Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOT117ES ENCODING THEM
This application is a continuation-in-part of Ser. No. Ob/XXX,XXX (converted
to a provisional application from non-provisional application Ser. No.
08/780,890},
filed January 9, 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
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.
SUBSTITUTE SHEET (RULE 26)
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 61 to nucleotide 642;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 89 to nucleotide 440;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BI164_1 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BI164_1 deposited under accession number ATCC 98290;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BI164_1 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BI164_1 deposited under accession number ATCC 98290;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
2 5 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
3 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:1 from nucleotide 61 to nucleotide 642; the nucleotide sequence of SEQ ID
N0:1 from
nucleotide 89 to nucleotide 440; the nucleotide sequence of the full-length
protein coding
sequence of clone BI164_1 deposited under accession number ATCC 98290; or the
nucleotide sequence of the mature protein coding sequence of clone BI164_1
deposited
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under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BI164_1 deposited under accession number ATCC 98290. 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 23 to amino
acid
127.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
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;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 23 to
amino acid 127;
(c) fragments of the amino acid sequence of SEQ ID N0:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BI164_1 deposited under accession number ATCC 98290;
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
2 0 of SEQ ID N0:2 from amino acid 23 to amino acid 127.
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;
2 5 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1625 to nucleotide 1750;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1484 to nucleotide 1729;
(d) a polynucleotide comprising the nucleotide sequence of the full-
3 0 length protein coding sequence of clone BK445_1 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BK445 1 deposited under accession number ATCC 98290;
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(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BK445_1 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BK445_1 deposited under accession number ATCC 98290;
(h) a polynucleotide encoding a protein comprising the amino acid
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;
(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 capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-{i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1625 to nucleotide 1750; the nucleotide sequence of SEQ
ID N0:3
from nucleotide 1484 to nucleotide 1729; the nucleotide sequence of the full-
length protein
coding sequence of clone BK445_1 deposited under accession number ATCC 98290;
or the
2 0 nucleotide sequence of the mature protein coding sequence of clone BK445_1
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BK445_1 deposited under accession number ATCC 98290. In yet other
preferred
embodiments) the present invention provides a polynucleotide encoding a
protein
2 5 comprising the amino acid sequence of SEQ ID N0:4 from amino acid 1 to
amino acid 35.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 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
amino acid 35;
(c) fragments of the amino acid sequence of SEQ ID N0:4; and
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(d) the amino acid sequence encoded by the cDNA insert of clone
BK445_1 deposited under accession number ATCC 98290;
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 i to amino acid 35.
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 99 to nucleotide 1058;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1 to nucleotide 644;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BP101 2 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BP101 2 deposited under accession number ATCC 98290;
(f) a polynucleotide comprising the nucleotide sequence of the mature
2 0 protein coding sequence of clone BP101 2 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BP101 2 deposited under accession number ATCC 98290;
(h) a polynucleotide encoding a protein comprising the amino acid
2 5 sequence of SEQ ID N0:6;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity;
(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 capable of hybridizing 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 99 to nucleotide 1058; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 1 to nucleotide 644; the nucleotide sequence of the full-
length protein
coding sequence of clone BP101 2 deposited under accession number ATCC 98290;
or the
nucleotide sequence of the mature protein coding sequence of clone BP101 2
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BP101 2 deposited under accession number ATCC 98290. 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 1 to amino
acid 182.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
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 1 to
amino acid 182;
(c) fragments of the amino acid sequence of SEQ ID N0:6; and
2 0 (d) the amino acid sequence encoded by the cDNA insert of clone
BP101 2 deposited under accession number ATCC 98290;
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 1 to amino acid 182.
2 5 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
3 0 N0:7 from nucleotide 237 to nucleotide 1184;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 207 to nucleotide 935;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CD124_3 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CD124_3 deposited under accession number ATCC 98290;
. (f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CDI24 3 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CD124_3 deposited under accession number ATCC 98290;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity;
(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 capable of hybridizing under stringent conditions
2 0 to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 237 to nucleotide 1184; the nucleotide sequence of SEQ ID
N0:7
from nucleotide 207 to nucleotide 935; the nucleotide sequence of the full-
length protein
coding sequence of clone CD124_3 deposited under accession number ATCC 98290;
or the
2 5 nucleotide sequence of the mature protein coding sequence of clone CD124_3
deposited
under accession number ATCC 98290. In other preferred embodiments, the
poiynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CD124 3 deposited under accession number ATCC 98290. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
3 0 comprising the amino acid sequence of SEQ ID NO:B from amino acid 1 to
amino acid 233.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to
amino acid 233;
(c) fragments of the amino acid sequence of SEQ ID N0:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CD124_3 deposited under accession number ATCC 98290;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:8 or the amino acid
sequence
of SEQ ID N0:8 from amino acid 1 to amino acid 233.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 142 to nucleotide 828;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:9 from nucleotide 1 to nucleotide 522;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CW924_1 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
2 5 cDNA insert of clone CW924_1 deposited under accession number ATCC 98290;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CW924_1 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
3 0 cDNA insert of clone CW924_1 deposited under accession number ATCC 98290;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity;
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(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 142 to nucleotide 828; the nucleotide sequence of SEQ ID
N0:9
from nucleotide 1 to nucleotide 522; the nucleotide sequence of the full-
length protein
coding sequence of clone CW924_1 deposited under accession number ATCC 98290;
or
the nucleotide sequence of the mature protein coding sequence of clone CW924_1
deposited under accession number ATCC 98290. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CW924_1 deposited under accession number ATCC 98290. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino
acid
127.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
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 NO:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to
2 5 amino acid 127;
(c) fragments of the amino acid sequence of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CW924_1 deposited under accession number ATCC 98290;
the protein being substantially free from other mammalian proteins. Preferably
such
3 0 protein comprises the amino acid sequence of SEQ ID NO:10 or the amino
acid sequence
of SEQ ID N0:10 from amino acid 1 to amino acid 127.
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
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 3 to nucleotide 1937;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 204 to nucleotide 414;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DF518 3 deposited under accession
number ATCC 98290;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone DF518 3 deposited under accession number ATCC 98290;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DF518 3 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DF518 3 deposited under accession number ATCC 98290;
(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
2 0 amino acid sequence of SEQ ID N0:12 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-{g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
2 S (1) a polynucleotide capable of hybridizing 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 3 to nucleotide 1937; the nucleotide sequence of SEQ ID
N0:11
from nucleotide 204 to nucleotide 414; the nucleotide sequence of the full-
length protein
3 0 coding sequence of clone DF518 3 deposited under accession number ATCC
98290; or the
nucleotide sequence of the mature protein coding sequence of clone DF518 3
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone DF518_3 deposited under accession number ATCC 98290. In yet other
preferred
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embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:12 from amino acid 67 to amino
acid
137.
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) the amino acid sequence of SEQ ID NO:I2 from amino acid 67 to
amino acid 137;
(c) fragments of the amino acid sequence of SEQ ID N0:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone
DF518_3 deposited under accession number ATCC 98290;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12 or the amino acid
sequence
of SEQ ID N0:12 from amino acid 67 to amino acid 137.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 137 to nucleotide 457;
(c), a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:13 from nucleotide 323 to nucleotide 457;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 82 to nucleotide 322;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DM406_1 deposited under accession
3 0 number ATCC 98290;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone DM406_1 deposited under accession number ATCC 98290;
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(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DM406_1 deposited under accession number
ATCC 98290;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DM406_1 deposited under accession number ATCC 98290;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:14;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity;
(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
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 137 to nucleotide 457; the nucleotide sequence of SEQ ID
N0:13
from nucleotide 323 to nucleotide 457; the nucleotide sequence of SEQ ID N0:13
from
nucleotide 82 to nucleotide 322; the nucleotide sequence of the full-length
protein coding
2 0 sequence of clone DM406_1 deposited under accession number ATCC 98290; or
the
nucleotide sequence of the mature protein coding sequence of clone DM406_1
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polvnucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone DM406_1 deposited under accession number ATCC 98290. In yet other
preferred
2 S embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:14 from amino acid 1 to amino
acid
62.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
3 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 N0:14;
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(b) the amino acid sequence of SEQ ID N0:14 from amino acid 1 to
amino acid 62;
(c) fragments of the amino acid sequence of SEQ ID N0:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone
DM406_1 deposited under accession number ATCC 98290;
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 62.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 312 to nucleotide 851;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 56 to nucleotide 470;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone EH189_1 deposited under accession
number ATCC 98290;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone EH189_1 deposited under accession number ATCC 98290;
(f} a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone EH189_1 deposited under accession number
ATCC 98290;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone EH189_1 deposited under accession number ATCC 98290;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:16;
(i) a polynucleotide encoding a protein comprising a fragment of the
. 3 0 amino acid sequence of SEQ ID N0:16 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
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(I) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 312 to nucleotide 851; the nucleotide sequence of SEQ ID
N0:15
from nucleotide 56 to nucleotide 470; the nucleotide sequence of the full-
length protein
coding sequence of clone EH189_1 deposited under accession number ATCC 98290;
or the
nucleotide sequence of the mature protein coding sequence of clone EH189_1
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone EH189_1 deposited under accession number ATCC 98290. 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
53.
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;
2 0 (b) the amino acid sequence of SEQ ID N0:16 from amino acid 1 to
amino acid 53;
(c) fragments of the amino acid sequence of SEQ ID N0:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone
EH189_1 deposited under accession number ATCC 98290;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid
sequence
of SEQ ID N0:16 from amino acid 1 to amino acid 53.
In one embodiment) the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
3 0 (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 20 to nucleotide 541;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 272 to nucleotide 541;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 1 to nucleotide 448;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone EH203_2 deposited under accession
number ATCC 98290;
(f} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone EH203_2 deposited under accession number ATCC 98290;
(g) a poiynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone EH203 2 deposited under accession number
ATCC 98290;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone EH203_2 deposited under accession number ATCC 98290;
(i) a polynucleotide encoding a protein comprising the amino acid
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;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
2 0 (a)-(h) above;
(1) a polvnucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
2 5 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:17 from nucleotide 20 to nucleotide 541; the nucleotide sequence of SEQ ID
N0:17
from nucleotide 272 to nucleotide 541; the nucleotide sequence of SEQ ID N0:17
from
nucleotide 1 to nucleotide 448; the nucleotide sequence of the full-length
protein coding
sequence of clone EH203_2 deposited under accession number ATCC 98290; or the
3 0 nucleotide sequence of the mature protein coding sequence of clone EH203 2
deposited
under accession number ATCC 98290. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone EH203_2 deposited under accession number ATCC 98290. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
CA 02276085 1999-06-30
WO 98130582 PCT/US98I00289
comprising the amino acid sequence of SEQ ID N0:18 from amino acid 1 to amino
acid
143.
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) the amino acid sequence of SEQ ID N0:18 from amino acid 1 to
amino acid 143;
(c) fragments of the amino acid sequence of SEQ ID N0:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone
EI-I203 2 deposited under accession number ATCC 98290;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:18 or the amino acid
sequence
of SEQ ID N0:18 from amino acid 1 to amino acid 143.
In certain preferred embodiments, the p.olynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
2 0 Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
2 5 polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
3 0 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
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effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature) can then be determined from such nucleotide sequence. The amino
acid
sequence of the protein encoded by a particular clone can also be determined
by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 0 As used herein a "secreted" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in ~~hich they are expressed. "Secreted" proteins also include without
limitation proteins
2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "BI164 1"
A polynucleotide of the present invention has been identified as clone
"BI164_I ".
BI164_1 was isolated from a human fetal kidney cDNA library using methods
which are
3 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the .basis of
computer
analysis of the amino acid sequence of the encoded protein. BI164_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"BI164_1 protein').
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The nucleotide sequence of BI164_l as presently determined is reported in SEQ
ID NO:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BI164_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:2.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BI164_1 should be approximately 800 bp.
The nucleotide sequence disclosed herein for BI164_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BI164_1 demonstrated at least some similarity with
sequences
identified as AA169106 (ms65e05.r1 Stratagene mouse embryonic carcinoma
(#937317)
Mus musculus cDNA clone 616448 5'), AA568401 (nfl6bll.sl NCI CGAP_Pr1 Homo
sapiens cDNA clone IMAGE:913917 similar to contains element TAR1 repetitive
element),
and AA579465 (nf29h01.s1 NCI CGAP_Prl Homo Sapiens cDNA clone IMAGE:915217).
Based upon sequence similarity, BI164_1 proteins and each similar protein or
peptide may
share at least some activity.
Clone "BK445 1"
A polynucleotide of the present invention has been identified as clone
"BK445_1".
BK445_1 was isolated from a human adult retina cDNA library using methods
which are
2 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BK445_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BK445_1 protein")
2 5 The nucleotide sequence of BK445_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 BK445_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
3 0 BK445_1 should be approximately 3400 bp.
The nucleotide sequence disclosed herein for BK445_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BK445_1 demonstrated at least some similarity with
sequences
identified as F08866 (H. sapiens partial cDNA sequence; clone c-2th07), M87889
(Human
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carcinoma cell-derived Alu RNA transcript, clone CD140), U52111 (Human Xq28
genomic
DNA in the region of the ALD locus containing the genes for creatine
transporter
{SLC6A8), CDM, adrenoleukodystrophy (ALD), Na+-isocitrate dehydrogenase
gamma),
and 269649 (Human DNA sequence from cosmid L69F7B, Huntington's Disease
Region,
chromosome 4p16.3 contains Huntington Disease (HD) gene). The predicted amino
acid
sequence disclosed herein for BK445_1 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
BK445_1 protein demonstrated at least some similarity to sequences identified
as L24521
(transformation-related protein [Homo sapiens]) and U22376 (alternatively
spliced
product using exon 13A [Homo sapiens]). Based upon sequence similarity,
BK445_1
proteins and each similar protein or peptide may share at least some activity.
The
nucleotide sequence of BK445 1 indicates that it may contain an Alu repetitive
element.
Clone "BP101 2"
A polynucleotide of the present invention has been identified as clone
"BP101_2".
BP101 2 was isolated from a human fetal kidney cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BP101 2 is a full-
length clone,
2 0 including the entire coding sequence of a secreted protein (also referred
to herein as
"BP101_2 protein")
The nucleotide sequence of BP101 2 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 BP101 2 protein corresponding to the
foregoing
2 5 nucleotide sequence is reported in SEQ ID N0:6. Another possible reading
frame and
predicted amino acid sequence encoded by BP101 2 is encoded by nucleotides 992-
1912
of SEQ ID N0:5 and is reported in SEQ ID N0:28; this alternative open reading
frame
could be joined to the reading frame reported in SEQ ID N0:6 if an insertion
or deletion
resulting in a frameshift was made in the sequence of SEQ ID N0:5. Amino acids
126 to
3 0 138 of SEQ ID N0:28 are a predicted leader/signal sequence, with the
predicted mature
amino acid sequence beginning at amino acid 139 of SEQ ID N0:28, or are a
transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BP101 2 should be approximately 2280 bp.
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The nucleotide sequence disclosed herein for BP101 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BP101 2 demonstrated at least some similarity with
sequences
identified as M87918 (Human carcinoma cell-derived Aiu RNA transcript, clone
NE51),
N74481 (za54d08.s1 Homo Sapiens cDNA clone 296367 3' similar to contains Alu
repetitive
element), and W03189 (za54d08.r1 Soares fetal liver spleen 1NFLS Homo sapiens
cDNA
clone 296367 5'). The predicted amino acid sequence disclosed herein for BP101
2 was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted BP101 2 protein demonstrated at least
some
similarity to sequences identified as L11672 (zinc finger protein [Homo
Sapiens]). Based
upon sequence similarity, BP101 2 proteins and each similar protein or peptide
may share
at least some activity. The nucleotide sequence of BP101 2 indicates that it
may contain
an Alu repetitive element.
Clone "CD124 3"
A polynucleotide of the present invention has been identified as clone "CD124
3".
CD124_3 was isolated from a human fetal brain cDNA library using methods which
are
selective far 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
2 0 analysis of the amino acid sequence of the encoded protein. CD124_3 is a
full-length
done, including the entire coding sequence of a secreted protein (also
referred to herein
as "CD124 3 protein").
The nucleotide sequence of CD124_3 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
2 5 predicted amino acid sequence of the CD124_3 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
CD124_3 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for CD124_3 was searched against the
3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CD124_3 demonstrated at least some similarity with
sequences
identified as AA123334 (mq10f01.r1 Barstead MPLRBl Mus musculus cDNA clone
578329
5'), AA215297 (zr94b03.r1 NCI_CGAP GCB1 Homo sapiens cDNA clone), AA361664
(EST71136 T-cell lymphoma Homo sapiens cDNA 5' end), M16362 (Mouse opa repeat
CA 02276085 1999-06-30
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mRNA, 3' end (MOUSE)), N56580 (SH2201F Homo sapiens cDNA clone), Q12515 (CSP-2
peptide from P. falciparum (clone 4)), W53105 (md14d02.r1 Soares mouse embryo
NbME13.5 14.5 Mus musculus cDNA clone 368355 5'), and X78609 (G.gallus genomic
DNA repeat region, clone 16E1). Based upon sequence similarity, CD124_3
proteins and
each similar protein or peptide may share at least some activity. The
nucleotide sequence
of CD124 3 indicates that it may contain a simple repeat sequence and a
chicken genomic
repeat sequence.
Clone "CW924 1"
A polynucleotide of the present invention has been identified as clone
"CW924_1".
CW924_1 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. CW924_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CW924_1 protein").
The nucleotide sequence of CW924_1 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 CW924_1 protein corresponding to the
foregoing
2 0 nucleotide sequence is reported in SEQ ID NO:10.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CW924_1 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for CW924_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. CW924_1 demonstrated at least some similarity with
sequences
identified as H86434 (ys93h08.r1 Homo sapiens cDNA clone 222399 5'), T15605
(IB1619
Infant brain, Bento Soares Homo sapiens cDNA 3'end similar to IB570 H. sapiens
brain
cDNA clone IB570), U17259 (Mus musculus p19 mRNA, complete cds (MOUSE)), and
W54334 {md05b08.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone
3 0 367479 5'). The predicted amino acid sequence disclosed herein for CW924_1
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted CW924_1 protein demonstrated at least
some
similarity to sequences identified as P50649 (Sequence encoded by brain
specific (Class III)
clone p1A75 ORF 1) and U17259 (p19 [Mus musculusJ). The predicted CW924_1
protein
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also demonstrated at least some similarity to mouse p21 protein. Based upon
sequence
similarity, CW924 1 proteins and each similar protein or peptide may share at
least some
activity. Amino acids 74 to 86 of SEQ ID N0:10 are a possible leader/signal
sequence,
with the predicted mature amino acid sequence beginning at amino acid 87, or
are a
transmembrane domain.
Clone "DF518 3"
A polynucleotide of the present invention has been identified as clone
"DF518_3".
DF518 3 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. DF518_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "DF518_3 protein").
The nucleotide sequence of DF518_3 as presently determined is reported in SEQ
ID NO:11. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the DF518_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 DF518_3 should be approximately 2650 bp.
The nucleotide sequence disclosed herein for DF518_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN /BLASTX and
FASTA search protocols. DF518 3 demonstrated at least some similarity with
sequences
identified as AA190696 (zp89a02.r1 Stratagene HeLa cell s3 937216 Homo Sapiens
cDNA
2 5 clone 627338 5'), H13983 (EST00009 Homo sapiens genomic clone C1-14 5'),
and T90095
(yd39d08.s1 Homo sapiens cDNA clone 110607 3'). Based upon sequence
similarity)
DF518_3 proteins and each similar protein or peptide may share at least some
activity.
Clone "DM406 1"
3 0 A polynucleotide of the present invention has been identified as clone
"DM406_1 ".
DM406_1 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. DM406_1 is a full-
length
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WO 98!30582 PCT/US98100Z89
clone, including the entire coding sequence of a secreted protein {also
referred to herein
as "DM406_1 protein")
The nucleotide sequence of DM406_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 DM406_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:14. 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.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
DM40b_1 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for DM406_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. DM406_1 demonstrated at least some similarity with
sequences
identified as D82016 (Human fetal brain cDNA 5'-end GEN-430610) and H12174
(ym17fO8.r1 Homo sapiens cDNA clone 48073 5'). Based upon sequence similarity,
DM406_1 proteins and each similar protein or peptide may share at least some
activity.
Clone "EH189 1"
A polynucleotide of the present invention has been identified as clone
"EH189_1".
2 0 EH189_1 was isolated from a human adult blood (peripheral blood
mononuclear cells
treated in z~iz~o with granulocyte-colony stimulating factor) 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. EH189_1 is a full-
length
2 5 clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "EH189_1 protein")
The nucleotide sequence of EH189_1 as presently determined is reported in SEQ
ID N0:15. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the EH189_1 protein corresponding to the
foregoing
3 0 nucleotide sequence is reported in SEQ ID N0:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
EH189_1 should be approximately 1450 bp.
The nucleotide sequence disclosed herein for EH189_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
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FASTA search protocols. EH189_1 demonstrated at least some similarity with
sequences
identified as AA681168 (vr75f04.s1 Knowles Softer mouse 2 cell Mus musculus
cDNA
clone 1134559 5'). Based upon sequence similarity, EH189_1 proteins and each
similar
protein or peptide may share at least some activity.
Clone "EH203 2"
A polynucleotide of the present invention has been identified as clone "EH203
2".
EH203_2 was isolated from a human adult blood (peripheral blood mononuclear
cells
treated in vivo with granulocyte-colony stimulating factor) 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. EH203_2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "EH203 2 protein")
The nucleotide sequence of EH203_2 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 EH203 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:18. Amino acids 1 to 84 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
2 0 amino acid 85, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
EH203_2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for EH203_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. EH203_2 demonstrated at least some similarity with
sequences
identified as AA482321 (ab15c05.r1 Stratagene lung (#937210) Homo Sapiens cDNA
clone
840872 5'), H56345 (yq97bI2.s1 Homo Sapiens cDNA clone 203711 3'), T92363
(ye19h06.s1
Homo Sapiens cDNA clone 118235 3'). Based upon sequence similarity, EH203_2
proteins
and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones BI164_l, BK445_l, BPI01 2, CD124 3, CW924_l, DF518_3, DM406_l,
EH189_l, and EH203_2 were deposited on January 8, 1997 with the American Type
Culture Collection as an original deposit under the Budapest Treaty and were
given the
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WO 98130582 PCTIUS98100289
accession number ATCC 98290, from which each clone comprising a particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
deposited material will be irrevocably removed upon the granting of the
patent, except
for the requirements specified in 37 C.F.R. ~ 1.808{b).
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 Fig. 1. 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
vectors in
which they were deposited.
2 0 Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of the
2 5 oligonucleotide probe that was used to isolate each ful I-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
BI164_1 SEQ ID N0:19
3 0 BK445_1 SEQ ID N0:20
BP101 2 SEQ ID N0:21
CD124 3 SEQ ID N0:22
CW924_1 SEQ ID N0:23
DF518_3 SEQ ID N0:24
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DM406_1 SEQ ID N0:25
EH189_1 SEQ ID N0:26
EH203_2 SEQ ID N0:27
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-''P ATP (specific
activity 6000
Ci / mmole) and T4 polynucleotide kinase using commonly employed techniques
for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
2 0 methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 Itl of the stock used to inoculate a sterile culture flask
containing 25 ml
2 5 of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
3 0 ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
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WO 98/30582 PCT/US98/00289
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 flg/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
5 a concentration greater than or equal to le+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
10 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,
1 S hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and
in R.S.
2 0 McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which
are incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valence of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein) such
a fusion
2 5 could be to the Fc portion of an IgG molecule. Other immunoglobulin
isotypes may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
3 0 listing by translation of the nucleotide sequence of each disclosed clone.
The mature form
of such protein may be obtained by expression of the disclosed full-length
polynucleotide
(preferably those deposited with ATCC) in a suitable mammalian cell or other
host cell.
The sequence of the mature form of the protein may also be determinable from
the amino
acid sequence of the full-length form.
27
CA 02276085 1999-06-30
WO 98130582 PCT/US98100289
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
gene (Albert and Morris, 1994, Trends Pharmacol. Sci.15(7): 250-254; Lavarosky
et al., 1997,
Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res.
Mol. Biol. 58: 1-
2 0 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
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
2 5 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
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
3 0 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.
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,
28
CA 02276085 1999-06-30
WO 98130582 PCT/US98100289
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present 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°io) and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60'%~ sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95°~a
identity) with that disclosed protein, where sequence identity is determined
by comparing
2 0 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
(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
2 5 preferably at least 95'% identity) with any such segment of any of the
disclosed proteins.
Species homologs of the disclosed polynucleotides and proteins are also
provided
by the present invention. As used herein, a "species homologue" is a protein
or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
3 0 polynucleotide, as determined by those of skill in the art. Species
homologs may be
isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source from the desired species.
29
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WO 98130582 PCTIUS98/00289
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
which also encode proteins which are identical, homologous, or related to that
encoded
by the polynucleotides .
S The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
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.
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StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)$ Buffer' 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 s 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 T"*; lxSSC Tp*; lxSSC
E RNA:RNA z 50 70C; lxSSC-or- 70C; 0.3xSSC
50C; lxSSC) 50% formamide
F RNA:RNA <50 T, *;1 xSSC TF*;1 xSSC
G DNA:DNA ~ 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50'%
formamide
Fi DNA:DNA <50 T""; 4xSSC TH*; 4xSSC
1 DNA:RNA a 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, SO/ formamide
J DNA:RNA <50 Ti'; 4xSSC T,*; 4xSSC
K RNA:RNA ~ 50 70C; 4xSSC -or- 67'C; lxSSC
50' C: 4xSSC, 50'7
formamide
L RNA:RNA <50 T, *; 2xSSC T,'; 2xSSC
M DNA:DNA ~ 50 50'C; 4xSSC -err- 50C; 2xSSC
40C; 6xSSC, 50,~~
formamide
N DNA:DNA <50 TN*; 6xSSC T~*; 6xSSC
O DNA:RNA ; 50 55C; 4xSSC -or- 55'C; 2xSSC
42C; 6xSSC) 50% formamide
P DNA:RNA <50 T~*; 6xSSC T,.'; 6xSSC
Q RNA:RNA ~ 50 60C; 4xSSC -or- 60' C; 2xSSC
45C; 6xSSC) 50/ formamide
2 R RNA:RNA <SO TH'; 4xSSC TR*; 4xSSC
0
s: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
hybridizing a poiynucleotide to a target polynucleotide of unknown sequence,
the hybrid length ~s assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
t: SSPE (lxSSPE is 0.15M NaCI, lOmM NaHZPO" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is O.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "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 (Naa] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
31
CA 02276085 1999-06-30
WO 98130582 PCT/US98/00289
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce
the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein
"operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
32
CA 02276085 1999-06-30
WO 98130582 PCT/L1S98/00289
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit}, and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987)) incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose0; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively) the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 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
33
CA 02276085 1999-06-30
WO 98/30582 PCTIi3S98/00289
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 transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 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
present invention.
34
CA 02276085 1999-06-30
WO 98/3!1582 PCTII1S98/00289
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
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"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise and-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
3 0 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
CA 02276085 1999-06-30
WO 98/30582 PCTIUS98100289
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L, and A.R. Kimmel
eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 0 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
2 5 can be added to the medium in or on which the microorganism is cultured.
C~okine 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
3 0 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
36
CA 02276085 1999-06-30
WO 98/30582 PCTIUS98I00289
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DAl,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and /or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Imnrunolog~~. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and
Sons, Toronto. 1994.
2 0 Assays for proliferation and differentiation of hematopoieHc and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
2 5 336:690-692) 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 Crrrrent
Protocols irr
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
3 0 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.
37
CA 02276085 1999-06-30
WO 98130582 PCT/US98100289
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.,
). Immunol.
140:508-512, 1988.
Immune Stimulating or Sup_pressin~ Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunode6ciency (SCID)),
e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV ) as well as
bacterial or fungal
2 0 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,
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
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
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,
3 0 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 (parricularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
38
CA 02276085 1999-06-30
WO 98130582 PCTIUS98I00289
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
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
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
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
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
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (c.~., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 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
39
CA 02276085 1999-06-30
WO 98/30582 PCTIUS98I00289
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 ef al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr mice or NZB hybrid
mice)
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
3 0 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
CA 02276085 1999-06-30
WO 98130582 PCT/US98/00289
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate) T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivn 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
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
2 5 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
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
3 0 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
(32
microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
41
CA 02276085 1999-06-30
WO 98130582 PCTIUS98/00289
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
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;
Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 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., ].
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092) 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: ht vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
j.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
42
CA 02276085 1999-06-30
WO 98/30582 PCTIUS98100289
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
3500,1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
S 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 169:1255-12b4, 1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; ltoh et al., Cell 66:233-243, 1991;
Zacharchuk,
journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al.) Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-I22, 1994; Galy et
al., Blood
85:2770-2778) 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity
2 5 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
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,
3 0 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
43
CA 02276085 1999-06-30
WO 98130582 PCT/US98/00289
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytoperua, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivn (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation {homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include)
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
2 0 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
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
2 5 Hematopoietic Celts. R.I. Freshney, et aI. eds. Vol pp. 265-268, Wiley-
Liss) lnc., 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
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et a1. eds.
Vol pp. 23-39)
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
3 0 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,
44
CA 02276085 1999-06-30
WO 98130582 PCT/US98100289
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and / or bone
growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
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
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regenerarion 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
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
3 0 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
CA 02276085 1999-06-30
WO 98130582 PCT/US98/00289
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
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,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 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
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers) ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
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)
3 0 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.
46
CA 02276085 1999-06-30
WO 98/30552 PCTlUS98100289
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 ).
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/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 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
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
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively) the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
(3 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,
3 0 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:
47
CA 02276085 1999-06-30
WO 98/30582 PCT/US98/00289
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
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
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
2 0 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
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376)
1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
48
CA 02276085 1999-06-30
WO 98!30582 PCTIUS98/00289
Hemostatic and Thrombol~tic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders {including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al.) J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991 ); Schaub,
Prostaglandins
35:467-474,1988.
Rece~tor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors)
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include) without limitation, cytokine receptors
and their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
49
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WO 98/30582 PCT/US98J110289
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,
2995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock)
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis)
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
2 0 from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 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
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 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
CA 02276085 1999-06-30
WO 98I3t1582 PCT/US98100289
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 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
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 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
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
3 0 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
51
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WO 98130582 PCTIUS98/00289
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
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
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 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
agents, including) without limitation, bacteria) viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 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;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 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
52
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WO 98130582 PCTIUS98100289
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 earner)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6) IL-7, IL-8, IL-
9, IL-10, IL-11,
2 0 IL-12, IL-13, IL-14, IL-15) IFN, TNFO, TNFl, 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
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
2 5 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
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
3 0 A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
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WO 98/30582 PCT/LTS98100289
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.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined) in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
2 0 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.
As used herein, the term "therapeutically effective amount" means the total
2 5 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
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
3 0 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
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CA 02276085 1999-06-30
WO 98/30582 PCTIUS98100289
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
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
composition of the invention may additionally contain a solid carrier such as
a gelatin or
2 0 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form) a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil) soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
2 5 physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
of the present invention) and preferably from about 1 to 50% protein of the
present
invention.
3 0 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
CA 02276085 1999-06-30
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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
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 ug to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
2 0 present invention will vary, depending on the severity of the disease
being treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
2 5 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
3 0 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 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
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CA 02276085 1999-06-30
WO 98/30582 PCTIUS98l00289
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
2 0 developing bone and cartilage and optimally capable of being resorbed into
the body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
2 5 application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
3 0 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-
57
CA 02276085 1999-06-30
WO 98130582 PGTIUS98/00289
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), 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
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
2 0 In further compositions, proteins of the invention may be combined with
other
agents beneficial to the treatment of the bone and /or cartilage defect,
wound, or tissue in
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGF-Vii), and insulin-like growth factor (IGF).
2 5 The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses) in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
3 0 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
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CA 02276085 1999-06-30
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the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and / or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced iri 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.
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
fE) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A> APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 719 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
CA 02276085 1999-06-30
WO 98/30582 PCTILTS98/00289
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:1:
GCTGTTTGTA TTTTGGCTGC TGCAGGAGCCATTTTAGAAATAAATATCTT CCTTCAATAG60
ATGAAAATGA AAATACAGAA AAAAGAGAAGCAGTTGTCAAATTTAAAAGT TTTGAATCAC120
TCCCCAATGT CTGATGCCTC TGTCAATTTTGACTACAAATCTCCATCCCC ATTTGACTGC180
AGCACTGATC AAGAAGAGAA AATTGAAGATGTTGCTAGTCACTGTCTGCC TCAGAAGGAC240
CTGTATACTG CTGAAGAGGA AGCTGCTACCCTTTTTCCTAGGAAAATGAC ATCCCATAAT300
GGGATGGAGG ACAGTGGAGG AGGAGGTACTGGAGTGAAGAAGAAACGGAA GAAAAAGGAG360
CCAGGAGACC AAGAGGGTGC AGCAAAGGGAAGCAAGGACAGAGAGCCCAA GCCAAAGAGG420
AAACGAGAAC CGAAAGAGCC AAAGGAACCCAGAAAGGCCAAGGAGCCGAA GAAGGCCAAG480
GAGCACAAGG AGCCGAAGCA AAAAGATGGGGCAAAGAAGGCACGGAAGCC CCGGGAGGCC540
TCGGGCACCA AGGAGGCCAA AGAGAAGAGGAGCTGCACTGACTCTGCAGC CAGGACGAAG600
TCCAGGAAGG CCAGCAAGGA GCAAGGACCAACCCCAGTGGF~~AAAAAAAA F,~~~AAAAAAA660
1~AAAAAAp,Ap, AAAAAAAAAA A,F~~AAAAAAAA,~~AAAP~AA P,AAAAAAAA719
P,F~APA
(2) INFORMATION FOR SEQ :
ID N0:2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Met Lys Ile Gln Lys Lys Glu Lys Gln Leu Ser Asn Leu Lys
1 5 10 15
Val Leu Asn His Ser Pro Met Ser Asp Ala Ser Val Asn Phe Asp Tyr
20 25 30
Lys Ser Pro Ser Pro Phe Asp Cys Ser Thr Asp Gln Glu Glu Lys Ile
35 40 45
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Glu Asp Val Ala Ser His Cys Leu Pro Gln Lys Asp Leu Tyr Thr Ala
50 55 60
Glu Glu Glu Ala Ala Thr Leu Phe Pro Arg Lys Met Thr Ser His Asn
65 70 75 80
Gly Met Glu Asp Ser Gly Gly Gly Gly Thr Gly Val Lys Lys Lys Arg
85 90 95
Lys Lys Lys Glu Pro Gly Asp Gln Glu Gly Ala Ala Lys Gly Ser Lys
100 105 110
Asp Arg Glu Pro Lys Pro Lys Arg Lys Arg Glu Pro Lys Glu Pro Lys
115 120 125
Glu Pro Arg Lys Ala Lys Glu Pro Lys Lys Ala Lys Glu His Lys Glu
130 135 140
Pro Lys Gln Lys Asp Gly Ala Lys Lys Ala Arg Lys Pro Arg Glu Ala
145 150 155 160
Ser Gly Thr Lys Glu Ala Lys Glu Lys Arg Ser Cys Thr Asp Ser Ala
165 170 175
Ala Arg Thr Lys Ser Arg Lys Ala Ser Lys Glu Gln Gly Pro Thr Pro
180 185 190
Val Glu
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2584 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
AGTGATTCTCCTGCCTCAGC CTCCCGAGTAGGTGGGATTATAGGCATGTG CCACTGTGCC60
CGGTTTATTTTTGTATTTTT AATAGAAATGGGGTTTCACCAGGCTGGTCT TGAACTCCTG120
ACCTCAAGTGATCCACCCGC CTCAGCCTCTCAAGGGACTAGGATTACAGG CAAGAGCCAC180
TGCGCCTGGCCTCTTTTAAC CATTTTGAATTGACTTTTGTGGATGGGGTA AGATAAGGGT240
CCAATTTCATTTCTTGGCAT GTGGTTATCTAGCTTTTTCAACACCATTTA TTAAAGAGAC300
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TATCCTTTCT TTATTGTGTACTTTTGACACCTTTGTTGATGATTAGTTGGCCACATAAAC360
GAGGATTTAT TTCTGGGATTCCTATTCTGTTCCATTGGTCTATATGTCTCCTTTTATGCC420
AGTACCATAC AGTTTTGATTACTGTAGGTCTGTAATATAATTTGAAATGAAGTGTGATGC480
CACCAGCTTT GTTCTTCTTGCTCAGGATCATTTTAGATATTCTGGGTCTTTTGGGTTCTG540
TATGGATGTT AGGAATTTTTTTTTTTTTTCTGTGAAAAATGTCATTGGAGTTTTGCTAGA600
GATTGCGTTG AATCTGTAGACTACTTTGTGTATTGCAGACATTTTGACAATAATGATTCT660
TCTGATCCTG AACATGGGATAGCTTTCCATTTATTTGTGTCATCTCCAGTTCTTTCATAT720
ATGTATTATA GCTTTTAGCATACACATCTTTCCCCTCCTTGGTAAAACTTATTCCTAAGT780
ATTTTATTCC TTTTGATGCTATTGTAAATGGGATTGCTTTCTTAATTTCTTTCTTGGATAB40
CTTCACTGTT AGTGAAGTGTAATTGATTTTTGTATGTTGATTTTGTATCCTGAAACTTTA900
CTGGATTCGT TTATTCATTCTAACAGTTTTTAGTGGCATATTTAGGGTTTTCTGTATATA960
GGATGATGTC ACCTGCAGAGACAATTTTACTTCTTCCTTTCTGATTTGGATGCCTTTTAT1020
TTCTTTTCCT TGCCTAATTTCTGTGGCTAGGATTTCTAGTACTATGTTGAATGTAAGTGA1080
TAAGTGTGGG CATCCTTGTCTGTCCTTGTCACATTCAGTTGTATGTGCAATACTGGAACC1140
ATCCAGCTCA TAGTCACGTAATTCCTCTTTGTGCAGCCTCATGTAAAATCACACCCCTAT1200
TCACATTTTT TTCTGTCTGTCTCCCACCTCTCCAGGTCCCTGCCCTGCAACTTTCAGTTA1260
CTTCAGACTC TCAGAAGACTAGCCTCTAAATACCTCAATTTATCTGCACTACTGTGGTC~.'1320
ACTTGAAATT CTCTTCCCTCTTCTGCAGT':'TGTAATGGCATTCAGGCCAAAAACTGTAG';380
AGTTTTAGGG CTCTCTTCATTTGCTTCTCTGCTCCTAGGGATCATAGTGCTGTGCTATCT1440
GTTGTCCAGC ATCTGACACAGTTGGTTTTATATTTTGTCTGGTTTTCTAGTTATTAAGCA1500
GGTTAAGTCT GGTTCCTGCTGTTCTATATAGCAGGAAGCAAAGG:'TCTCTCATGAATTT~.'156C
TAGCATAATA ACATAAGCGTGGATTCCAAAAATTCATTACACAGATCCTGGAAATCTGT".'1620
TACTATGTTT TATACATTCTGTGAAAGCTGGAGAATATATATTTGTTTTACTATGTGTAT1680
CGCAAATAAA ATATCAATTTCATGTGCCAGAGAATTGAATACATTATCAAAGGGTCATTT1740
CATAACGTTA TAATCACCATCCATCCTGTAAGTCCTTTGA TTTCTATGGA1800
AATTTCTTCT
TCTAACATTG ATCAATAACGAATTCCTAAGCATCTATATTAGTTAGCCCAGTGTTTCTCA1860
AAGTCCACCG CATGTAAAAATCATCTAGGGAATCTTTCTA TCTGATTTAG1920
AAATGTGGAT
TAGGTCTGGG GTGAGGTCTTCCCTTTTGCAATTCCAGCAA GATGCTGGTA1980
GTTCCCATGG
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CTGGTTAATG AAGTCACTTA GAGTAGCAAG GCCTAGTATT TACAATGAGC2040
GAGCCTGTTT
ACGTTTTGGG TATCATCAAA ACAAGAGAAT ATGGTGTGGG TGGAAGGTTG2100
CTAAAAATGT
TGTTACTTGA TATTTCAAAA AGATCATTGA TCTCAGAGAT ATGTTGGATT2160
AATCAGAAAA
TCATCACATT TATTCTATGG CTTAATGTTG AATATATAAT AAGAGAGATA2220
TTTTATTTTG
CCACAAATCT TTAGCTATTA ATATTTAGGG CCTTAATTCA TATTGTCTCA2280
ATGTTAATGT
AATGTGATTC ATTCCCTCTA TAGGTATACT TTCATGCCTA CAAAACCATC2340
AAAACAGGCA
TTCCTAACTT TCTTTCCTCT TTTTAGTCTC AGTGACAGTG GTTCTCAAAC2400
TTCCTGAACC
ATTTTGGTCT GAGGACCCCT TTATGGCCTT GAAGAGCTTT TATTTATGAA2460
AAAAATGATC
GGTAATACCT GCTAATACTT GTTGTAATCT AGAAATGTAA AAATTTTTAT2520
ATTAACATTG
TAATTCACTT AAAATATCAA TAAACCCATT P~~~AAAAAAA P,~~AAAAAAAA2
ATTTGCTAAT 5
8
0
AAAA 2584
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Phe Tyr Thr Phe Cys Glu Ser Trp Arg Ile Tyr Ile Cys Phe Thr
1 5 10 15
Met Cys Ile Ala Asn Lys Ile Ser Ile Ser Cys Ala Arg Glu Leu Asn
20 25 30
Thr Leu Ser Lys Gly His Phe Ile Thr Leu
35 40
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2233 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:5:
CTTTCCCATC TTTTGCAAAGAAACTTTTGATCTCTCTGTG CAAAGATGTTCCCTTTCAAG60
TTAAGTGTGT GGCCTGCCACAAGACACTGCGTTCCCACAT GGAGCTCACTGCCCATTTCA120
GAGTTCATTG TCGAAATGCTGGACCTGTAGCTGTAGCTGA GAAGAGCATTACCCAGGTTG180
CAGAGAAATT CATATTAAGAGGTTATTGTCCAGATTGCAA TCAAGTCTTTGTGGATGAAA240
CCAGCACCCA AAATCATAAGCAGAATTCAGGACACAAAGT CCGAGTCATTAACTCAGTGG300
AAGAATCAGT CTTACTCTATTGCCACAGCAGCGAAGGGAA CAAGGATCCTTCTTCTGACT360
TGCATTTATT GTTGGATCAATCAAAATTTTCATCACTTAA AAGAACCATGTCTATTAAAG420
AATCTAGCTC ACTGGAGTGCATTGCCATTCCAAAAAAGAA GATGAATTTAAAAGATAAAA480
GCCATGAAGG TGTTGCTTGTGTCCAGAAAGAAAAATCAGT AGTTAAAACCTGGTTCTGTG540
AATGCAATCA GCGATTCCCAAGTGAAGATGCAGTAGAAAA GCATGTTTTCTCAGCAAACA600
CAATGGGTTA TAAATGTGTGGTCTGTGGAAAGGTATGTGA TGATTCAGGGGTCATTCGTT660
TACACATGAG CCGGATTCACGGAGGGGCACATTTAAATAA CTTTCTTTTCTGGTGTCGGA720
CATGCAAAAA GGAGTTAACAAGGAAAGATACTATCATGGC ACATGTGACTGAATTTCATA780
ATGGACACAG ATATTTTTATGAGATGGATGAGGTAGAAGG TGAAACTTTGCCATCATCCT84C
CTACAACATT GGATAATTTGACTGCTAACAAGCCTTCATC AGCTATTACTGTTATTGATC900
ATTCCCCGGC AAATAGTTCTCCGAGGGGTAAATGGCAATG CCGGATTTGTGAAGATATGT960
TTGATTCCCA GGAATATGTAAACAGCACTGCATGTCTTTG GCAAGCCACAAGTTTCATAG102C
ATACAGCTGT GCTCACTGCAGAAAGCCTTTTCATAAGATA GAAACATTGTACCGACATTGlOBC
CCAAGATGAG CATGACAATGAGATAAAGATTAAATACTTC TGTGGGCTTT.GTGATCTTAT1140
CTTTAATGTG GAAGAAGCATTTTTGAGTCATTATGAGGAG CACCACAGCATAGATTATGT2200
ATTTGTGTCA GAAAAAP.CTGAAACTTCAATTAAAACCGAA GATGATTTTCCAGTAATAGA1260
GACCAGTAAC CAGTTAACTTGTGGTTGCCGTGAGAGTTAC ATCTGTAAAGTCAACAGAAA1320
AGAAGATTAT AGCAGATGTCTCCAAATCATGCTGGATAAA GGAAAACTGTGGTTTCGCTG1380
CAGTTTATGT TCGGCAACAGCACAGAATTTAACCGACATG AACACTCATATCCATCAAGT1440
GCACAAAGAA AAGAGTGATGAGGAGGAGCAGCAGTATGTA ATCAAGTGTGGCACCTGCAC1500
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CAAAGCATTT CATGATCCTG AGAGTGCACA CATAGAAAAC ATTGCTTCTT1560
GCAGCATTTC
ACAGAAACCC AGTGTGGCTC ATTTTGGATC AACCTGTACA AGTTTACTGC1620
TGAAAAATCA
TAGTGCCTCA CATACAGAGA GAAAACTGAA AACTATTCAA AAAGTTTAGA1680
ACAGGCAATA
CATGGAGAAA GGAGTTGAGA ATGACCTAAG ATAGAGGAAG AAATTGTTGA1740
CTATCAGAAT
GCTTCCAGAT TTGGATTACC TGCGAACCAT GTCTTTGTAG ATTTTGATAA1800
GACTCATATA
CTGGTCAAAC TTTTTTGGTC ATCTACCAGG CAAGGAACAT TTATTTGGGG1860
GCATCTAAAC
CTTTCAAGGT ACGGTTAATA AGAAAAACAA TTTCCCACCT CTTAGAATAT1920
AAGAAAACTT
AGTTCAGTTT AAAAGGCTCC TCTTAAACCT AACATAGGAA CATAATTGGA1980
TCCTGGATAG
ATATTGTTCT TTTGTATTAT AGTTACTCTG TTTCTTTACA TGCCTCTGTG2040
CTAGAATCGT
GTAAATGTTA GCTCTATCTG GTGACTCAAA CTTTTGGCCG GGCGCGGTGG2100
GTTTATGGAT
CTCACGTCTG TAATCCCAGA ACTTTGGAAG ACGGATCACG AGGTCAGAAG2160
GCTGAGGCGG
ATCGAGACCA TCCTGGCTAA CATGGTGAAA ACTAAAAAAA P~~AAAAAAAA2220
CCCCGTCTCT
AAAAAAAAAA AAA 2233
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 320 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Glu Leu Thr Ala His Phe Arg Val His Cys Arg Asn Ala Gly Pro
1 5 10 15
Val Ala Val Ala Glu Lys Ser Ile Thr Gln Val Ala Glu Lys Phe Ile
20 25 30
Leu Arg Gly Tyr Cys Pro Asp Cys Asn Gln Val Phe Val Asp Glu Thr
35 40 45
Ser Thr Gln Asn His Lys Gln Asn Ser Gly His Lys Val Arg Val Ile
50 55 60
Asn Ser Val Giu Glu Ser Val Leu Leu Tyr Cys His Ser Ser Glu Gly
65 70 75 80
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Asn Lys Asp Pro Ser Ser Asp Leu His Leu Leu Leu Asp Gln Ser Lys
85 90 95
Phe Ser Ser Leu Lys Arg Thr Met Ser Ile Lys Glu Ser Ser Ser Leu
100 105 110
Glu Cys Ile Ala Ile Pro Lys Lys Lys Met Asn Leu Lys Asp Lys Ser
115 120 125
His Glu Gly Val Ala Cys Val Gln Lys Glu Lys Ser Val Val Lys Thr
130 135 140
Trp Phe Cys Glu Cys Asn Gln Arg Phe Pro Ser Glu Asp Ala Val Glu
145 150 155 160
Lys His Val Phe Ser Ala Asn Thr Met Gly Tyr Lys Cys Val Val Cys
165 170 175
Gly Lys Val Cys Asp Asp Ser Gly Val Ile Arg Leu His Met Ser Arg
180 185 190
Ile His Gly Gly Ala His Leu Asn Asn Phe Leu Phe Trp Cys Arg Thr
195 200 205
Cys Lys Lys Glu Leu Thr Arg Lys Asp Thr Ile Met Ala His Val Thr
210 215 220
Glu Phe His Asn Gly His Arg Tyr Phe Tyr Glu Met Asp Glu Val Glu
225 230 235 240
Gly Glu Thr Leu Pro Ser Ser Ser Thr Thr Leu Asp Asn Leu Thr Ala
245 250 255
Asn Lys Pro Ser Ser Ala Ile Thr Val Ile Asp His Ser Pro Ala Asn
260 265 270
Ser Ser Pro Arg Gly Lys Trp Gln Cys Arg Ile Cys Glu Asp Met Phe
275 280 285
Asp Ser Gln Glu Tyr Val Asn Ser Thr Ala Cys Leu Trp Gln Ala Thr
290 295 300
Ser Phe Ile Asp Thr Ala Val Leu Thr Ala Glu Ser Leu Phe Ile Arg
305 310 315 320
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1285 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:7:
GGAAAGCCATTTCTAGCACA GAAGCAGTTC TAAACAACCGATTCATTCGA GTCTTGTGGC60
ATAGGGAAAATAATGAGCAA CCGACACTAC AGTCCTCAGCACAGCTGCTC CTGCAACAAC120
AGCAAACACTTAGTCACCTC TCACAGCAGC ACCATCACCTGCCACAGCAT CTACATCAGC180
AGCAGGTGCTAGTGGCCCAG TCTGCTCCTT CAACAGTGCACGGAGGTATC CAGAAGATGA240
TGAGCAAACCACAGACATCA GGTGCATATG TTCTTAACAAAGTTCCTGTT AAACATCGTC300
TTGGACATGCAGGTGGTGCT GGAGAAGATT GCCAGATATTTTCAACTCCA GGCCATCCAA360
AAATGATTTACAGCTCCTCA AACTTAAAGA CACCTTCAAAGCTCTGTTCA GGGTCTAAAT420
CTCATGATGTTCAAGAAGTG CTTAAAAAAA AACAGGAAGCAATGAAGTTA CAACAAGATA480
TGAGGAAAAAAAGACAGGAA GTGTTAGAAA AGCAAATAGAATGCCAAAAG ATGTTAATAT540
CCAAGTTAGAAAAAAACAAA AACATGAAAC CAGAAGAAAGAGCAAATATA ATGAAGACTT600
TGAAAGAGCTTGGAGAGAAG ATCTCACAAT TAAAAGATGAATTAAAAACA TCTTCTGCAG660
TCTCCACACCATCTAAAGTG AAGACAAAAA CGGAGGCCCAGAAGGAGTTA TTAGATACTG720
AACTGGACCTCCACAAGAGG CTGTCCTCAG GAGAAGACACCACAGAATTA CGGAAAAAAC780
TCAGTCAGTTACAGGTTGAG GCTGCACGGT TAGGTATTTTACCTGTGGGT CGAGGAAAGA840
CCATGTCCTCTCAAGGTCGA GGAAGAGGCC GAGGGCGTGGAGGAAGAGGA AGGGGCTCAC900
TAAATCACATGGTGGTGGAC CATCGTCCCA AAGCACTAACAGTTGGAGGA TTCATTGAGG960
AAGAAAAAGAAGACTTGCTT CAGCATTTCT CAACCGCAAACCAAGGGCCA AAATTTAAAG1020
ACCGTCGGCTACAGATATCA TGGCACAAGC CCAAGGTACCATCTATATCC ACTGAGACTG1080
AAGAAGAAGAAGTCAAGGAG GAGGAAACAG AAACCTCAGATTTGTTTTTG CCTGATGATG1140
ACGATGAAGATGAAGATGAA TATGAGTCTC GCTCATGGCGAAGATGAAAT CTGATGCTAG1200
CTGTATAATTTTTAGGAATA TTGTTTAGAA GAACAACTTTTAAAAATTAT TTAAAAGAAG2260
TCAATGAGCCP~~AAAAAAAA AAAAA 1285
(2) INFORMATION
FOR SEQ
ID N0:8:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Met Ser Lys Pro Gln Thr Ser Gly Ala Tyr Val Leu Asn Lys Val
1 5 10 15
Pro Val Lys His Arg Leu Gly His Ala Gly Gly Ala Gly Glu Asp Cys
20 25 30
Gln Ile Phe Ser Thr Pro Gly His Pro Lys Met Ile Tyr Ser Ser Ser
35 40 45
Asn Leu Lys Thr Pro Ser Lys Leu Cys Ser Gly Ser Lys Ser His Asp
50 55 60
Val Gln Glu Val Leu Lys Lys Lys Gln Glu Ala Met Lys Leu Gln Gln
65 70 75 80
Asp Met Arg Lys Lys Arg Gln Glu Val Leu Glu Lys Gln Ile Glu Cys
85 90 95
Gln Lys Met Leu Ile Ser Lys Leu Glu Lys Asn Lys Asn Met Lys Pro
100 105 110
Glu Glu Arg Ala Asn Ile Met Lys Thr Leu Lys Glu Leu Gly Glu Lys
115 120 125
Ile Ser Gln Leu Lys Asp Glu Leu Lys Thr Ser Ser Ala Val Ser Thr
130 135 140
Pro Ser Lys Val Lys Thr Lys Thr Glu Ala Gln Lys Glu Leu Leu Asp
145 150 155 160
Thr Glu Leu Asp Leu His Lys Arg Leu Ser Ser Gly Glu Asp Thr Thr
165 170 175
Glu Leu Arg Lys Lys Leu Ser Gln Leu Gln Val Glu Ala Ala Arg Leu
180 185 190
Gly Ile Leu Pro Val Gly Arg Gly Lys Thr Met Ser Ser Gln Gly Arg
195 200 205
Gly Arg Gly Arg Gly Arg Gly Gly Arg Gly Arg Gly Ser Leu Asn His
210 215 220
Met Val Val Asp His Arg Pro Lys Ala Leu Thr Val Gly Gly Phe Ile
225 230 235 240
Glu Glu Glu Lys Glu Asp Leu Leu Gln His Phe Ser Thr Ala Asn Gln
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245 250 255
Gly Pro Lys Phe Lys Asp Arg Arg Leu Gln Ile Ser Trp His Lys Pro
260 265 270
Lys Val Pro Ser Ile Ser Thr Glu Thr Glu Glu Glu Glu Val Lys Glu
275 280 285
Glu Glu Thr Glu Thr Ser Asp Leu Phe Leu Pro Asp Asp Asp Asp Glu
290 295 300
Asp Glu Asp Glu Tyr Glu Ser Arg Ser Trp Arg Arg
305 310 3I5
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2346 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii1 MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:9:
CACGCGAGGCGCTGTCCTTTCAGCACCACAAGCTCGGGCT~:~AGGAGGGhGGACTCCTGGC60
CGTCCTCCTCCTCTTCAAATTGGCTTGAATCTGCTCTGACCCCCCACGAGTGCAGCACAG120
TCTGGGAAGAAAGGCGTAAGGATGGTGAAGCTGAACAGTAACCCCAGCGAGAAGGGAACC180
AAGCCGCCTTCAGTTGAGGATGGCTTCCAGACCGTCCCTCTCATCACTCCCTTGGAGGTT240
AATCACTTACAGCTGCCTGCTCCAGAAAAGGTGATTGTGAAGACAAGAACGGAATATCAG300
CCGGAACAGAAGAACAAAGGGAAGTTCCGGGTGCCGAAAATCGCTGAATTTACGGTCACC360
ATCCTTGTCAGCCTGGCCCTAGCTTTCCTTGCGTGCATCGTGTTCCTGGTGGTTTACAAA920
GCCTTCACCTATGATCACAGCTGCCCAGAGGGATTCGTCTATAAGCACAAACGCTGTATC480
CCAGCCTCCCTGGATGCTTACTACTCCTCCCAGGACCCCAATTCCAGAAGCCGCTTCTAC540
ACAGTCATCAGCCACTACAGCGTGGCCAAGCAGAGCACTGCCCGGGCCATCGGGCCGTGG600
CTGTCAGCAGCCGCTGTCATCCATGAGCCCAAGCCGCCCAAGAACCCAGGGCCACTAAAG660
GCCTGCCCCAGCCAAAATGGGGGGCGGGGTGGAAAGGAGGACCCCCATTGGCTAACCCAA720
GCTCCAGTTACAAAACAACACTGTACTCCTGGGATATGGGGGCGGGGGCGGGGCAGGGCA780
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GGGTGGGGGG AAAAACGCAC CAAAAACGTGGTGTGTGCTGGAGTTGTCTG 840
AACCGATATT
TCTTTTTGTT CCTTGGTATTGTTGATTCGTCGCCGAGTCAGGCTCATGTACAAAGGCATG900
TTTCGTGTTG ATTGTTCCCATGTAAGATATTTTTAAAGCCACTGCTTATTCTTTGTTAGG960
AAAATGTAAC AGCAGAAAAGGAAAGAAACAAAGAACATGAACAAAAAGCATTAAACTGGC1020
TCCATCAGAA GACGTTGAAGGGCAGTGAAGAGCACAGACTCTGTGGGCTTCTTAGATAAG1080
AAAACGTAGC TTCAGTGGGGGCTCCAGGGTTGCAGAGTATGAGTGACACAGACCGGGACT1140
ATTCCATTAG CCTGTGGTCTGCAGGGTAGGCCCGCAGGAAATGAGGAATGGCCGAGCTGG1200
AGAGAAGAGC TGATTTTGGCATTACTAAGCCCAGAACGCACATAACCCATAGTGAAATGT1260
GCTGGCCTCT GGTGCATTTTGCAAGATGAGCACAAACTTTCTGGGCCTCCATCCTAGGAC1320
CTGGGCAGAC CCACATGGCCTGGGCTTTGAATGCCCACCCTGCGACGGTGGGTTTTGCAT1380
CAGCAAACGC TGAGGAGTGGGCAGATTTTTTTTGTTTTTTGCTTGCATTTTTTAGATCCA1440
CACCTGGATA CTGCCCATGTTGACGAGACAGCAGCAGGGGGAGAGGGAGGGAAGGAAGGT1500
GCGGCTGCAA GAAGGAAGGCACGGGACAGGCATGTGACACTAGGCCACAAGCGATAAGCA1560
CAGGCACCTG ACTTTTAAGTTTTTGTTTGTTTGTTGTTTCCCAAAGTGCTGATAACAATA1620
ACAACAACAA TAGGATTCCAACCAGGAGCCTCAAGTGACAGCCAGGAAGAGACCTGAAGG1680
TTGGGGCCAC CACAATGCCAAATCGTTTCTAAAGGAAGCTGAAAAATGGGACTGTCTTTT1740
GCCCAC'~TCG TTGTGTTAAAAGGGGACATTTGTCCAAACTCCCCAACCGAGTTTTAGAAG1800
CTCCTGACAA GGAGGCAGCATCCAGCCTTGACCAGGCCTCCCAGTTCCCTGGAACCGTAT1860
CAGGCATTCG CCTGCCTCTCACAAATGTTTCAGGGAGGCCAGTTCTGCAGGGTGTCAGCT1920
CCAGGACCCA CAGGGCCAGAACCAGCTGGGAGAATTGGTTATTTGAGATGTGGTACTGCT1980
TCCTCACAAG TCTCCCACAGGCCATGTAAAGGGTATTTTTTTGTGGCTTGCTGTGTTGCT204C
GAGATCATCG TATGCAACAGCTGGGTAATAAGACTAGCATAGCTCAAACTATCCTGCCAA2100
ACGCTCTCAT ATGATTTTTCCTCCCTTCTCCCCCAACCTCCAATCACCCTGAGTCACCTG2160
TAAATTCATT TGTCATTCAAAGCGGAATAACAAGTTGTCCCTAGCAAAACCGCTGAGCGC2220
TTTATAATTT TGTGGTGTATTTTTGTCAGTAGGTAGCAGAGGCGGAAGTATTTTTTGGTG2280
TAATTCTTGA
AATTTTCTGACAGGAAACAAATAAAGATAGATGTGTCTGAGP~AAAAAAAA2340
AAAAAA 2346
(2? INFORMATION FOR SEQ 0:
ID NO:1
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(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 229 amino acids
(B) TYPE: amino acid
{C) STRANDEDNESS:
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Met Val Lys Leu Asn Ser Asn Pro Ser Glu Lys Gly Thr Lys Pro Pro
1 5 10 15
Ser Val Glu Asp Gly Phe Gln Thr Val Pro Leu Ile Thr Pro Leu Glu
20 25 30
Val Asn His Leu Gln Leu Pro Ala Pro Glu Lys Val Ile Val Lys Thr
35 40 45
Arg Thr Glu Tyr Gln Pro Glu Gln Lys Asn Lys Gly Lys Phe Arg Val
5C 55 60
Pro Lys Ile Ala Glu Phe Thr Val Thr Ile Leu Val Ser Leu Ala Leu
65 70 75 80
Ala Phe Leu Ala Cys Ile Val Phe Leu Val Val Tyr Lys Ala Phe Thr
85 90 95
Tyr Asp His Ser Cys Pro Glu Gly Phe Val Tyr Lys His Lys Arg Cys
100 105 110
Ile Pro Ala Ser Leu Asp Ala Tyr Tyr Ser Ser Gln Asp Pro Asn Ser
115 120 125
Arg Ser Arg Phe Tyr Thr Val Ile Ser His Tyr Ser Val Ala Lys Gln
130 135 140
Ser Thr Ala Arg Ala Ile Gly Pro Trp Leu Ser Ala Ala Ala Val Ile
145 150 155 160
His Glu Pro Lys Pro Pro Lys Asn Pro Gly Pro Leu Lys Ala Cys Pro
165 170 175
Ser Gln Asn Gly Gly Arg Gly Gly Lys Glu Asp Pro His Trp Leu Thr
180 185 190
Gln Ala Pro Val Thr Lys Gln His Cys Thr Pro Gly Ile Trp Gly Arg
195 200 205
Gly Arg Gly Arg Ala Gly Trp Gly Glu Lys Arg Thr Lys Asn Val Val
210 215 220
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Cys Ala Gly Val Val
225
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2104 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:11:
GGATGAGTCT CAAGATGGAC AACCGGGATGTTGCAGGAAAGGCTAACCGGTGGTTTGGGG60
TTGCTCCCCC TAAATCTGGA AAAATGAACATGAACATCCTTCACCAGGAAGAGCTCATCG120
CTCAGAAGAA ACGGGAAATT GAAGCCAAAATGGAACAGAAAGCCAAGCAGAATCAGGTGG180
CCAGCCCTCA GCCCCCACAT CCTGGCGAAATCACAAATGCACACAACTCTTCCTGCATTT240
CCAACAAGTT TGCCAACGAT GGTAGCTTCTTGCAGCAGTTTCTGAAGTTGCAGAAGGCAC300
AGACCAGCAC AGACGCCCCG ACCAGTGCGCCCAGCGCCCCTCCCAGCACACCCACCCCCA360
GCGCTGGGAA GAGGTCCCTG CTCATCAGCAGGCGGACAGGCCTGGGGCTGGCCAGCCTGC420
CGGGCCCTGT GAAGAGCTAC TCCCACGCCAAGCAGCTGCCCGTGGCGCACCGCCCGAGTG480
~.CTTCCAGTC CCCTGACGAG GACGAGGAGGAGGACTATGAGCAGTGGCTGGAGATCAAAG540
TTTCACCCCC AGAGGGAGCC GAGACTCGGAAAGTGATAGAGAAATTGGCCCGCTTTGTGG600
CAGAAGGAGG CCCCGAGTTA GAAAAAGTAGCTATGGAGGACTACAAGGATAACCCAGCAT66U
TTGCATTTTT GCACGATAAG AATAGCAGGGGATTCCTCTACTACAGGAAGAAGG':GGCTG7~~
AGATAAGAAA GGAAGCACAG AAGTCGCAGGCAGCCTCTCAGAAAGTTTCACCCCCAGAGG780
ACGAAGAGGT CAAGAACCTT GCAGAAAAGTTGGCCAGGTTCATAGCGGACGGGGGTCCCG840
AGGTGGAAAC CATTGCCCTC CAGAACAACCGTGAGAACCAGGCATTCAGCTTTCTGTATG900
AGCCCAATAG CCAAGGGTAC AAGTACTACCGACAGAAGCTGGAGGAGTTCCGGAAAGCCA960
AGGCCAGCTC CACAGGCAGC TTCACAGCACCTGATCCCGGCCTGAAGCGCAAGTCCCCTC1020
CTGAGGCCCT GTCAGGGTCC TTACCCCCAGCCACCACCTGCCCCGCCTCGTCCACGCCTG1080
CGCCCACTAT CATCCCTGCT CCAGCTGCCCCCGGGAAGCCAGCCTCCGCAGCCACCGTGA1140
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AGAGGAAGCGGAAGAGCCGG TGGGGGCCTG 1200
AAGAGGATAA GGTAGAGCTC
CCACCTGCTG
AACTGGTGCAGAGGGACGTG GATGCCTCTCCCTCGCCTCTGTCAGTTCAG GACCTCAAGG1260
GGCTCGGCTATGAGAAGGGG AAGCCTGTGGGTCTAGTGGGCGTCACAGAG CTTTCAGACG1320
CCCAGAAGAAGCAGCTGAAG GAGCAGCAGGAGATGCAGCAGATGTACGAC ATGATCATGC1380
AGCACAAGCGGGCCATGCAG GACATGCAGCTGCTGTGGGAGAAGGCAGTC CAACAGCACC1440
AGCACGGCTATGACAGTGAT GAGGAGGTGGACAGCGAGCTGGGCACCTGG GAGCACCAGC1500
TGCGGCGCATGGAGATGGAT AAGACCAGGGAATGGGCCGAGCAGCTGACA AAGATGGGCC1560
GGGGCAAGCACTTCATCGGA GACTTCCTGCCTCCAGACGAGCTGGAAAAG TTTATGGAGA1620
CCTTCAAGGCCCTGAAGGAG GGCCGTGAGCCTGACTACTCAGAGTACAAG GAGTTCAAGC1680
TGACTGTGGAGAACATCGGC TACCAGATGTTGATGAAGATGGGCTGGAAG GAGGGCGAGG1740
GGCTGGGCTCAGAGGGCCAG GGCATCAAGAACCCAGTGAACAAGGGCACC ACCACAGTGG1800
ACGGCGCTGGCTTCGGCATT GACCGGCCGGCGGAGCTCTCCAAGGAGGAC GACGAGTATG1860
AGGCGTTCCGCAAGAGGATG ATGCTGGCCTACCGCTTCCGGCCCAACCCC CTGAACAATC1920
CCAGACGGCCTTACTACTGA GTGTTCTGGAAATACATACTTTCTGAATGA CCAACCGTCC1980
CTGGACTGTGGAATGTTCCG GCCTGCATTTCTGCCCACCCCTTCCGTTGT CACGAGTGCC2040
GTGCCGTGTAATAAAGTCCC AGTGCTCATCCF,AAAAAAAAP,AAAAAA.AAA AP,AAAAAAAA2100
AAAA 2104
(2) INFORMATION
FOR
SEQ
ID N0:12:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 645 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) OLECULE TYPE: protein
M
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Ser Leu Lys Met Asp Asn Arg Asp Val Ala Gly Lys Ala Asn Arg
1 5 10 15
Trp Phe Gly Val Ala Pro Pro Lys Ser Gly Lys Met Asn Met Asn Ile
20 25 30
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Leu His GlnGluGlu LeuIle AlaGlnLys LysArgGlu IleGluAla
35 40 45
Lys Met GluGinLys AlaLys GlnAsnGln ValAlaSer ProGlnPro
50 55 60
Pro His ProGlyGlu IleThr AsnAlaHis AsnSerSer CysIleSer
65 70 75 80
Asn Lys PheAlaAsn AspGly SerPheLeu GlnGlnPhe LeuLysLeu
85 90 95
Gln Lys AlaGlnThr SerThr AspAlaPro ThrSerAla ProSerAla
100 105 110
Pro Pro SerThrPro ThrPro SerAlaGly LysArgSer LeuLeuIle
115 120 125
Ser Arg ArgThrGly LeuGly LeuAlaSer LeuProGly ProValLys
130 135 140
Ser Tyr SerHisAla LysGln LeuProVal AlaHisArg ProSerVal
145 250 155 160
Phe Gln SerProAsp GluAsp GluGluGlu AspTyrGlu GlnTrpLeu
165 170 175
Glu Ile LysValSer ProPro GluGlyAla GluThrArg LysValIle
180 185 190
Glu Lys LeuAlaArg PheVal AlaGluGly GlyProGlu LeuGluLys
195 200 205
Val Aia MetGluAsp TyrLys AspAsnPro AlaPheAla PheLeuHis
210 215 220
Asp Lys AsnSerArg GlyPhe Leu,TyrTyr ArgLysLys ValAlaGlu
225 230 235 240
Ile Arg LysGluAla GlnLys SerGlnAla AlaSerGln LysValSer
245 250 255
Pro Pro GluAspGlu GluVaI LysAsnLeu AlaGluLys LeuAlaArg
260 265 270
Phe Ile AlaAspGly GlyPro GluValGlu ThrIleAla LeuGlnAsn
275 280 285
Asn Arg GluAsnGln AlaPhe SerPheLeu TyrGluPro AsnSerGln
290 295 300
Gly Tyr LysTyrTyr ArgGln LysLeuGlu GluPheArg LysAlaLys
305 310 315 320
Ala Ser Ser Thr Gly Ser Phe Thr Ala Pro Asp Pro Gly Leu Lys Arg
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325 330 335
Lys Ser Pro Pro Glu Ala Leu Ser Gly Ser Leu Pro Pro Ala Thr Thr
340 345 350
Cys Pro Ala Ser Ser Thr Pro Ala Pro Thr Ile Ile Pro A1a Pro Ala
355 360 365
Ala Pro Gly Lys Pro Ala Ser Ala Ala Thr Val Lys Arg Lys Arg Lys
370 375 380
Ser Arg Trp Gly Pro Glu Glu Asp Lys Val Glu Leu Pro Pro Ala Glu
385 390 395 400
Leu Val Gln Arg Asp Val Asp Ala Ser Pro Ser Pro Leu Ser Val Gln
405 410 415
Asp Leu Lys Gly Leu Gly Tyr Glu Lys Gly Lys Pro Val Gly Leu Val
420 425 430
Gly Val Thr Glu Leu Ser Asp Ala Gln Lys Lys Gln Leu Lys Glu Gln
435 440 445
Gln Glu Met Gln Gln Met Tyr Asp Met Ile Met Gln His Lys Arg Ala
450 455 460
Met Gln Asp Met Gln Leu Leu Trp Glu Lys Ala Val Gln Gln His Gln
465 470 475 480
His Gly Tyr Asp Ser Asp Glu Glu Val Asp Ser Glu Leu Gly Thr Trp
485 490 495
Glu His Gln Leu Arg Arg Met Glu Met Asp Lys Thr Arg Giu Trp Ala
500 505 510
Glu Gln Leu Thr Lys Met Gly Arg Gly Lys His Phe Ile Gly Asp Phe
515 520 525
Leu Pro Pro Asp Glu Leu Glu Lys Phe Met Glu Thr Phe Lys Ala Leu
530 535 540
Lys Glu Gly Arg Glu Pro Asp Tyr Ser Glu Tyr Lys Glu Phe Lys Leu
545 550 555 560
Thr Val Glu Asn Ile Gly Tyr Gln Met Leu Met Lys Met Gly Trp Lys
565 570 575
Glu Gly Glu Gly Leu Gly Ser Glu Gly Gln Gly Ile Lys Asn Pro Val
580 585 590
Asn Lys Gly Thr Thr Thr Val Asp Gly Ala Gly Phe Gly Ile Asp Arg
595 600 605
Pro Ala Glu Leu Ser Lys Glu Asp Asp Glu Tyr Glu Ala Phe Arg Lys
610 615 620
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Arg Met Met Leu Ala Tyr Arg Phe Arg Pro Asn Pro Leu Asn Asn Pro
625 630 635 640
Arg Arg Pro Tyr Tyr
645
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1642 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:13:
CAAGTTTTGTTGTGCTGATTTAACAGCCTGTGAATTCTGCAAACACGATCGTGAAAAAAT 60
GCCAATCTGTCCTGTGTAAGVCCTGTGTGAAGTTTTGACTTTAATCTACCAGATCACTCC 120
TTCACCCTCCATAAAGATGTCTGAACCTGACACTTCCTCAGGATTTTCGGGAAGTGTGGA 180
GAATGGAACTTTTCTTGAGCTGTTTCCCACATCCCTGTCCACGTCAGTGGACCCATCCTC 240
AGGCCACCTGTCAAACGTCTACATCTATGTGTCCATATTCCTCAGCCTTTTAGCGTTTCT 300
GCTTCTGCTTTTAATCATTGCCCTCCAGAGGCTCAAAAATATCATCTCCTCCAGTTCCTC 360
CTACCCAGAGTATCCAAGCGACGCTGGAAGTTCTTTCACCAATTTGGAAGTCTGCAGCAT 420
TTCCTCTCAGAGGTCCACTTTTTCAAACCTTTCATCCTGAGGAAAATGGAAGAGTCCTTG 480
AGTGTGGCAGCAGTTTTGACATCCCCTTACGGAAGTGTCCCGTGAGGCATTGCCTCATGA 540
AAGAAATGATCCTTTTGGTGTAGACCTGCTTCTCCTTCTCCTTTTTCTCTGATTTCTTTT 600
CTGTTCATGATGCTTTTCATTTGGGGATGGAGACACCGATGTTGGTGGAAATGTGTGCAA 660
ACCCCAAGGTGCAGAATTTCACACAAATGGCTTGATGAATCTAGACTGGGCTTCTTCAGG 720
TAAGTCAGTTCATTCTACTTTGTTGGACGCCGTAGACTCATCTGAGGTGGCCTCTCCGTG 780
GATGCTGGACATGGACTCGCACTTCATTTCTTTTACAAAGCCGTGAAATCAACTGAGCCT B40
GCAGAAACTGGCAAAATCAAGTCTGACCTATGTAGAAGTTATTTTCCATATTTAAAAGAT 900
AAAGTGGAGACACCAAACTTAAAAAGGAAGAGAGTCAATTTAGATTTAATGTATGACATT 960
TCTAAAACTGAGGGTAAATATATGCTAAATATTTTCTTTAACTTCACTTTAACAAGTAAA 1020
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AATCACACTT ATTGATGAAT GTAAGTCATTTGGGAAAGTTTTGGAAGAGT TTACATTTTA1080
ATAGCAAGAC AAACATGTAT TATGATGGAGCTTGTGATGTATTTCTGCCT CCTGGAGTTT1140
TATTTTGTTT TTCTGCTTAT GTTTTAGTCATAATGCCACAAAGTTGTGGA ATTTTTGTGA1200
TTAATTGCTG TTACTAGTAC TAAGAGAAGCACCAGAAGAGATGCCAAGAA GTTTTTATAT1260
GAATAATTTC TATCAGTGAG AATTAAGCATATGGAAAATATTCATTTAGT TGTATTTTAT1320
ACAGTAATAA CTCTTAGCTG TCGTGTAAGTTCCTTTATTCGGTTTCATAG TCTTTATAAT1380
TTTAGTGCAG AATTATATTA AGCCTCCAAGATGTCTGATATTTGTTCACT CACATTAGGT1440
TGTAAAACTT AGAAACTAAA TTGCAAATATATGTGTTATTATATACTCCA CGAATGTTGC1500
GTCTCTGATA ATTAGTTGTT GTATGTTAACATAATACTCTACATTAGGAT TTCAGGATGT1560
GAGTTTGTAT TAAAAATTGT AGGCACTCCAAAAAAAAAAAAAAAAAAAAA P,I~.AAAAAAAA1620
P.,AAAAAAAAA P.,F~~AAAAAAA 16
AA 4
2
(2) INFORMATION FOR SEQ
ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Ser Glu Pro Asp Thr Ser Ser Gly Phe Ser Gly Ser Val Glu Asn
1 5 10 15
Gly Thr Phe Leu Glu Leu Phe Pro Thr Ser Leu Ser Thr Ser Val Asp
20 25 30
Pro Ser Ser Gly His Leu Ser Asn Val Tyr Ile Tyr Val Ser Ile Phe
35 40 45
Leu Ser Leu Leu Ala Phe Leu Leu Leu Leu Leu Ile Ile Ala Leu Gln
50 55 60
Arg Leu Lys Asn Ile Ile Ser Ser Ser Ser Ser Tyr Pro Glu Tyr Pro
65 70 75 80
Ser Asp Ala Gly Ser Ser Phe Thr Asn Leu Glu Val Cys Ser Ile Ser
85 90 95
7g
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Ser Gln Arg Ser Thr Phe Ser Asn Leu Ser Ser
100 105
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1365 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID N0:15:
CGCAGGAAGC AGAAGAGCAGAGCGAGGACG ACGACGAGGATACAGAAGAGGAACAGGGGG60
AAGAAAAGGA AAAGGGAGCGCAGGAGAAAA GGAGGGGGAAGAGAGTCCGTTTTGCAGAAG120
ATGAAGAAAA GAGTGAAAATTCCTCGGAGG ACGGTGACATAACGGATAAGAGTCTTTGTG180
GAAGTGGTGA AAAGTACATCCCACCTCATG TGAGGCAAGCTGAGGAGACAGTGGACTTCA240
AGAAAAAGGA AGAACTAGAAAGGCTGAAGA AACATGTAAAAGGTCTACTTAACAGGTTGA300
GTGAACCCAA CATGGCTTCCATCAGTGGGC AGCTGGAGGAACTGTACATGGCCCACAGCA360
GAAAGGACAT GAATGACACCCTGACCTCCG CTCTCATGGGTGCCTGCGTCACTGCCTCGG420
CCATGCCCAG CAGACTGATGATGGAGCATG TTCTCTTAGTCAGCATCCTTCACCACACAG480
TTGGAATCGA GGTCGGTGCCCACTTTCTGG AGGCAGTGGTGAGGAAGTTCGATGCCATCT540
ATAAATACGG AAGCGAAGGGAAAGAGTGTG ACAACCTGTTCACCGTCATTGCCCATTTAT600
ACAACTTCCA CGTGGTACAGTCTCTCCTCA TCTTCGACATTTTGAA.AAAACTGATTGGAA660
CTTTCACCGA AAAAGATATTGAACTGATCT TGTTAATGCTGAAAAACGTGGG'~T'.~:".~CA':720
TGAGGAAAGA TGATGCTTTATCACTTAAGG AATTGATCACTGAAGCCCAGACCAAAGCCA780
GCGGGGCAGG CAGCGAGTTTCAGGACCAGA CCAGGGTACGCGTGCGACGCTTGATCTGCT840
TCCTAAGTCC CTAAAGCTCACAAACTGGCC AGAACCTAAAAATCAGTATCTGGGATTCGG900
TTTATGCTAG AGACGATGTTGGCCCTGAAG AACAATGACATGCGCAAAATTCCAGGCTAT960
GACCCCGAGC CCGTGGAGAAGCTGAGGAAA CTGCAGAGAGCTTTGGTCCGCAACGCCGGC1020
TCAGGTTCTG AGACGCAGCTTCGCGTCTCC TGGGACAGTGTCTTGAGTGCGGAGCAGACG1080
GGTCGCTGGT GGATTGTGGGGTCCGCCTGG AGTGGGGCCCCGATGATCGACAACAGTCAC1140
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CATACGCACC TGCAGAAGCA GYTTGTGGGG ACGGTAGGGA CACCCATGYT CAAGGYTGCC 1200
AGGCAGAGGC ACCCCCCTGT GTGGTGTGTG GTCCTGGCTT TACCTGGAGC AGCTCTCTTA 1260
CTGTTCTTGA ATGGTCACTG AAATGTACAA GGTTTATCTG GAGGCCTTAC AGAAATTGCT 1320
ATTAATATTA CATTGTGATA TAATTATTCC F,~~,4AAAAAAA AAAAA 1365
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
{B) TYPE: amino acid
{C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Ala Ser Ile Ser Gly Gln Leu Glu Glu Leu Tyr Met Ala His Ser
1 5 10 15
Arg Lys Asp Met Asn Asp Thr Leu Thr Ser Ala Leu Met Gly Ala Cys
20 25 30
Val Thr Ala Ser Ala Met Pro Ser Arg LEU Met Met Glu His Val Leu
35 40 45
Leu Val Ser Ile Leu His His Thr Val Gly Ile Glu Val Gly Ala His
50 55 60
Phe Leu Glu Ala Val Val Arg Lys Phe Asp Ala Ile Tyr Lys Tyr Gly
65 70 75 80
Ser Glu Gly Lys Glu Cys Asp Asn Leu Phe Thr Val Ile Ala His Leu
85 90 95
Tyr Asn Phe His Val Val Gln Ser Leu Leu Ile Phe Asp Ile Leu Lys
100 105 110
Lys Leu Ile Gly Thr Phe Thr Glu Lys Asp Ile Glu Leu Ile Leu Leu
115 120 125
Met Leu Lys Asn Val Gly Phe Ser Leu Arg Lys Asp Asp Ala Leu Ser
130 135 ~ 140
Leu Lys Glu Leu Ile Thr Glu Ala Gln Thr Lys Ala Ser Gly Ala Gly
145 150 155 160
Ser Glu Phe Gln Asp Gln Thr Arg Val Arg Val Arg Arg Leu Ile Cys
165 170 175
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Phe Leu Ser Pro
180
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1310 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:17:
CAAGCACCAGGAAGTCAAGATGCAAGCACCAGCCTTCAGGGACAAGAAACAGGGGGTCTC 60
AGCCAAGAATCAAGGTGCCCATGACCCAGACTATGAGAATATCACCTTGGCCTTCAAAAA 120
TCAGGACCATGCAAAGGGTGGTCATTCACGACCCACGAGCCAAGTCCCAGCCCAGTGCAG 180
GCCGCCCTCAGACTCCACCCAGGTCCCCTGCTGGTTGTACAGAGCCATCCTGAGCCTGTA 240
CATCCTCCTGGCCCTGGCCTTTGTCCTCTGCATCATCCTGTCAGCCTTCATCATGGTGAA 300
GAATGCTGAGATGTCCAAGGAGCTGCTGGGCTTTAAAAGGGAGCTTTGGAATGTCTCAAA 360
CTCCGTACAAGCATGCGAAGAGAGACAGAAGAGAGGCTGGGATTCCGTTCAGCAGAGCAT 42C
CACCATGGTCAGGAGCAAGATTGATAGATTAGAGACGACATTAGCAGGCATAAAAAACAT 480
TGACACAAAGGTACAGAAAATCTTGGAGGTGCTGCAGAAAATGCCACAGTCCTCACCTCA 540
ATAAATGAGAGGACATTGTGGCAGCCAAAGCCACAACTTGGAAGATGGGGCTGCACCTGC 600
CAACGAAGACGGGAAATGACCCCCCCCCCCAGCCTAGTGTGAACCTGCCCCTCGTCCCAC 660
GTATAGAAAAACCTCGAGTCATGGTGAATGAGTGTCTCGGAGTTGCTCGTGTGTGTGTAC 72G
ACCTGCGTGCGTGTGTGTGCGTGTGTGCGCGTGTGTTCGTGTATGTGCGTGTGTGCGTGC 780
GCGTGTGTGTGCATTTTGCAAAGGGTGGACATTTCAGTGTATCTCCCAGAAAGGTGAKGA 840
ATGAATAGGACTGAGAGTCACAGTGAATGTGGCATGCATGCCTGTGTCATGWGACATATG 900
TGAGTCTCGGCATGTCACGGTGGGTGGCTGTGTYTGAGCACCTCCAGCAGATGTCACTCT 960
GAGTGTGGGTGTTGGTGACATGCATTGCACGGGCCTGTCTCCCTGTTTGTGTAAACATAC 1020
TAGAGTATACTGCGGCGTGTTTTCTGTCTACCCATGTCATGGTGGGGGAGATTTATCTCC 1080
GWACATGTGGGTGTCGCCATGTGTGCCCTGTCACTATCTGTGGCTGGGTGAACGGCTGTG 2140
gl
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TCATTATGAG TGTGCCGAGT TATGCCACCC TGTGTGCTCA GGGCACATGC ACACAGACAT 1200
TTATYTYTGC ACTCACATTT TGTGACTTAT GAAGATAAAT AAAGTCAAGG GAAAACAGCG 1260
TCMAAAAAAA P~~AAAAAAA.A P~AAAAAAAAA A,F~P.AAAAAAA AAAAAAAAAA 1310
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Gln Ala Pro Ala Phe Arg Asp Lys Lys Gln Gly Val Ser Ala Lys
1 5 10 15
Asn Gln Gly Ala His Asp Pro Asp Tyr Glu Asn Ile Thr Leu Ala Phe
20 25 30
Lys Asn Gln Asp His Ala Lys Gly Gly His Ser Arg Pro Thr Ser Gln
35 40 45
Val Pro Ala Gln Cys Arg Pro Pro Ser Asp Ser Thr Gln Val Pro Cys
50 55 60
Trp Leu Tyr Arg Ala Ile Leu Ser Leu Tyr Ile Leu Leu Ala Leu Ala
65 70 75 80
Phe Val Leu Cys Ile Ile Leu Ser Ala Phe Ile Met Val Lys Asn Ala
85 ~ 90 95
Glu Met Ser Lys Glu Leu Leu Gly Phe Lys Arg Glu Leu Trp Asn Val
100 105 110
Ser Asn Ser Val Gln Ala Cys Glu Glu Arg Gln Lys Arg Gly Trp Asp
115 120 125
Ser Val Gln Gln Ser Ile Thr Met Val Arg Ser Lys Ile Asp Arg Leu
130 135 140
Glu Thr Thr Leu Ala Gly Ile Lys Asn Ile Asp Thr Lys Val Gln Lys
145 150 155 160
Ile Leu Glu Val Leu Gln Lys Met Pro Gln Ser Ser Pro Gln
165 170
(2) INFORMATION FOR SEQ ID N0:19:
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(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 N0:19:
GNGGCATCAGA CATTGGGGAG TGATTCAA 29
(2) INFORMATION FOR SEQ ID N0:20:
(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 N0:20:
CNTGAGAGAAC CTTTGCTTCC TGCTATAT 29
(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
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
CNATGCACTCC AGTGAGCTAG ATTCTTTA 29
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
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(Ay 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 N0:22:
TNAATTGTGAG ATCTTCTCTC CAAGCTCT 29
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iiy MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GNCGGTCTGGA AGCCATCCTC AACTGAAG 29
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(Cy STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
ANCTCTTCCTG GTGAAGGATG TTCATGTT 29
{2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
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(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 N0:25:
CNTTATGGAGG GTGAAGGAGT GATCTGGT 29
(2) INFORMATION FOR SEQ ID N0:26:
(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:
ANGTCCACTGT CTCCTCAGCT TGCCTCAC 29
(2) INFORMATION FOR SEQ ID N0:27:
(~) 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 N0:27:
GNTTGCATCTT GACTTCCTGG TGCTTGTA 29
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 307 amino acids
(B) TYPE: amino acid
gs
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Met Ser Leu Ala Ser His Lys Phe His Arg Tyr Ser Cys Ala His Cys
1 5 10 15
Arg Lys Pro Phe His Lys Ile Glu Thr Leu Tyr Arg His Cys Gln Asp
20 25 30
Glu His Asp Asn Glu Ile Lys Ile Lys Tyr Phe Cys Gly Leu Cys Asp
35 40 45
Leu Ile Phe Asn Val Glu Glu Ala Phe Leu Ser His Tyr Glu Glu His
50 55 60
His Ser Ile Asp Tyr Val Phe Val Ser Glu Lys Thr Glu Thr Ser Ile
65 70 75 80
Lys Thr Glu Asp Asp Phe Pro Val Ile Glu Thr Ser Asn Gln Leu Thr
85 90 95
Cys Gly Cys Arg Glu Ser Tyr Ile Cys Lys Val Asn Arg Lys Glu Asp
100 105 110
Tyr Ser Arg Cys Leu Gln Ile Met Leu Asp Lys Gly Lys Leu Trp Phe
115 120 125
Arg Cys Ser Leu Cys Ser Ala Thr Ala Gln Asn Leu Thr Asp Met Asn
130 135 140
Thr His Ile His Gln Val His Lys Glu Lys Ser Asp Glu Glu Glu Gln
145 150 155 160
Gln Tyr Val Ile Lys Cys Gly Thr Cys Thr Lys Ala Phe His Asp Pro
165 I70 175
Glu Ser Ala Gln Gln His Phe His Arg Lys His Cys Phe Leu Gln Lys
180 185 190
Pro Ser Val Ala His Phe Gly Ser Glu Lys Ser Asn Leu Tyr Lys Phe
195 200 205
Thr Ala Ser Ala Ser His Thr Glu Arg Lys Leu Lys Gln Ala Ile Asn
210 215 220
Tyr Ser Lys Ser Leu Asp Met Glu Lys Gly Val Glu Asn Asp Leu Ser
225 230 235 240
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Tyr Gln Asn Ile Glu Glu Glu Ile Val Glu Leu Pro Asp Leu Asp Tyr
245 250 255
Leu Arg Thr Met Thr His Ile Val Phe Val Asp Phe Asp Asn Trp Ser
260 265 270
Asn Phe Phe Gly His Leu Pro Gly His Leu Asn Gln Gly Thr Phe Ile
275 280 285
Trp Gly Phe Gln Gly Thr Val Asn Lys Lys Asn Lys Arg Lys Leu Phe
290 295 300
Pro Thr Ser
305
g7
