACGATCGC720
ATCCTGCGGG
TGGAAGCCAT TGACGGTGAC TGCTCCCCCC AGTACAGCCA GATCTGCTAC780
TATGAGATTC
TCACACCCAA CACCCCTTTC CTCATTGACA ATGACGGGAA CATTGAGAAC840
ACAGAGAAGC
TGCAGTACAG TGGTGAGAGG CTCTATAAGT TTACAGTGAC AGCTTATGAC900
TGTGGGAAGA
AGCGGGCAGC AGATGATGCT GAGGTGGAGA TTCAGGTGAA~GCC.CACCTGT960
AAACCCAGCT
GGCAAGGCTG GAACAAAAGG ATCGAATATG CACCAGGTGC TGGGAGCTTG1020
GCTTTGTTCC
CTGGTATCCG CCTGGAGACC TGTGATGAAC CACTCTGGAA CATTCAGGCC1080
ACCATAGAGC
TGCAGACCAG CCATGTGGCC AAGGGCTGTG ACCGTGACAA CTACTCAGAG1140
CGGGCGCTGC
GGAAACTCTG TGGTGCTGCC ACTGGGGAGG TGGATCTGTT GCCCATGCCT1200
GGCCCCAATG
CCAACTGGAC AGCAGGACTC TCGGTGCACT ACAGCCAGGA CAGCAGCCTG1260
ATCTACTGGT
TCAATGGCAC CCAGGCTGTG CAGGTGCCCC TGGGTGGCCC CAGTGGGCTG1320
GGCTCTGGGC
CCCAGGACAG CCTCAGTGAC CACTTCACCC TGTCCTTCTG GATGAAGCAT1380
GGCGTAACTC
2 CCAACAAGGG CAAGAAGGAA GAGGAAACCA TCGTATGTAA CACTGTCCAG1440
5 AATGGTGAGC
CTCCCCTCCA GGCACTAGCC AGAGGGGGAA ACTGGCTTCT TGTCCCGCCT1500
CTGTCACTGC
CCAGTGTGTG ACTGTGAACA GGTCACTTCC CCTCTCTTCA TTTGTGAGGT1560
GCAAGTGCCA
GGTGTGATAT GCCTTGATTC TGTGCTTTAT CCCCAACATG ACATGTTGGA1620
TCGTAAAAAA
P~~~AAAAAAA AAAAAAAAAA P,AAAAAAAAA AA,AAAAAAAA AA,AAAAAAAA16
p,~~AAAAAAAp, 8
0
3 AAAAAAP.AAA AAAA 16
5 9
4
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
4 (A) LENGTH: 428 amino acids
0
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
4 (ii) MOLECULE TYPE: protein
5
50 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Thr Leu Leu Leu Leu Pro Leu Leu Leu Ala Ser Leu Leu Ala~Ser
1 5 10 15
5 5 Cys Ser Cys Asn Lys Ala Asn Lys His Lys Pro Trp Ile Glu Ala Glu
86
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20 25 30
Tyr Gln Gly Ile Val Met Glu Asn Asp Asn Thr Val Leu Leu Asn Pro
35 40 45
Pro Leu Phe Ala Leu Asp Lys Asp Ala Pro Leu Arg Tyr Ala Gly Glu
50 55 60
Ile Cys GlyPheArgLeuHis GlySerGlyValProPhe GluAlaVal
65 70 75 80
Ile Leu AspLysAlaThrGly GluGlyLeuIleArgAla LysGluPro
85 90 95
Val Asp CysGluAlaGlnLys GluHisThrPheThrIle GlnAlaTyr
100 105 110
Asp Cys GlyGluGlyProAsp GlyAlaAsnThrLysLys SerHisLys
115 120 125
Ala Thr ValHisValArgVal AsnAspValAsnGluPhe AlaProVal
130 135 140
Phe Val GluArgLeuTyrArg AlaAlaValThrGluGly LysLeuTyr
2 145 150 155 160
5
Asp Arg IleLeuArgValGlu AlaIleAspGlyAspCys SerProGln
165 270 175
3 Tyr Ser GlnIleCysTyrTyr GluIleLeuThrProAsn ThrProPhe
0
180 185 190
Leu Ile AspAsnAspGlyAsn IleGluAsnThrGluLys LeuGlnTyr
195 200 205
35
Ser Gly GluArgLeuTyrLys PheThrValThrAlaTyr AspCysGly
210 215 220
Lys Lys ArgAlaAlaAspAsp AlaGluValGluIleGln ValLysPro
40 225 230 235 240
Thr Cys LysProSerTrpGln GlyTrpAsnLysArgIle GluTyrAla
245 250 255
4 Pro Gly AlaGlySerLeuAla LeuPheProGlyIleArg LeuGluThr
5
260 265 270
Cys Asp Glu Pro Leu Trp Asn Ile Gln Ala Thr Ile Glu Leu Gln Thr
275 280 285
Ser His Val Ala Lys Gly Cys Asp Arg Asp Asn Tyr Ser Glu Arg Ala
290 295 300
Leu Arg Lys Leu Cys Gly Ala Ala Thr Gly Glu Val Asp Leu Leu Pro
305 310 315 320
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Met Pro Gly Pro Asn Ala Asn Trp Thr Ala Gly Leu Ser Val His Tyr
325 330 335
Ser Gln Asp Ser Ser Leu Ile Tyr Trp Phe Asn Gly Thr Gln Ala Val
340 345 350
Gln Val Pro Leu Gly Gly Pro Ser Gly Leu Gly Ser Gly Pro Gln Asp
355 360 365
Ser Leu Ser Asp His Phe Thr Leu Ser Phe Trp Met Lys His Gly Val
370 375 380
Thr Pro Asn Lys Gly Lys Lys Glu Glu Glu Thr Ile Val Cys Asn Thr
385 390 395 400
Val Gln Asn Gly Glu Pro Pro Leu Gln Ala Leu Ala Arg Gly Gly Asn
405 410 415
Trp Leu Leu Val Pro Pro Leu Ser Leu Pro Ser Val
420 425
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
2 5 (A) LENGTH: 1309 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
3 O (ii) MOLECULE TYPE: cDNA
3 S (xi)
SEQUENCE
DESCRIPTION:
SEQ ID N0:17:
CTGGTCCTCC TTTGCAGAGG TGGTGCGGAGCTCCTGTTTGACGGTATTAA GAAACATCGA60
GTCACTTTGC CTGGACAGGA GGAACCCTGGGACATCCGGAACCTGCTCAT CTGGATCAAG120
40
AAGAATTTGC TAAAAGAGCG GCCAGAGTTGTTCATCCAGGGAGACAGCGT GCGGCCAGGA180
ATTCTGGTGC TGATTAACGA TGCCGACTGGGAGCTACTGGGTGAGCTGGA CTACCAGCTT240
4 5 CAGGACCAGGACAGCGTCCT CTTCATCTCCACTCTGCACGGCGGCTGAGG GCCCTTCTCT300
GGGCCTGGGC ACCCTTAGAG GGGAGAACGAAGCAATCAGACATCCCCTTG GGCCCTGCTT360
CCAGGTCTCC CTGTCCCCCT TGCCTGCCTT CTTCCCTGCT CTGTCCCCTA AGCTCCCTCC 420
AGGCAGGGAA AAGAGGCCAG GTGCTAAAAA TGAGCCTTTC TCAAGCACGT GAGCAGCGGA 480
AGGCAGACAG GCGCCAGAGC CCAGCACTCC CTTTTCCAGC AGCTGTGGTG GGGGAGGGTT 540
5 5 CCCCTCCAGT TTGTCAAGAG TTGAAGGAGG CTCTGTGGCC AGGTGACCTG GCTGCCTTCC 600
gg
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ACTCCTTGTA CCTCAGTCTA AACATGGAGT GGCCGCTGACAGCCCCAGAG660
AAGGCGCTCC
CCAGCGTCTT CATGGGGAAG ATGAATGGAC CTGAGTAGCTCCCTCCCTAC720
GAAGGAAGGC
CCAAAGACTG GAGGCTTCTC AGCCTCAATT TCCCTGTCTGGGCTCTGCCT780
TACAGCTGAG
GTCCCCCACT GCTATCAGTA TGGAACCCCA GCTGGGGTCCGCCGACTCCC840
CCTATTGAGT
CCCACCGCCA GCAGCTGCTC CTCCAGCCAC ACCCTTCCTGCCYTAGCCCT900
CTCCCCCCAC
TGACCCTGGC TGGCCTGCCC CGCTCCACAG GCCACCAGATGACCCTCCCC960
GGGCTCCTGA
AGGCTGCTTA CAGCTCATTC TGCTGGGGGT AGAGATGAGGAGTTAAACCT1020
GGAGGGAGTA
1 TGGACTAGCA AGTAGAAGCC TGGGGGGATG CGTGTGCCTCTCCACAACTG1080
5 AGTTTCCTCC
AATATAGTGG CTGAAAACTG GGGAGATACT TGATGGCGCGTTCTCTCCCT1140
AATGTCCGTT
TCCCACCTCC TGCAGGAAGC AGGACGGGGC AGGCAGCACCAGTGCTTTGC1200
TGGTAGGCAC
CCCTCCTCCC CTTCCCTTCT GGAAGTCTTG GGGCCTCAGTGCCGGCCTTG1260
GCTTGCAACA
GGCAAATAAA AGACTAGGTT GTTTACTAAA AAAAAAAAAA 1309
AAAAAAAAA
2 (2) INFORMATION FOR SEQ ID N0:18:
5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids
(B) TYPE: amino acid
3 (C) STRANDEDNESS:
O
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Ala Arg Met Ser Val Phe Ser Pro Phe Pro Pro Pro Ala Gly Ser
1 5 10 15
Arg Thr Gly Gln Ala Ala Pro Gly Arg His Ser Ala Leu Pro Leu Leu
20 25 30
Pro Phe Pro Ser Gly Ser Leu Gly Ala Ser Val Leu Ala Thr Ala Gly
35 40 45
Leu Gly Gln Ile Lys Asp
50
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
5 5 (A) LENGTH: 1740 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
1O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GGCCCGTGCG CTCCATCAAC CACGCCAGCC TCATCTCTGC ACTCTCCCGG60
GACTATCGCA
ACCTGAAGCC CAGTGCTGTT GCCCCACAGA GAAAGATGCC ACTGGATGAC120
ACCAAACTGA
TTATCCACCA GACACTCAGC GTCTTAGAAG ATATTGTGGA GAATATCTCG180
GGGGAGTCCA
CCAAGTCTCG ACAGATTTGC TACCAGTCGC TGCAGGAATC TGTTCAGGTC240
TCCCTGGCCC
2 TCTTTCCAGC TTTTATCCAT CAGTCAGATG TGACTGATGA GATGCTGAGC300
O TTCTTCCTCA
CTCTGTTTCG AGGCCTTAGA GTACAGATGG GTGTGCCTTT CACTGAGCAA360
ATCATACAGA
CTTTCCTCAA CATGTTTACC AGAGAGCAGT TAGCCGAGAG CATCCTCCAC420
GAGGGCAGCA
CAGGCTGCCG GGTGGTGGAG AAGTTTCTGA AGATCCTGCA GGTGGTGGTC480
CAGGAGCCAG
GCCAGGTGTT CAAGCCCTTC CTCCCCAGCA TCATCGCCCT GTGCATGGAG540
CAAGTGTATC
3 CCATCATTGC CGAGCGTCCC TCCCCTGATG TGAAGGCCGA GCTGTTTGAG600
O CTCCTTTTCC
GGACGCTCCA TCACAACTGG AGGTACTTCT TCAAGTCCAC CGTGCTGGCC660
AGTGTCCAGA
GGGGGATCGC TGAGGAGCAG ATGGAGAATG AGCCCCAGTT CAGTGCCATC720
ATGCAGGCTT
TCGGACAGTC CTTTCTCCAG CCCGACATCC ACCTTTTTAA ACAAAATCTC780
TTCTACTTGG
AGACTCTCAA CACCAAGCAG AAGCTGTACC ACAAGAAGAT CTTCCGGACT840
GCCATGCTGT
4 TCCAGTTTGT GAACGTGCTG CTCCAGGTCC TGGTCCACAA GTCCCATGAT900
O CTTCTGCAGG
AGGAGATTGG CATCGCCATC TACAACATGG CCTCAGTCGA CTTTGATGGC960
TTCTTTGCCG
CCTTCCTCCC AGAGTTCCTG ACCAGCTGTG ATGGTGTGGA TGCCAACCAG1020
AAAAGTGTGC
TGGGGCGGAA TTTCAAGATG GATCGGGACC TGCCCTCATT CACCCAGAAT1080
GTGCACAGGC
TGGTCAACGA CCTGCGCTAC TACAGACTCT GCAACGACAG CCTGCCCCCT1140
GGCACTGTGA
5 AGCTCTAGGC CTGCTACTGC CTGGGGACAC GGACTTCTGC TGCTGCCACC1200
O TGCGCCAGCC
CTACCTTCCA CCACAGATGT CTCCCAGATG GGCCTTGGTC ACACTCCTTG1260
GCTTCTCCCA'
CCGCAAGCAA CGCTGCCTGC CTCTGCCGCT CCTCCACATC TTGCCGCTGC1320
CCAGCAGAGC
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TGGCTTCTGG GTCCACCTGA GCACTGGACGGTGCTCCCAG GGCGTTGGAG CAGGCGGAGG1380
GGTGTGTGGC CAGGTACTAG GAGGCACCAGGAAATCCCGC GGGGTGGCCC ATGCAGACCA2440
GGCGCACGTG GCTCATGGGG CAGAATTGCCAAGGACAGCT CACGACAGTG CMACCTTCTC1500
ACCATTCCAG CCAAGGAGAG ATGTGACGTTGGAAMTGYTY TGGCAMTTYT GTCAAGCCTC1560
CCCCGCCCCA ATTGCCTTGA RATYTYTGCTCTTTGTCAGA GATTTGCAAA GACTCAMGTT1620
TTTGTTGTTT TCTCATCATT CCATTGTGATACTAAGAAAC TAAGAAGCTT AATGAAAAGA1680
AATAAAATGC CTATGTTGTT GTTCTAGGRRAAAAAAAAAA AAAAAAAAAA A,~~i~AAAAAAA1740
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 amino acids
2 0 (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Met Pro Leu Asp Asp Thr Lys Leu Ile Ile His Gln Thr Leu Ser Val
1 5 10 15
Leu Glu Asp Ile Val Glu Asn Ile Ser Gly Glu Ser Thr Lys Ser Arg
20 25 30
Gln Ile Cys Tyr Gln Ser Leu Gln Glu Ser Val Gln Val Ser Leu Ala
35 40 45
Leu Phe Pro Ala Phe Ile His Gln Ser Asp Val Thr Asp Glu Met Leu
55 60
Ser Phe Phe Leu Thr Leu Phe Arg Gly Leu Arg Val Gln Met Gly Val
65 70 75 80
Pro Phe Thr Glu Gln Ile Ile Gln Thr Phe Leu Asn Met Phe Thr Arg
85 90 95
Glu Gln Leu Ala Glu Ser Ile Leu His Glu Gly Ser Thr Gly Cys Arg
100 105 110
Val Val Glu Lys Phe Leu Lys Ile Leu Gln Val Val Val Gln Glu~Pro
115 120 125
Gly Gln Val Phe Lys Pro Phe Leu Pro Ser Ile Ile Ala Leu Cys Met
91
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130 135 140
Glu Gln Val Tyr Pro Ile Ile Ala Glu Arg Pro Ser Pro Asp Val Lys
145 150 155 160
Ala Glu Leu Phe Glu Leu Leu Phe Arg Thr Leu His His Asn Trp Arg
165 170 175
Tyr Phe Phe Lys Ser Thr Val Leu Ala Ser Val Gln Arg Gly Ile Ala
180 185 190
Glu GluGlnMetGluAsn GluProGlnPheSerAla IleMetGlnAla
195 200 205
Phe GlyGlnSerPheLeu GlnProAspIleHisLeu PheLysGlnAsn
210 215 220
Leu PheTyrLeuGluThr LeuAsnThrLysGlnLys LeuTyrHisLys
225 230 235 240
Lys IlePheArgThrAla MetLeuPheGlnPheVal AsnValLeuLeu
245 250 255
Gln ValLeuValHisLys SerHisAspLeuLeuGln GluGluIleGly
260 265 270
Ile AlaIleTyrAsnMet AlaSerValAspPheAsp GlyPhePheAla
275 280 285
3 Ala PheLeuProGluPhe LeuThrSerCysAspGly ValAspAlaAsn
0
290 295 300
Gln Lys Ser Val Leu Gly Arg Asn Phe Lys Met Asp Arg Asp Leu Pro
305 310 315 320
Ser Phe Thr Gln Asn Val His Arg Leu Val Asn Asp Leu Arg Tyr Tyr
325 330 335
Arg Leu Cys Asn Asp Ser Leu Pro Pro Gly Thr Val Lys Leu
340 345 350
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
5 0 (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ANTGTCTTGA CTACAAGCTC CACGGGGGC
29
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS: _
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
2 0 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
TNGCCAAGGA GAAAGCGAGG CAGACAAGG 29
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3 0 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
4 0 ANATCGACTC TTTGCATCGC ACATTTTGT 29
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
93
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CNTTCTTCGG ACTTATGTTT GAATCTATC 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs _
(B) TYPE: nucleic acid
1 0 (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
CNTTCCTCTT AGATCTCAGT ATCCACCTC 29
(2) INFORMATION FOR SEQ ID N0:26:
2 5 {i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
CNCAGACAGG GGAGATAACA ATGAGGTGC 29
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
4 5 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
94
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WO 98/56805 PCT/US98/12076
TNCTATAGGT GACTTCACCC TGTCAGGAG 29
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
TNTTACAGGA GCAGGACGCG AGCAGAGAG 29
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
2 5 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
3 0 (A) DESCRIPTION: /desc = "oligonucleotide"
3 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ANTCAGTTGT GGAGGAGGAA ACTGAGGCA 2g
(2) INFORMATION FOR SEQ TD N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
CNTCGAAACA GAGTGAGGAA GAAGCTCAG 2g
CA 02293825 1999-12-10
WO 98/56805 PCTNS98/12076
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
Z5 (xi) SEQUENCE N0:31:
DESCRIPTION:
SEQ
ID
Met ValAlaTrpArgSer AlaPheLeuValCysLeu AlaPheSerLeu
1 5 10 15
2 Ala ThrLeuValGlnArg GlySerGlyAspPheAsp AspPheAsnLeu
0
20 25 30
Glu AspAlaValLysGlu ThrSerSerValLysGln ProTrpAspHis
35 40 45
25
Thr ThrThrThrThrThr AsnArgProGlyThrThr ArgAlaProAla
50 55 60
Lys ProProGlySerGly LeuAspLeuAlaAspAla LeuAspAspGln
30 65 70 75 80
Asp AspGlyArgArgLys ProGlyIleGlyGlyArg GluArgTrpAsn
85 90 95
3 His ValThrThrThrThr LysArgProValThrThr ArgAlaProAla
5
100 105 110
Asn ThrLeuGlyAsnAsp PheAspLeuAlaAspAla LeuAspAspArg
115 120 125
40
Asn AspArgAspAspGly ArgArgLysProIleAla GlyGlyGlyGly
130 135 140
Phe SerAspLysAspLeu GluAspIleValGlyGly GlyGluTyrLys
45 145 150 155 160
Pro AspLysGlyLysGly AspGlyArgTyrGlySer AsnAspAspPro
165 170 175
50 Gly SerGlyMetValAla GluProGlyThrIleAla GlyValAlaSer
180 185 190
Ala LeuAlaMetAlaLeu IleGlyAlaValSerSer TyrIleSerTyr
195 200 205
55
96
CA 02293825 1999-12-10
WO 98/56805 PCT/US98112076
Gln Gln Lys Lys Phe Cys Phe Ser Ile Gln Gln Gly Leu Asn Ala Asp
210 215 220
Tyr Val Lys Gly Glu Asn Leu Glu Ala Val Val Cys Glu Glu Pro Gln
225 230 235 240
Val Lys Tyr Ser Thr Leu His Thr Gln Ser Ala Glu Pro Pro Pro Pro
245 250 255
Pro Glu Pro Ala Arg Ile
260
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE
DESCRIPTION:
SEQ
ID
N0:32:
Met HisValLeu GluThrLeuAspLeuLeu ValLeuArgAlaAspLys
1 5 10 15
Gly LysAspAla ArgLeuPheValPheArg LeuSerAlaLeuGlnLys
20 25' 30
Gly LeuGluGly LysGlnAlaGlyLysSer ArgSerAspCysArgGlu
35 40 45
Asn LysLeuGlu LysThrLysGlyCysHis LeuTyrAlaIleAsnThr
50 55 60
His HisSerArg GluLeuArgIleValVal AlaIleArgAsnLysLeu
65 70 75 80
Leu LeuIleThr ArgLysHisAsnLysPro SerGlyValThrSerThr
85 90 95
Ser LeuLeuSer ProLeuSerGluSerPro ValGluGluPheGlnTyr
100 105 110
Ile ArgGluIle CysLeuSerAspSerPro MetValMetThrLeuVal
115 120 125
Asp GlyProAla GluGluSerAspAsnLeu IleCysValAlaTyrArg
130 135 140
5 His GlnPheAsp ValValAsnGluSerThr GlyGluAlaPheArgLeu
5
97
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
145 150 155 160
His His Val Glu Ala Asn Arg Val Asn Phe Val Ala Ala Ile Asp Val
165 170 175
Tyr Glu Asp Gly Glu Ala Gly Leu Leu Leu Cys Tyr Asn Tyr Ser Cys
180 185 190
Ile Tyr Lys Lys Val Cys Pro Phe Asn Gly Gly Ser Phe Leu Val Gln
195 200 205
Pro Ser Ala Ser Asp Phe Gln Phe Cys Trp Asn Gln Ala Pro Tyr Ala
210 215 220
Ile Val Cys Ala Phe Pro Tyr Leu Leu Ala Phe Thr Thr Asp Ser Met
225 230 235 240
Glu Ile Arg Leu Val Val Asn Gly Asn Leu Val His Thr Ala Val Val
245 250 255
Pro Gln Leu Gln Leu Val Ala Ser Arg Ser Asp Ile Tyr Phe Thr Ala
260 265 270
Thr Ala Ala Val Asn Glu Val Ser Ser Gly Gly Ser Ser Lys Gly Ala
275 280 285
Ser AlaArgAsnSerProGln ThrProProGlyArg AspThrProVal
290 295 300
3 Phe ProSerSerLeuGlyGlu GlyGluIleGlnSer LysAsnLeuTyr
0
305 310 315 320
Lys IleProLeuArgAsnLeu ValGlyArgSerIle GluArgProLeu
325 330 335
Lys SerProLeuValSerLys ValIleThrProPro ThrProIleSer
340 345 350
Val GlyLeuAlaAlaIlePro ValThrHisSerLeu SerLeuSerArg
355 360 365
Met GluIleLysGluIleAla SerArgThrArgArg GluLeuLeuGly
370 375 380
Leu SerAspGluGlyGlyPro LysSerGluGlyAla ProLysAlaLys
385 390 395 400
Ser LysProArgLysArgLeu GluGluSerGlnGly GlyProLysPro
405 410 415
Gly Ala Val Arg Ser Ser Ser Ser Asp Arg Ile Pro Ser Gly Ser Leu
420 425 430
Glu Ser Ala Ser Thr Ser Glu Ala Asn Pro Glu Gly His Ser Ala Ser
435 440 445
98
CA 02293825 1999-12-10
WO 98/56805 PCT/US98/12076
Ser Asp Gln Asp Pro Val Ala Asp Arg Glu Gly Ser Pro Val Ser Gly
450 455 460
Ser Ser Pro Phe Gln Leu Thr Ala Phe Ser Asp Glu Asp Ile Ile Asp
465 470 475 480
Leu Lys
99
