Note: Descriptions are shown in the official language in which they were submitted.
CA 02280968 1999-08-10
WO 98/3b067 PCTIUS98/02767-
10
SECRETED PROTEII~TS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of Ser. No. 60/XXX,XXX (converted
to
a provisional application from non-provisional application Ser. No.
08/800,418), filed
February 14, 1997, which is incorporated by reference herein.
2 0 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.
2 5 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
3 0 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
3 5 techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 128 to nucleotide 1006;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 182 to nucleotide 362;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BG481_1 deposited under accession
number ATCC 98331;
{e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BG481_1 deposited under accession number ATCC 98331;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BG481_1 deposited under accession number
ATCC 98331;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BG481 1 deposited under accession number ATCC 98331;
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 polynucleotlde 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 128 to nucleotide 1006; the nucleotide sequence of SEQ ID
N0:1
from nucleotide 182 to nucleotide 362; the nucleotide sequence of the full-
length protein
coding sequence of clone BG481_1 deposited under accession number ATCC 98331;
or the
nucleotide sequence of the mature protein coding sequence of clone BG481_1
deposited
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under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BG481_1 deposited under accession number ATCC 98331. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 1 to amino
acid 78.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
In other embodiments, the present invention.provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 1 to
amino acid 78;
(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
BG481_1 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 1 to amino acid 78.
2 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
{b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:3 from nucleotide 250 to nucleotide 846;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 361 to nucleotide 846;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1 to nucleotide 564;
3 0 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BJ9_1 deposited under accession number
ATCC 98331;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BJ9_1 deposited under accession number ATCC 98331;
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(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BJ9_1 deposited under accession number ATCC
98332;
{h) a polynucleotide encoding the mature protein encoded, by the
cDNA insert of clone BJ9_1 deposited under accession number ATCC 98331;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(I) 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
N0:3 from nucleotide 250 to nucleotide 846; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 361 to nucleotide 846; the nucleotide sequence of SEQ ID N0:3 from
nucleotide 1 to nucleotide 564; the nucleotide sequence of the full-length
protein coding
2 0 sequence of clone Bj9_1 deposited under accession number ATCC 98331; or
the nucleotide
sequence of the mature protein coding sequence of clone BJ9_1 deposited under
accession
number ATCC 98331. In other preferred embodiments, the polynucleotide encodes
the
full-length or mature protein encoded by the cDNA insert of clone BJ9_1
deposited under
accession number ATCC 98331. In yet other preferred embodiments, the present
2 5 invention provides a polynucleotide encoding a protein comprising the
amino acid
sequence of SEQ ID N0:4 from amino acid 1 to amino acid 105.
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
3 0 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 1 to
amino acid 105;
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(c) fragments of the amino acid sequence of SEQ ID N0:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone BJ9_1
deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid
sequence
. of SEQ ID N0:4 from amino acid 1 to amino acid 105.
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 354 to nucleotide 674;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BK34 3 deposited under accession
number ATCC 98331;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BK34_3 deposited under accession number ATCC 98331;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BK34 3 deposited under accession number
2 0 ATCC 98331;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BK34_3 deposited under accession number ATCC 98331;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
2 5 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleoHde of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
3 0 of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 354 to nucleotide 674; the nucleotide sequence of the
full-length
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protein coding sequence of clone BK34_3 deposited under accession number ATCC
98331;
or the nucleotide sequence of the mature protein coding sequence of clone BK34
3
deposited under accession number ATCC 98331. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BK34 3 deposited under accession number ATCC 98331.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5 or SEQ ID N0:7.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6; and
(c) the amino acid sequence encoded by the cDNA insert of clone
BK34 3 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:8;
{b} a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:B from nucleotide 823 to nucleotide 960;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:8 from nucleotide 931 to nucleotide 960;
2 5 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BP883_2 deposited under accession
number ATCC 98331;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BP883 2 deposited under accession number ATCC 98331;
3 0 (f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BP883_2 deposited under accession number
ATCC 98331;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BP883_2 deposited under accession number ATCC 9$331;
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(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:9;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:9 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 polynucleoHde 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
N0:8 from nucleotide 823 to nucleotide 960; the nucleotide sequence of SEQ ID
N0:8 from
nucleotide 931 to nucleotide 960; the nucleotide sequence of the full-length
protein coding
sequence of clone BP883_2 deposited under accession number ATCC 98331; or the
nucleotide sequence of the mature protein coding sequence of clone BP883_2
deposited
under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BP883 2 deposited under accession number ATCC 98331. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
2 0 comprising the amino acid sequence of SEQ ID N0:9 from amino acid 1 to
amino acid 16.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:8.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 5 consisting of:
(a) the amino acid sequence of SEQ ID N0:9;
(b) the amino acid sequence of SEQ ID N0:9 from amino acid 1 to
amino acid 16;
(c) fragments of the amino acid sequence of SEQ ID N0:9; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
BP883_2 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:9 or the amino acid
sequence
of SEQ ID N0:9 from amino acid 1 to amino acid 16.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:10 from nucleotide 84 to nucleotide 1016;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:10 from nucleotide 786 to nucleotide 1016;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:10 from nucleotide 619 to nucleotide 899;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CI363_1 deposited under accession
number ATCC 98331;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CI363_1 deposited under accession number ATCC 98331;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CI363_1 deposited under accession number
ATCC 98331;
(h) a polynucleotide encoding the mature protein encoded by the
2 0 cDNA insert of clone CI363_1 deposited under accession number ATCC 98331;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:11;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:11 having biological activity;
2 5 (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
3 0 to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:10 from nucleotide 84 to nucleotide 1016; the nucleotide sequence of SEQ ID
N0:10
from nucleotide 786 to nucleotide 1016; the nucleotide sequence of SEQ ID
N0:10 from
nucleotide 619 to nucleotide 899; the nucleotide sequence of the full-length
protein coding
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sequence of clone CI363_1 deposited under accession number ATCC 98331; or the
nucleotide sequence of the mature protein coding sequence of clone CI363_1
deposited
under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CI363_1 deposited under accession number ATCC 98331. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:11 from amino acid 180 to
amino acid
272.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:10.
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 N0:11;
(b) the amino acid sequence of SEQ ID N0:11 from amino acid 180 to
amino acid 272;
(c) fragments of the amino acid sequence of SEQ ID NO:11; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CI363_1 deposited under accession number ATCC 98331;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:11 or the amino acid
sequence
of SEQ ID N0:11 from amino acid 180 to amino acid 272.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 S (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 88 to nucleotide 561;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:12 from nucleotide 142 to nucleotide 561;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 1 to nucleotide 554;
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(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone C0806_1 deposited under accession
number ATCC 98331;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C0806_1 deposited under accession number ATCC 98331;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone C0806_1 deposited under accession number
ATCC 98331;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C0806_1 deposited under accession number ATCC 98331;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:13;
(j) a polynucleoHde encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:13 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
2 0 to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:I2 from nucleotide 88 to nucleotide 561; the nucleotide sequence of SEQ ID
N0:12
from nucleotide 142 to nucleotide 561; the nucleotide sequence of SEQ ID N0:12
from
nucleotide 1 to nucleotide 554; the nucleotide sequence of the full-length
protein coding
2 5 sequence of clone C0806_1 deposited under accession number ATCC 98331; or
the
nucleotide sequence of the mature protein coding sequence of clone C0806_1
deposited
under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone C0806_1 deposited under accession number ATCC 98331. In yet other
preferred
3 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:13 from amino acid 112 to
amino acid
156.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:12.
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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 N0:13;
(b) the amino acid sequence of SEQ ID N0:13 from amino acid 112 to
. amino acid 156;
(c) fragments of the amino acid sequence of SEQ ID N0:13; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C0806_1 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:13 or the amino acid
sequence
of SEQ ID N0:13 from amino acid 112 to amino acid 156.
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
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 787 to nucleotide 945;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 NO:14 from nucleotide 853 to nucleotide 945;
{d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 324 to nucleotide 945;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CT24_3 deposited under accession
2 5 number ATCC 98331;
(f} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CT24 3 deposited under accession number ATCC 98331;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CT24_3 deposited under accession number
3 0 ATCC 98331;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CT24 3 deposited under accession number ATCC 98331;
(i} a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:15;
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{j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 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
N0:14 from nucleotide 787 to nucleotide 945; the nucleotide sequence of SEQ ID
N0:14
from nucleotide 853 to nucleotide 945; the nucleotide sequence of SEQ ID N0:14
from
nucleotide 324 to nucleotide 945; the nucleotide sequence of the full-length
protein coding
sequence of clone CT24_3 deposited under accession number ATCC 98331; or the
nucleotide sequence of the mature protein coding sequence of clone CT24_3
deposited
under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CT24_3 deposited under accession number ATCC 98331.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:14.
2 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:15;
(b) fragments of the amino acid sequence of SEQ ID N0:15; and
2 5 (c) the amino acid sequence encoded by the cDNA insert of clone
CT24_3 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:15.
In one embodiment, the present invention provides a composition comprising an
3 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 562 to nucleotide 738;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 1 to nucleotide 729;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ER366_3 deposited under accession
number ATCC 98331;
{e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ER366_3 deposited under accession number ATCC 98331;
{f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone ER366_3 deposited under accession number
ATCC 98331;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone ER366_3 deposited under accession number ATCC 98331;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:17;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:17 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
2 0 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
N0:16 from nucleotide 562 to nucleotide 738; the nucleotide sequence of SEQ ID
N0:16
2 5 from nucleotide 1 to nucleotide 729; the nucleotide sequence of the full-
length protein
coding sequence of clone ER366_3 deposited under accession number ATCC 98331;
or the
nucleotide sequence of the mature protein coding sequence of clone ER366_3
deposited
under accession number ATCC 98331. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
3 0 clone ER366_3 deposited under accession number ATCC 98331. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:17 from amino acid 1 to amino
acid
56.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:16.
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 N0:17;
(b) the amino acid sequence of SEQ ID N0:17 from amino acid 1 to
amino acid 56;
(c) fragments of the amino acid sequence of SEQ ID N0:17; and
(d) the amino acid sequence encoded by the cDNA insert of clone
ER366_3 deposited under accession number ATCC 98331;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:17 or the amino acid
sequence
of SEQ ID N0:17 from amino acid 1 to amino acid 56.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
2 0 disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
2 5 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.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
3 0 specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
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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.
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
2 0 sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
2 5 Clone "BG481 1"
A polynucleotide of the present invention has been identified as clone "BG481
1".
BG481 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
3 0 analysis of the amino acid sequence of the encoded protein. BG481_1 is a
full-length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BG481_1 protein").
The nucleotide sequence of BG481_1 as presently determined is reported in SEQ
ID NO:1. What applicants presently believe to be the proper reading frame and
the
IS
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predicted amino acid sequence of the BG481 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
BG481_1 should be approximately 2600 bp.
The nucleotide sequence disclosed herein for BG481_i was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BG481 1 demonstrated at least some similarity with
sequences
identified as AB002351 (Human mRNA for KIAA0353 gene, partial cds), 841215
(yf84h04.s1 Homo sapiens cDNA clone 29428 3'), W26369 (26f5 Human retina cDNA
randomly primed sublibrary Homo sapiens cDNA), and W27653 (36e7 Human retina
cDNA randomly primed sublibrary Homo sapiens cDNA). The predicted amino acid
sequence disclosed herein for BG481_l was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
BG481_1 protein demonstrated at least some similarity to sequences identified
as
AB002351 (KIAA0353 [Homo sapiens]}. Based upon sequence similarity, BG481_l
proteins and each similar protein or peptide may share at least some activity.
Clone "B19 1"
A polynucleotide of the present invention has been identified as clone
"BJ9_1".
2 0 BJ9_1 was isolated from a human adult ovary (pool of retinoic-acid-
treated, activin-
treated, and untreated PA-1 teratocarcinoma cells) 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. BJ9_1 is a full-
length clone,
2 5 including the entire coding sequence of a secreted protein (also referred
to herein as
"Bj9_1 protein")
The nucleotide sequence of Bj9_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 BJ9_1 protein corresponding to the foregoing
nucleotide
3 0 sequence is reported in SEQ ID N0:4. Amino acids 25 to 37 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 38.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BJ9_1 should be approximately 1100 bp.
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The nucleotide sequence disclosed herein for BJ9_1 was searched against the
GenBank and GeneSeq_nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BJ9_1 demonstrated at least some similarity with
sequences
identified as AA114043 (zm29f09.r1 Stratagene pancreas (#937208) Homo sapiens
cDNA
clone 527081 5') and H27069 (y116a06.r1 Homo sapiens cDNA clone 158386 5'
similar to
gb I M87903 I HUMALNE37 Human carcinoma cell-derived Alu RNA transcript,
(rRNA);
gb:M87338 ACTIVATOR 140 KD SUBUNIT (HUMAN);contains Alu repetitive element).
Based upon sequence similarity, BJ9_1 proteins and each similar protein or
peptide may
share at least some activity. The nucleotide sequence of BJ9_1 indicates that
it may
contain Alu and CAAA repeat sequences.
Clone "BK34 3"
A polynucleotide of the present invention has been identified as clone
"BK34_3".
BK34 3 was isolated from a human adult retina cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BK34_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"BK34_3 protein")
2 0 The nucleotide sequence of the 5' portion of BK34_3 as presently
determined is
reported in SEQ ID N0:5. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:6. The predicted amino acid
sequence of
the BK34_3 protein corresponding to the foregoing nucleotide sequence is
reported in SEQ
ID NO:6. Additional nucleotide sequence from the 3' portion of BK34_3,
including the
2 5 polyA tail, is reported in SEQ ID N0:7.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BK34_3 should be approximately 1350 bp.
The nucleotide sequence disclosed herein for BK34 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
3 0 FASTA search protocols. BK34 3 demonstrated at least some similarity with
sequences
identified as H94111 (yw58h12.s1 Soares placenta 8to9weeks 2NbHP8to9W Homo
sapiens
cDNA clone 256487 3'). Based upon sequence similarity, BK34_3 proteins and
each similar
protein or peptide may share at least some activity.
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WO 98/36067 PCT/US98/02767
Clone "BP883 2"
A polynucleotide of the present invention has been identified as clone
"BP883_2".
BP883_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. BP883_2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"BP883 2 protein")
The nucleotide sequence of BP883_2 as presently determined is reported in SEQ
ID N0:8. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BP883 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:9. Amino acids 24 to 36 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 37, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BP883_2 should be approximately 1445 bp.
The nucleotide sequence disclosed herein for BP883_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BP883_2 demonstrated at least some similarity with
sequences
2 0 identified as AA164399 (zo96a10.s1 Stratagene ovarian cancer (#937219)
Homo sapiens
cDNA clone 594714 3'), H04678 (yj10a10.r1 Homo sapiens cDNA clone), H17605
(ym36h02.r1 Homo sapiens cDNA clone 50386 5'), I25658 (Sequence 19 from patent
US
5552281), N47764 {yy55e08.r1 Homo sapiens cDNA clone 277478 5'), Q61070 (Human
brain Expressed Sequence Tag EST01127), Q72530 (Osteoclast-specific/related
expressed
2 5 gene clone 241B), 837216 (yh96f12.r1 Homo sapiens cDNA clone 137615 5'),
and W56113
(zc56g06.r1 Soares parathyroid tumor NbHPA Homo sapiens). Based upon sequence
similarity, BP883 2 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts a potential transmembrane
domain
at the carboxyl terminus of the BP883_2 protein sequence reported as SEQ ID
N0:9.
Clone "CI363 1"
A polynucleotide of the present invention has been identified as clone
"CI363_1 ".
CI363_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
18
CA 02280968 1999-08-10
WO 98136067 PCT/US98/02767
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CI363_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"CI363_1 protein").
The nucleotide sequence of CI363_1 as presently determined is reported in SEQ
ID N0:10. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CI363_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:11. Amino acids 222 to 234 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 235, or are a transmembrane domain. Amino acids 133 to 145 are
another
possible leader/signal sequence, with the predicted mature amino acid sequence
beginning at amino acid 146, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CI363_1 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for CI363_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CI363_1 demonstrated at least some similarity with
sequences
identified as AA024102 (mh98c08.r1 Soares mouse placenta 4NbMP13.5 14.5 Mus
musculus cDNA clone 458990 5' similar to PIR:S19586 519586 N-methyl-D-
aspartate
2 0 receptor glutamate-binding chain - rat), AA413815 (vc67b06.s1 Knowles
Solter mouse 2
cell Mus musculus cDNA clone 779603 5'), H06014 (y176e04.r1 Homo sapiens cDNA
clone
43696 5' similar to SP:S19586 519586 N-METHYL-D-ASPARTATE RECEPTOR
GLUTAMATE-BINDING CHAIN}, N70951 (za34a02.s1 Homo sapiens cDNA clone 294410
3'), 873412 (yj92fi2.r1 Homo sapiens cDNA clone), and 561973 (NMDA receptor
2 5 glutamate-binding subunit). The predicted amino acid sequence disclosed
herein for
CI363_1 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted CI363_1 protein demonstrated
at least
some similarity to sequences identified as 561973 (NMDA receptor glutamate-
binding
subunit [rats, Peptide, 516 aa] [Rattus sp.]} and V01555 (BWRFl reading frame
12 [Human
3 0 herpesvirusJ). Based upon sequence similarity, CI363_1 proteins and each
similar protein
or peptide may share at least some activity. The TopPredII computer program
predicts
seven potential transmembrane domains within the CI363_1 protein sequence,
centered
around amino acids 110, 130, 180, 200, 230, 250, and 290 of SEQ ID N0:11,
respectively.
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_ WO 98/36067 PCT/US98/02767
Clone "C0806 1 "
A polynucleotide of the present invention has been identified as clone
"C0806_1 ".
C0806_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. C0806_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "C0806_1 protein").
The nucleotide sequence of C0806_1 as presently determined is reported in SEQ
ID N0:12. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the C0806_1 protein corresponding to the
foregoing
nucleotide sequence is reported, in SEQ ID N0:13. Amino acids 6 to 18 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 19, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
C0806_1 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for C0806_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. C0806_1 demonstrated at least some similarity with
sequences
2 0 identified as AA075444 (zm87d06.r1 Stratagene ovarian cancer (#937219}
Homo Sapiens
cDNA clone 544907 5' similar to WP:F22E10.5 CE05695 PHOSPHOTRANSFERASE),
F0882I (H. sapiens partial cDNA sequence; clone c-2tc04), H19601 (yn59g02.r1
Homo
sapiens cDNA clone 172754 5'), 839687 (yc97cO8.s1 Homo sapiens cDNA clone
24193 3'),
844048 (yg22b11.s1 Homo Sapiens cDNA clone 33076 3'), T19387 (Human gene
signature
2 5 HUMGS00411), T49354 (ya74gOl.r1 Homo Sapiens cDNA clone 67440 5'), U12735
(Glycine
max aminoalcoholphosphotransferase (AAPT1) mRNA, complete cds), and Y08486
(M.musculus Sycp3 gene). The predicted amino acid sequence disclosed herein
for
C0806_1 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted C0806_1 protein demonstrated
at least
3 0 some similarity to sequences identified as U12735
(aminoalcoholphosphotransferase
[Glycine max]), Y08486 (synaptonemal complex protein [Mus musculus]), 250797
(F22E10.5 (Caenorhabditis elegans]), and 267882 (F22E10.5 (Caenorhabditis
elegans]).
Two amino acid sequence motifs were identified in the predicted C0806_1
protein: a
CDP-alcohol phosphatidyltransferase motif and a cytochrome b/b6 motif. Based
upon
CA 02280968 1999-08-10
WO 98136067 PCT/US98/02767
sequence similarity, C0806_1 proteins and each similar protein or peptide rnay
share at
least some activity.
Clone "CT24 3"
A polynucleotide of the present invention has been identified as clone "CT24
3".
CT24_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. CT24_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"CT24_3 protein").
The nucleotide sequence of CT24_3 as presently determined is reported in SEQ
ID
N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CT24_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15. Amino acids 10 to 22 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 23, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing
clone
CT24_3 should be approximately 2000 bp.
2 0 The nucleotide sequence disclosed herein for CT24_3 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CT24_3 demonstrated at least some similarity with
sequences
identified as AA344698 (EST50612 Gall bladder I Homo sapiens cDNA 5' end),
H48143
(yp80h01.s1 Homo sapiens cDNA clone 193777 3'), N72077 (yz97c06.s1 Homo
sapiens
2 5 cDNA clone 290986 3'), T95902 (ye47e08.s1 Homo sapiens cDNA clone 120902
3'), T96003
(ye47e08.r1 Homo Sapiens cDNA clone 120902 5'), and 245277 (H. sapiens partial
cDNA
sequence; clone c-21d02). Based upon sequence similarity, CT24_3 proteins and
each
similar protein or peptide may share at least some activity
3 0 Clone "ER366 3"
A polynucleotide of the present invention has been identified as clone
"ER366_3".
ER366_3 was isolated from a human fetal brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
21
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_ WO 98/36067 PCT/US98/0276?
analysis of the amino acid sequence of the encoded protein. ER366_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ER366_3 protein")
The nucleotide sequence of ER366_3 as presently determined is reported in SEQ
ID N0:16. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the ER366_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:17. Amino acids 12 to 24 are a
possible
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 25, or are part of a potential transmembrane domain that includes
the amino-
terminal 20 amino acids of SEQ ID N0:17.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ER366 3 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for ER366_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. No hits were found in the database. The nucleotide and
amino
acid sequences of ER366_3 indicate that it may contain one or more Alu or MIR
repeat
sequences.
Deposit of Clones
2 0 Clones BG481_l, BJ9_l, BK34 3, BP883_2, CI363_l, C0806_l, CT24_3, and
ER366_3 were deposited on February 14,1997 with the American Type Culture
Collection
as an original deposit under the Budapest Treaty and were given the accession
number
ATCC 98331, from which each clone comprising a particular polynucleotide is
obtainable.
All restrictions on the availability to the public of the deposited material
will be
2 5 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
3 0 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,
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_ WO 98/36067 PCT/US98/027b7
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.
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
oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
BG481_1 ~ SEQ ID N0:18
BJ9_1 SEQ ID N0:19
BK34_3 SEQ ID N0:20
2 0 BP883 2 SEQ ID N0:21
CI363_1 SEQ ID N0:22
C0806_1 SEQ ID N0:23
CT24_3 SEQ ID N0:24
ER366 3 SEQ ID N0:25
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
3 0 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;
23
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(b) It should be designed to have a Tm of approx. 80 ° C {assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-3zP ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 pl of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
~g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
2 0 colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
2 5 a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
3 0 filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
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The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, ef al., Bio/Technology 10, 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature form
of such protein may be obtained by expression of the disclosed full-length
polynucleotide
2 0 (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.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
2 5 are transcribed to produce the mRNAs from which cDNA polynucleotide
sequences are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
3 0 be isolated in accordance with known methods using the sequence
information disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
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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,
Biockem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res.
Mol. Biol. 58: I-
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
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
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
2 0 can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al., 2994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
2 5 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
3 0 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
26
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_WO 98/36067 PCT/US98/02767
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
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
polynucleotide, as determined by those of skill in the art. Species homologs
may be
2 0 isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source from the desired species.
Preferably,
species homologs are those isolated from mammalian species.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
2 5 which also encode proteins which are identical, homologous, or related to
that encoded
by the polynucleotides .
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
3 0 reduced stringency conditions, more preferably stringent conditions, and
most preferably
highly stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown W the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at Ieast as
27
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_ WO 98/36067 PCT/US98/02767
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
{bp)~ Buffer' and Buffer'
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA 2 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC T~*; lxSSC
E RNA:RNA s 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 T~*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 T,i*; 4xSSC TH*; 4xSSC
I DNA:RNA s 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA s 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC T~ *; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
2 P DNA:RNA <50 TP*; 6xSSC T,,*; 6xSSC
0
Q RNA:RNA z 50 60C; 4XSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
#: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
2 5 hybridizing a polynucleotide to a target polynucleotide of unknown
sequence, the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
t: SSPE (lxSSPE is 0.15M NaCI, lOmM NaH~P04, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
3 0 (lxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and
wash buffers; washes are
performed for 15 minutes after hybridization is complete.
*TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
28
CA 02280968 1999-08-10
WO 98/36067 PCT/US98102767
C bases). For hybrids between 18 and 49 base pairs in length, Tm{°C) =
81.5 + 16.6(log~o[Na']) + 0.41 (%G+C) -
(600/N), where N is the number of bases in the hybrid, and [Na'1 is the
concentration of sodium ions in the
hybridization buffer ([Na'] for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, j., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Moleciclar 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,
1 S 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
linked" means that the isolated polynucleotide of the invention and an
expression control
2 5 sequence are situated within a vector or cell in such a way that the
protein is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
protein. Mammalian host cells include, for example, monkey COS cells, Chinese
Hamster
3 0 Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells,
human Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
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WO 98/36067 PCT/US98/02767
Alternatively, it may be possible to produce the protein in lower eukaryotes
such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. {the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987), incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
2 0 The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein 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 5 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
3 0 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
CA 02280968 1999-08-10
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InVitrogen, respectively. The protein can also be tagged with an epitope and
subsequently purified by, using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography~(RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of 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,
2 0 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 5 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
3 0 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.
3I
CA 02280968 1999-08-10
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Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be useful
for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
presentinvention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
2 0 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
2 5 information to derive PCR primers for genetic fingerprinting; as a probe
to "subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
3 0 immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that
described in Gyuris et aL, Cell 75:791-803 (1993)) to identify polynucleotides
encoding the
32
CA 02280968 1999-08-10
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other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
2 0 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. ICimmel
eds.,1987.
Nutritional Uses
2 5 Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
or amino acid supplement, use as a carbon source, use as a nitrogen source and
use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
3 0 or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
can be added to the medium in or on which the microorganism is cultured.
33
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_ WO 98/36067 PCTIUS98I02767
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, 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
1 S 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 ai., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
2 0 133:327-342, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 2992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
2 5 Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994;
and
Measurement of mouse and human Interferon~~y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
3 0 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky,
P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
34
CA 02280968 1999-08-10
_ WO 98136067 PCT/US98102767
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Suppressin~~ Activity
2 0 A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
2 5 as effecting the cytolytic activity of NK cells and other cell
populations. These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
3 0 herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various
fungal infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
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WO 98/36067 PCT/US98/02767
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
2 0 the tolerizing agent has ceased. Operationally, tolerance can be
demonstrated by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
2 5 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
3 0 the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand{s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
36
CA 02280968 1999-08-10
WO 98136067 PCT/US98102767
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow ef al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
2 0 Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 5 Administration of reagents which block costimulaNon of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
3 0 the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
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marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
2 0 costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 5 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
3 0 transfected tumor cells are returned to the patient to result in
expression of the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigen{s) on the surface of the tumor cell provides the necessary
38
CA 02280968 1999-08-10
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costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and p2
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
2 0 limitation, those described in: Current Protocols in Immunology, Ed by J.
E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;
Handa et al.,
2 5 J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-
3500,1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et aL, J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-5I2, 1988;
Bertagnolli et al.,
3 0 Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Th1 /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
39
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antibody production, Mond, J.j. and Brunswick, M. In Current Protocols in
Immunology.
j.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by j. E. CoIigan, 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. Imrnunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
254:5071-5079, 2995; Porgador et al., Journal of Experimental_Medicine 182:255-
260, 1995;
2 5 Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
2 0 proteins that prevent apoptosis after superantigen induction and proteins
that regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 2991;
Zacharchuk,
Journal of Immunology 245:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 5 Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation; those described in: Antica et al.,
Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis ReQUlating Activity
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
CA 02280968 1999-08-10
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lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiahon/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and ali of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
2 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 5 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
3 0 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
41
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Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
I62, 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
bums,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
2 0 well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 5 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
3 0 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
42
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circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
2 0 peripheral nervous system diseases and neuropathies, as well as mechanical
and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 5 Parkinsori s disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
3 0 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
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It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) Hssue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
2 0 Assays for wound healing activity include, without limitation, those
described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
2 5 Activin /Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
activities. Inhibins are characterized by their ability to inhibit the release
of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
3 0 invention, alone or in heterodimers with a member of the inhibin a family,
may be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
44
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~i group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
United States Patent 4,798,885. A protein of the invention may also be useful
for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-
782,1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobiiize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
2 0 treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 5 population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
of cells can be readily determined by employing such protein or peptide in any
known
assay for cell chemotaxis.
The activity of a protein of the invention may; among other means, be measured
3 0 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
CA 02280968 1999-08-10
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include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,
1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol.153: 1762-
1768,1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
2 0 described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick
et al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Rece~tor/Ligand Activity
2 5 A protein of the present invention may also demonstrate activity as
receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
3 0 cellular adhesion molecules (such as selectins, integrins and their
ligands) and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
development of cellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
46
,.
CA 02280968 1999-08-10
- WO 98/36067 PCT/US98/02767
limitation, fragments of receptors and ligands} may themselves be useful as
inhibitors of
receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
2 0 suppressing production of other factors which more directly inhibit or
promote an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
2 5 lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
3 0 Cadherin /Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss
or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
47
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_ WO 98/36067 PCT/US98/02767
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
2 0 suppressor role in carcinomas derived from other tissue typea. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
2 5 Cancer cells have also been shown to express cadherins of a different
tissue type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
3 0 reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
48
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CA 02280968 1999-08-10
_ WO 98/36067 PCTIUS98/02767
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
2 0 A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
3 0 agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
49
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WO 98/36067 PCT/US98/OZ767
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproiiferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
{such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
2 0 salts, buffers, stabilizers, solubilizers, and other materials well known
in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
2 5 such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-
8, IL-9, IL-10, IL-11,
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
3 0 pharmaceutical composition to produce a synergistic effect with protein of
the invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
CA 02280968 1999-08-10
_ WO 98/36067 PCT/US98102767
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-sHmulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
2 0 The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
2 5 monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposornal forniulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
3 0 As used herein, the term "therapeutically effective amount" means the
total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
51
CA 02280968 1999-08-10
_ WO 98136067 PCT/US98/027G7
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokirte(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
2 0 cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
2 5 composition of the invention may additionally contain a solid carrier such
as a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
3 0 may be added. The liquid form of the pharmaceutical composition may
further contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. 4'Jhen administered
in liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
52
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CA 02280968 1999-08-10
. WO 98/36067 PCT/US98/027b7
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
2 0 response. Larger doses of protein of the present invention may be
administered until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 IZg
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 lzg to
about 1 mg)
2 5 of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
3 0 will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
53
CA 02280968 1999-08-10
- WO 98/36467 PCT/US98/02'167
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. 85 2149-2154 (1963); J.L. Krstenansky, et
al., FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
2 0 tissue damage. Topical administration may be suitable for wound healing
and tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
2 5 composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
3 0 The choice of matrix material is based on biocompaHbility,
biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciurnphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
54
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CA 02280968 1999-08-10
_WO 98/36067 PCT/US98/02767
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose {CMC). Other preferred sequestering agents include
hyaluronic
2 0 acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
2 5 are prevented from infiltrating the matrix, thereby providing the protein
the opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
3 0 (EGF), platelet derived growth factor (PDGF), transforming growth factors
(TGF-a and
TGF-(3), and insulin-like growth factor (IGF}.
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
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The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
2 0 Treated cells can then be introduced in vivv for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
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WO 98/36067 PCT/US98/027b7
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: 25
(iv} CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) 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 NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2647 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
57
i
CA 02280968 1999-08-10
_ WO 98/36067 PCT/LTS98/02767
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTCCCCGAAA GGTTTGCAGA 60
CGCCTGTGAA GGATGCTGGT
GGTGGGACCG GTAGAGAGGC
AGAAGCAAGA GAGCTACGGTTCAGGTTGGGCACCAGTGAT GCCACTGGTT CTCTGCAAGG120
CGATTCCATG ACAGAAACCGTAGCAGAAAACATCGTTACC AGTATCCTGA AGCAGTTCAC180
CCAGTCTCCA GAGACAGAAGCATCTGCTGATTCTTTTCCA GACACAAAAG TCACTTACGT240
GGACAGGAAA GAGCTTCCTGGGGAAAGGAAAACAAAGACT GAAATAGTTG TGGAGTCTAA300
ACTGACTGAG GATGTTGATGTTTCCGATGAAGCTGGCCTG GACTACCTTT TAAGCAAGGA360
TATTAAGGAA GTGGGGCTGAAAGGCAAGTCAGCCGAGCAG ATGATAGGAG ACATCATCAA420
CCTCGGCCTG AAAGGGAGGGAGGGGAGAGCAAAGGTCGTC AACGTGGAGA TCGTGGAGGA480
GCCCGTGAGT TATGTCAGCGGGGAGAAGCCGGAGGAGTTT TCCGTCCCAT TCAAAGTGGA540
GGAGGTCGAA GATGTGTCGCCAGGCCCCTGGGGGTTGGTT AAGGAGGAGG AAGGTTATGG600
AGAAAGCGAT GTCACATTCTCAGTTAATCAGCATCGAAGG ACCAAGCAGC CCCAGGAGAA660
CACGACTCAC GTGGAAGAAGTGACAGAGGCAGGTGATTCA GAGGGCGAGC AGAGTTATTT720
TGTGTCCACT CCAGATGAACACCCCGGGGGGCACGACAGA GATGACGGCT CGGTGTACGG780
GCAGATCCAC ATCGAGGAGGAATCCACCATCAGGTACTCT TGGCAGGATG AAATCGTGCA840
GGGGACTCGA AGGAGGACACAGAAGGACGGTGCAGTGGGC GAGAAGGTTG TGAAGCCCTT900
GGATGTCCCA GCGCCCTCTCTGGAGGGGGACCTGGGTTCC ACTCACTGGA AAGAACAAGC960
TAGAAGCGGT GAATTTCATGCCGAACCCACAGTCAGAAAA AGAAATTAAA ATACCCCACG1020
AATTCCACAC CTCCATGAAGGGCATCTCCTCCAAGGAGCC CCGGCAGCAG CTGGTGGAGG1080
TCATCGGGCA GCTGGAGGAAACCCTTCCCGAGCGCATGAG GGAGGAGCTG TCCGCCCTCA1140
CCAGAGAGGG GCAGGGTGGGCCGGGGAGCGTTTCCGTGGA TGTCAAGAAG GTCCAGGGTG1200
CTGGTGGCAG TTCCGTGACCCTGGTTGCTGAAGTCAACGT CTCACAAACT GTGGATGCCG1260
ATCGGTTAGA CCTGGAGGAGCTGAGCAAAGATGAGGCCAG TGAGATGGAG AAGGCTGTGG1320
AGTCGGTGGT TCGGGAGAGCCTGAGCAGGCAACGCAGCCC AGCGCCTGGC AGCCCAGATG1380
AGGAAGGTGG AGCGGAGGCCCCGGCTGCTGGCATTCGCTT CAGGCGTTGG GCCACCCGGG1440
58
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CA 02280968 1999-08-10
WO 98136067 PCT/US98102767
AGCTGTACAT CCCTTCAGGC GAGAGCGAGGTTGCTGGTGGGGCCTCTCAC AGCTCGGGAC1500
AGCGCACTCC CCAGGGCCCA GTGTCGGCCACTGTGGAGGTCAGCAGCCCC ACAGGCTTTG1560
CCCAGTCACA GGTGCTGGAG GATGTGAGCCAGGCTGCAAGGCACATAAAA CTCGGCCCCT1620
CTGAAGTCTG GAGGACTGAG CGAATGTCATATGAAGGACCCACTGCAGAA GTGGTGGAGA1680
TGGATGTGAG TAACGTAGAG GCGATCCGCAGCCGGACACAGGAAGCGGGA GCTCTCGGTG1740
TGTCTGACCG TGGTTCCTGG AGAGACGCGGACAGTAGGAATGACCAGGCA GTTGGTGTGA1800
GCTTTAAGGC CTCTGCTGGG GAAGGAGACCAGGCCCACAGAGAACAGGGC AAGGAGCAGG1860
CCATGTTTGA TAAGAAGGTG CAGCTCCAGAGAATGGTAGACCAAAGGTCG GTGATTTCAG1920
ATGAAAAGAA AGTTGCCCTC CTCTATCTAGACAATGAGGAGGAGGAGAAT GATGGGCATT1980
GGTTTTAATA AGCAGAAACA TTTTGTTTTAATGGCAGCCTGTTGGCGACG TGCCAACATC2040
CAAAGGCCTT AACTTATTTT AAGAGGCCGAGGGAGTCTATGAAAATCTCC CCTTTTTTAC2100
TTTTTTAAAG AGTACTCCCG GCATGGTCAATTTCCTTTATAGTTAATCCG TAAAGGTTTC2160
CAGTTAATTC ATGCCTTAAA AGGCACTGCAATTTTATTTTTGAGTTGGGA CTTTTACAAA2220
ACACTTTTTT CCCTGGAGTC TTCTCTCCACTTCTGGAGATGAATTTCTAT GTTTTGCACC2280
TGGTCACAGA CATGGCTTGC ATCTGTTTGAAACTACAATTAATTATAGAT GTCAAAACAT2340
TAACCAGATT AAAGTAATAT ATTTAAGAGTAAATTTTGCTTGCATGTGCT AATATGAAAT2400
AACAGACTAA CATTTTAGGG GAAAAATAAATACAATTTAGACTCTAAAAA GTCTTTTCAA2460
AAAGAAATGG GAAATAGGCA GACTGTTTATGTTAAAAA.AATTCTTGCTAA ATGATTTCAT2520
CTTTAGGAAA AAATTACTTG CCATATAGAGCTAAATTCATCTTAAGACTT GAATGAATTG2580
CTTTCTATGT ACAGAACTTT AAACAATATAGTATTTATGGCGAGGAAAAA F,~~~.AAAAAAA2640
AAAAAAA 2647
(2) INFORMATION FOR EQ ID :
S N0:2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 293 aminoacids
(B) TYPE: amino acid
(C) STRANDE DNESS:
(D) TOPOLOG Y: linear
(ii) MOLECULE TYP E: protein
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CA 02280968 1999-08-10
- WO 98/36067 PCT/US98/02767
(xiy SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Thr Glu Thr VaI Ala Glu Asn Ile Val Thr Ser Ile Leu Lys Gln
1 5 10 15
Phe Thr Gln Ser Pro Glu Thr Glu Ala Ser Ala Asp Ser Phe Pro Asp
20 25 30
Thr Lys Val Thr Tyr Val Asp Arg Lys Glu Leu Pro Gly Glu Arg Lys
35 40 45
Thr Lys Thr Glu Ile Val Val Glu Ser Lys Leu Thr Glu Asp Val Asp
50 55 60
Val Ser Asp Glu Ala Gly Leu Asp Tyr Leu Leu Ser Lys Asp Ile Lys
65 70 75 80
Glu Val Gly Leu Lys Gly Lys Ser Ala Glu Gln Met Ile Gly Asp Ile
85 90 95
Ile Asn Leu Gly Leu Lys Gly Arg Glu Gly Arg Ala Lys Val Val Asn
100 105 110
Val Glu Ile Val Glu Glu Pro Val Ser Tyr Val Ser Gly Glu Lys Pro
115 120 125
Glu Glu Phe Ser Val Pro Phe Lys Val Glu Glu Val Glu Asp Val Ser
130 135 140
Pro Gly Pro Trp Gly Leu Val Lys Glu Glu Glu Gly Tyr Gly Glu Ser
145 150 155 160
Asp Val Thr Phe Ser Val Asn Gln His Arg Arg Thr Lys Gln Pro Gln
165 170 175
Glu Asn Thr Thr His Val Glu Glu Val Thr Glu Ala Gly Asp Ser Glu
180 185 190
Gly Glu Gln Ser Tyr Phe Val Ser Thr Pro Asp Glu His Pro Gly Gly
195 200 205
His Asp Arg Asp Asp Gly Ser Val Tyr Gly Gln Ile His Ile Glu Glu
210 215 220
Glu Ser Thr Ile Arg Tyr Ser Trp Gln Asp Glu Ile Val Gln Gly Thr
225 230 235 240
Arg Arg Arg Thr Gln Lys Asp Gly Ala Val Gly Glu Lys Val Val Lys
245 250 255
Pro Leu Asp Val Pro Ala Pro Ser Leu Glu Gly Asp Leu Gly Ser Thr
260 265 270
His Trp Lys Glu Gln Ala Arg Ser Gly Glu Phe His Ala Glu Pro Thr
275 280 285
r
CA 02280968 1999-08-10
_ WO 98/36067 PCT/US98/02767
Val Arg Lys Arg Asn
290
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1073 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
CACCTGCCTCGGCCTCCCAGAGTGCTGAGATTACAGGCGTGAGCCTCCGCGCCCGGCCCC60
CTTGCAGTTCTCTCTGATTTGGTTTGTTCTGTCTCAGGCTTCTGTGGCAGGACTGGCCCA120
GGGAGGAGGAAGCCAGCAGCACACCTGGGGAATGGGGTCCCGGCCGGGAGGCTTGGCCTC180
TGGGCGACCTCGTCCTGTTTTGTTTGTTTGTTTGTTTGTTTTTTTAAAGGTAAACCTCCT240
GGGCCGCAGATGGCAAAGGGAGTGCCTGGGCCTGGTGACCCAGGGCTGGATCCACCCCTG300
CGGAGCCC'?'GGGCCAGGCAGGTGTCTGCTGCTCACCTGGCTCTGGAGGGCTGCCCTGCAG360
CTGGGCCTGGGGACAGGTCGGCTGTGGGGCAGCTCAGTACCCTCCCTGAGGCTCACGGTG420
GCTCCGAGCATGAGCTCTGCCTCCTGGGCGAGACCCAGCAGTGGACAGCACGGTCCTCAC480
ACCCAGCTCCCTGCACACCCAGGCCAGCCACCCCTCCCGCTCGTGCACAGGCACGCAGAT540
GCGCTCACACGTACACACACACAAATGCACGCCCACTTGCACATGCTCACGCACACGTTC600
ACACATGCACACTCACGCTCACACATGCTGTCACGCATACACACACGCACATACTCCTGC660
ACATGTTCCCATGCATGTGTGTGCACTCGGACCGAGCATCTCCCACGCACCTCTACCCCA720
CCCCAAGCACCTCTCTCCCCCCATGCACCTCTCCCCAACAACACACACAGCCCCCTGCAC780
CGCCCGCCCCCCGCCCCCACCAAGGCCCCAGCCTCTGGCCATCAGTCCTGGTGCCAGAGC840
TTTGCGTGAAGTTCGGGCCGCAGAGTGGCCCGCTGGGACTCCCATGTGCTGCCGTCTGAT900
GTGCTCAGATGGGCTCATCGTTGGTTCGTTTTTACTGTATATTTATAGTAATAAAATCAT960
GCAGCAAAAAAAAAAAAAAAAAAAAAAAAAP~~?~AAAAAAAP~~.AAAAAAAAF,~~.AAAAAAAA102
0
AAAAAA.AAAAP,~~~AAAAAAAP~~~AAAAA.AAp,~~AAAAAAAA1~,~1~AAAAAA.AAAAA 10
7
3
(2) INFORMATION EQ ID
FOR S N0:4:
61
CA 02280968 1999-08-10
_ WO 98/35067 PCTNS98/027b7
(i) SEQUENCE CHARACTERISTICS: -
(A) LENGTH: 199 amino acids _
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ala Lys Gly Val Pro Gly Pro Gly Asp Pro Gly Leu Asp Pro Pro
1 5 10 15
Leu Arg Ser Pro Gly Pro Gly Arg Cys Leu Leu Leu Thr Trp Leu Trp
20 25 30
Arg Ala Ala Leu Gln Leu Gly Leu Gly Thr Gly Arg Leu Trp Gly Ser
35 40 45
Ser Val Pro Ser Leu Arg Leu Thr Val Ala Pro Ser Met Ser Ser Ala
50 55 60
Ser Trp Ala Arg Pro Ser Ser Gly Gln His Gly Pro His Thr Gln Leu
65 70 75 g0
Pro Ala His Pro Gly Gln Pro Pro Leu Pro Leu Val His Arg His Ala
85 90 95
Asp A1a Leu Thr Arg Thr His Thr Gln Met His Ala His Leu His Met
100 105 110
Leu Thr His Thr Phe Thr His Ala His Ser Arg Ser His Met Leu Ser
215 120 125
Arg Ile His Thr Arg Thr Tyr Ser Cys Thr Cys Ser His Ala Cys Val
130 135 140
Cys Thr Arg Thr Glu His Leu Pro Arg Thr Ser Thr Pro Pro Gln Ala
145 150 . 255 160
Pro Leu Ser Pro His Ala Pro Leu Pro Asn Asn Thr His Ser Pro Leu
165 270 175
His Arg Pro Pro Pro Ala Pro Thr Lys Ala Pro Ala Ser Gly His Gln
180 185 190
Ser Trp Cys Gln Ser Phe Ala
195
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
62
,.
CA 02280968 1999-08-10
._ WO 98136067 PCT/US98/02767
(A) LENGTH: 674 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:5:
GGAAATTCCAGCGCTGCTTC ATCTTCCAGTGCTGATAATTATGGCATTAG CCATCCTGAG60
TTTCTGCTATGGTGCTGGAA AATCAGTTCATGTGCTGAGACATATAGGCG GTCCTGAGAG120
CGAACCTCCCCAGGCACTTC GGCCACGGGATAGAAGACGGCAGGAGGAAA TTGATTATAG180
ACCTGATGGTGGAGCAGGTG ATGCCGATTTCCATTATAGGGGCCAAATGG GCCCCACTGA240
GCAAGGCCCTTATGCCAAAA CGTATGAGGGTAGAAGAGAGATTTTGAGAG AGAGAGATGT300
TGACTTGAGATTTCAGACTG GCAACAAGAGCCCTGAAGTGCTCCGGGCAT TTGATGTACC360
AGACGCAGAGGCACGAGAGC ATCCCACGGTGGTACCCAGTCATAAATCAC CTGTTTTGGA420
TACAAAGCCCAAGGAGACAG GTGGAATCCTGGGGGAAGGCACACCGAAAG AAAGCAGTAC480
TGAAAGCAGCCAGTCGGCCA AGCCTGTCTCTGGCCAAGACACATCAGGGA ATACAGAAGG540
TTCACCCGCAGCGGAAAAGG CCCAGCTCAAGTCTGAAGCCGCAGGCAGCC CAGACCAAGG600
CAGCACATACAGCCCCGCAA GAGGTGTGGCTGGACCACGTGGACAGGATC CGGTCAGCAG660
CCCCTGTGGCTAGA 674
(2) INFORMATION
FOR
SEQ
ID N0:6:
(i) S EQUENCE CHARACTERISTICS:
(A} LENGTH: 107 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) OLECULE TYPE: protein
M
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Tyr Gln Thr Gln Arg His Glu Ser Ile Pro Arg Trp Tyr Pro Val
2 5 10 15
Ile Asn His Leu Phe Trp Ile Gln Ser Pro Arg Arg Gln Val Glu Ser
63
CA 02280968 1999-08-10
WO 98/36067 PCT/US98/02767
20 25 30
Trp Gly Lys Ala,His Arg Lys Lys Ala Val Leu Lys Ala Aia Ser Arg
35 40 45
Pro Ser Leu Ser Leu Ala Lys Thr His Gln Gly Ile Gln Lys Val His
50 55 60
Pro Gln Arg Lys Arg Pro Ser Ser Ser Leu Lys Pro Gln Ala Ala Gln
65 70 75 gp
Thr Lys Ala Ala His Thr Ala Pro Gln Glu Val Trp Leu Asp His Val
85 90 95
Asp Arg Ile Arg Ser Ala Ala Pro Val Ala Arg
100 105
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
AACAATGTTT AAATGGAATA ATGTTGAATG TTTACAGGCT GTAAGAACTA TTGTATACAC 60
AAAATAATAC ACAAAGTTTG TACTTTGTGT ACAAATACAA ATTTGTACTT TGTGTACAAA 120
TAATACAAAA AGTTTGTATA CACAAAAAAA F,~1AAAAAAAA p,AAAAAAAA 16 9
{2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1210 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GGTTCTGTGC TCCTGAGATT AGTTCAGATG GTCTAACCAT TGTTCTATAT GTGCATTTTA 60
64
. , ,
CA 02280968 1999-08-10
_ WO 98136067 PCT/US98/02767
GTTAATATTG TGTATTAAAG GATAAGTCTT 120
AATGCTCAAA GTATGTTAAA AATAGATGTA
GTAAATCAGT CCCTTTGTGA ATGTCCTTTT AGGAAGGCCT GTCCTCTGGG180
GTTAGTTTTT
AGTGACCTTT ATTAGTCCAC CCCTTGGAGC GTACTTAGTC ACGGGGATGG240
TAGACATCCT
TGGAAGAGGG AGAAGAGGAA GGGTGAAGGG TGCTAGTATC TCCATATCTA300
AAGGGCTCTT
GACGATGGTT TTAGATGATA ACCACAGGTC TTTTTAGTAA AGTGCCTGTG360
TACAAGAGCG
TTCATTGTGG ACAAAGTTAT TATTTTGCAA TTACGAATGG GGTGACAACT420
CATCTAAGCT
TATGATAAAA ACTAGAGCTA GTGAATTAGC ATACCTTTGT TATAATTGAT480
CTATTTGTAA
AGGATACATC TTGGACATGG AATTGTTAAG GCAGTGTATG TCAGGACTTG540
CCACCTCTGA
TTCATTAGGT TGGCAGCAGA GGGGCAGAAG GGTAGAGATG TATGCAGATG600
GAATTATACA
TGTCCATATA TGTCCATATT TACATTTTGA TGTATGCATC TCTTGGCTGT660
TAGCCATTGA
ACTATAAGAA CACATTAATT CAATGGAAAT AATATTTTAA TGGTATAGAT720
ACACTTTGCT
CTGCTAATGA ATTCTCTTAA AAACATACTG CTGTGTGTTT CATTTTAAAT780
TATTCTGTTG
TGAGCATTAA GGGAATGCAG CATTTAAATC CAATGCTTTT ATCTAGAGGC840
AGAACTCTGC
GTGTTGCCAT TTTTGTCTTA TATGAAATTT AAAGGCAGGA TTACATCTTT900
CTGTCCCAAG
TTTTTTTTTT TTAGCAGTTT GAGTTGGTGT TGGTTATCAG AATACTCATA960
AGTGTATTCT
TAGCTTTGGG ATTTTGAATT GGTAAATATT GAAAAATCAT GATACATACT1020
CATGATGTGT
GTACAGTCTC AGTCCCATAA AATTGGATGT CACAGGATCT AGAAGAATAT1080
TGTGCCTACA
GTCAAACTAT AAACTGCTTG TGATTGTGAA CTTTGCTTGT GTTTTTCAAT1140
TGACTTTGTT
TTCCTATAAT GCACATACTA ACTTTTAAAA ATTTTAAAAG CCTGTAAAAA1200
AATAAAGGTT
1210
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CA 02280968 1999-08-10
WO 98/36067 PCT/US98J02767
Met Leu Leu Ser Arg Gly Val Leu Pro Phe Leu Ser Tyr Met Lys Phe
1 5 10 15
Leu Ser Gln Glu Arg Gln Asp Tyr Ile Phe Phe Phe Phe Phe Ser Ser
20 25 30
Leu Ser Trp Cys Ser Val Phe Leu Val Ile Arg Ile Leu Ile
35 40 45
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2309 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:10:
GCAACACTCCGAGGCCAGGAACGCTCCGTCTGGAACGGCGCAGGTCCCAGCAGCTGGGGT 60
TCCCCGTCAGCCCGTGAGCGGCCATGTCCAACCCCAGCGCCCCACCACCATATGAAGACC 120
GCAACCCCCTGTACCCAGGCCCTCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGC 180
CAGGAGGGTATCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCT 240
ACCCACAGCCCATGCCCCCCACCCACCCGATGCCCATGAACTACGGCCCAGGCCATGGCT 300
ATGATGGGGAGGAGAGAGCGGTGAGTGATAGCTTCGGGCCTGGAGAGTGGGATGACCGGA 360
AAGTGCGACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCA 420
CTGTGGCCATCATTGCTATCTTCACCTTTGTGGAACCTGTCAGCGCCTTTGTGAGGAGAA 480
ATGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCGTTGTCACCTACCTGATCCTTGCCT 540
GCTGCCAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTACTT 600
TTGCCATGGGCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCA 660
TTGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTCAGACCA 720
AGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGTGA 780
CTGGGATTGTCACTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTCCACATGCTCT 840
ATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGGCTTACGACACACAGCTGGTCC 900
TGGGGAACCGGAAGCACACCATCAGCCCGGAGGACTACATCACTGGCGCCCTGCAGATTT 960
66
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_ WO 98/36067 PCT/US98/02767
ACACAGACATCATCTACATC TTCACCTTTGTGCTGCAGCTGATGGGGGAT CGCAATTAAG1020
GAGCAAGCCCCCATTTTCAC CCGATCCTGGGCTCTCCCTTCCAAGCTAGA GGGCTGGGCC1080
CTATGACTGTGGTCTGGGCT TTAGGCCCCTTTCCTTCCCCTTGAGTAACA TGCCCAGTTT1140
CCTTTCTGTCCTGGAGACAG GTGGCCTCTCTGGCTATGGATGTGTGGGTA CTTGGTGGGG1200
ACGGAGGAGCTAGGGACTAA CTGTTGCTCTTGGTGGGCTTGGCAGGGACT AGGCTGAAGA1260
TGTGTCTTCTCCCCGCCACC TACTGTATGACACCACATTCTTCCTAACAG CTGGGGTTGT1320
GAGGAATATGAAAAGAGCCT ATTCGATAGCTAGAAGGGAATATGAAAGGT AGAAGTGACT1380
TCAAGGTCACGAGGTTCCCC TCCCACCTCTGTCACAGGCTTCTTGACTAC GTAGTTGGAG1440
CTATTTCTTCCCCCAGCAAA GCCAGAGAGCTTTGTCCCCGGCCTCCTGGA CACATAGGCC1500
ATTATCCTGTATTCCTTTGG CTTGGCATCTTTTAGCTCAGGAAGGTAGAA GAGATCTGTG1560
CCCATGGGTCTCCTTGCTTC AATCCCTTCTTGTTTCAGTGACATATGTAT TGTTTATCTG1620
GGTTAGGGATGGGGGACAGA TAATAGAACGAGCAAAGTAACCTATACAGG CCAGCATGGA1680
ACAGCATCTCCCCTGGGCTT GCTCCTGGCTTGTGACGCTATAAGACAGAG CAGGCCACAT1?40
GTGGCCATCTGCTCCCCATT CTTGAAAGCTGCTGGGGCCTCCTTGCAGGC TTCTGGATCT1800
CTGGTCAGAGTGAACTCTTG CTTCCTGTATTCAGGCAGCTCAGAGCAGAA AGTAAGGGGC1860
AGAGTCATACGTGTGGCCAG GAAGTAGCCAGGGTGAAGAGAGACTCGGTG CGGGCAGGGA1920
GAATGCCTGGGGGTCCCTCA CCTGGCTAGGGAGATACCGAAGCCTACTGT GGTACTGAAG1980
ACTTCTGGGTTCTTTCCTTC TGCTAACCCAGGGAGGGTCCTAAGAGGAAG GTGACTTCTC2040
TCTGTTTGTCTTAAGTTGCA CTGGGGGATTTCTGACTTGAGGCCCATCTC TCCAGCCAGC2100
CACTGCYTTCTTTGTAATAT TAAGTGCCTTGAGCTGGAATGGGGAAGGGG GACAAGGGTC2160
AGTCTGTCGGGTGGGGGCAG AAATCAAATCAGCCCAAGGATATAGTTAGG ATTAATTACT2220
TAATAGAGAAATCCTAACTA TATCACACAAAGGGATACAACTATAAATGT AATAAAGTTT2280
ATGTCTAGAAGTTAAAAAAA Pu~AAAAAAA 2309
(2) INFORMATION
FOR SEQ
ID N0:11:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 311 acids
amino
(B) TYPE: amino
acid
(C} STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE
TYPE:
protein
67
CA 02280968 1999-08-10
_ WO 98/3b067 PCTIUS98/027b7
(xi?SEQUENCE
DESCRIPTION:
SEQ
ID
N0:11:
MetSer AsnProSer AlaPro ProProTyr Glu ArgAsnPro Leu
Asp
1 5 10 15
TyrPro GlyProPro ProPro GlyGlyTyr Gly ProSerVal Leu
Gln
20 25 30
ProGly GlyTyrPro AlaTyr ProGlyTyr Pro ProGlyTyr Gly
Gln
35 40 45
HisPro AlaGlyTyr ProGln ProMetPro Pro HisProMet Pro
Thr
50 55 60
Met Asn Tyr Gly Pro Gly His Gly Tyr Asp Gly Glu Glu Arg Ala Val
65 70 75 gp
Ser Asp Ser Phe Gly Pro Gly Glu Trp Asp Asp Arg Lys Val Arg His
85 90 95
Thr Phe Ile Arg Lys Val Tyr Ser Ile Ile Ser Val Gln Leu Leu Ile
100 105 110
Thr Val Ala Iie Ile Ala Ile Phe Thr Phe Vai Glu Pro Val Ser Ala
115 120 125
Phe Val Arg Arg Asn Val Ala Val Tyr Tyr Val Ser Tyr Ala Val Phe
130 135 140
Val Val Thr Tyr Leu Ile Leu Ala Cys Cys Gln Gly Pro Arg Arg Arg
145 150 155 160
Phe Pro Trp Asn Ile Ile Leu Leu Thr Leu Phe Thr Phe Ala Met Gly
165 170 175
Phe Met Thr Gly Thr Ile Ser Ser Met Tyr Gln Thr Lys Ala Val Ile
180 185 190
Ile Ala Met Ile Ile Thr Ala Val Val Ser Ile Ser Val Thr Ile Phe
195 200 205
Cys Phe Gln Thr Lys Val Asp Phe Thr Ser Cys Thr Gly Leu Phe Cys
210 215 220
Val Leu Gly Ile Val Leu Leu Val Thr Gly Ile Val Thr Ser Ile Val
225 230 235 240
Leu Tyr Phe Gln Tyr Val Tyr Trp Leu His Met Leu Tyr Ala Ala Leu
245 250 255
Gly Ala Ile Cys Phe Thr Leu Phe Leu Ala Tyr Asp Thr Gln Leu Val
260 265 270
68
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Leu Gly Asn Arg Lys His Thr Ile Ser Pro Glu Asp Tyr Ile Thr Gly
275 280 - 285
Ala Leu Gln Ile Tyr Thr Asp Ile Ile Tyr Ile Phe Thr Phe Val Leu
290 295 300
Gln Leu Met Gly Asp Arg Asn
305 310
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1212 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:12:
CACCGCTACAGCGCGGCGGGCGTCTCGCTGCTCGAGCCGCCGCTGCAGCTCTACTGGACC 60
TGGCTGCTCCAGTGGATCCCGCTCTGGATGGCCCCCAACTCCATCACCCTGCTGGGGCTC 120
GCCGTCAACGTGGTCACCACGCTCGTGCTCATCTCCTACTGTCCCACGGCCACCGAAGAG 180
GCACCATACTGGACATACCTTTTATGTGCACTGGGACTTTTTATTTACCAGTCACTGGAT 240
GCTATTGATGGGAAACAAGCCAGAAGAACAAACTCTTGTTCCCCTTTAGGGGAGCTCTTT 300
GACCATGGCTGTGACTCTCTTTCCACAGTATTTATGGCAGTGGGAGCTTCAATTGCCGCT 360
CGCTTAGGAACTTATCCTGACTGGTTTTTTTTCTGCTCTTTTATTGGGATGTTTGTGTTT 420
TATTGCGCTCATTGGCAGACTTATGTTTCAGGCATGTTGAGATTTGGAAAAGTGGATGTA 480
ACTGAAATTCAGATAGCTTTAGTGATTGTCTTTGTGTTGTCTGCATTTGGAGGAGCAACA 540
ATGTGGGACTATACGTTTTCTTGAACAGCAGTTGACCAACAGATTCCTATTCTAGAAATA 600
AAATTGAAGATCCTTCCAGTTCTTGGATTTCTAGGGCACCAGTGTCTTGTCACCTGGACT 660
CCACATAGGACTAATTATTATACTGGCAATAATGATCTATAAA.AAGTCAGCAACTGATGT 720
GTTTGAAAAGCATCCTTGTCTTTATATCCTAATGTTTGGATGTGTCTTTGCTAAAGTCTC 780
ACAAAAATTAGTGGTAGCTCACATGACCAAAAGTGAACTATATCTTCAAGACACTGTCTT 840
TTTGGGGCCAGGTCTTTTGTTTTTAGACCAGTACTTTAATAACTTTATAGACGAATATGT 900
TGTTCTATGGATGGCAATGGTGATTTCTTCATTTGATATGGTGATATACTTTAGTGCTTT 960
69
i
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_ WO 98/36067 PCT/US98/02767
GTGCCTGCAA ATTTCAAGAC ACCTTCATCT AAATATATTC AAGACTGCAT GTCATCAAGC 1020
ACCTGAACAG GTTCAAGTTC TTTCTTCAAA GAGTCATCAG AATAACATGG ATTGAAGAGA 1080
CTTCCGAACA CTTGCTATCT CTTGCTGCTG CTGTTTCATG GAAGGAGATA TTAAACATTT 1140
GTTTAATTTT TATTTAAGTG TTATACCTAT TTCAGCAAAT AAAATATTTC ATTGCTTAAA 1200
~'~' ~ 1212
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Met Ala Pro Asn Ser Ile Thr Leu Leu Gly Leu Ala Val Asn Val Val
1 5 10 15
Thr Thr Leu Val Leu Ile Ser Tyr Cys Pro Thr Ala Thr Glu Glu Ala
20 25 30
Pro Tyr Trp Thr Tyr Leu Leu Cys Ala Leu Gly Leu Phe Ile Tyr Gln
35 40 45
Ser Leu Asp Ala Ile Asp Gly Lys Gln Ala Arg Arg Thr Asn Ser Cys
50 55 60
Ser Pro Leu Gly Glu Leu Phe Asp His Gly Cys Asp Ser Leu Ser Thr
65 70 75 80
Val Phe Met Ala Val Gly Ala Ser Ile Ala Ala Arg Leu Gly Thr Tyr
85 ~ 90 95
Pro Asp Trp Phe Phe Phe Cys Ser Phe Ile Gly Met Phe Val Phe Tyr
100 105 110
Cys Ala His Trp Gln Thr Tyr Val Ser Gly Met Leu Arg Phe Gly Lys
115 120 125
Val Asp Val Thr Glu Ile Gln Ile Ala Leu Val Ile Val Phe Val Leu
130 135 140
Ser Ala Phe Gly Gly Ala Thr Met Trp Asp Tyr Thr Phe Ser
145 150 155
r
CA 02280968 1999-08-10
WO 98/36067 PCT/US98/02767
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1831 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:14:
AGACAACTAAGCAACATCTGAATACAAATGTAAAGTATGCTGGCTCAGTGTATCTTTTAG60
GTCACTAAAGAGGACACATCAGTAAATAAATTGTTTTCAGTGTAATATTTTTACATAAAA120
ATARATTTCCCCCCCAAAATTTAARATTTATTTARATTCTTGAGACATTTTTTTATGAAA180
AGGAATTAATAACATACATCATCAAAACTTTTCTTAGGAGCGTTGAAACAATATAAGAAG240
GTTACAGAAGGCCTTCTAAAATATGAATTCCCATCTGTATGAGGTACTTCTTTCCAACTC300
AGTGACTTTTTGCCCAGAGACTTGGAAGACTGGGCAATGTTTTCTT.TCCCCTTTCCCAAC360
TCCTGGAGACAAGGCCCTAGGAGCAATTTCCTCCAGTAGGGCAGGCCGTAGTCCTGCTCT420
GTCTTACTTCCCACTGCAGCAGGAACATGTCCTCGCCTCTGATTCCTTCTGTACCAAGCC480
ATTGGTCCATTGAGGTCAATTGAGGACTCACTGTAAATTTTAAATCTGTTAATAAAGCAA540
GGATATTGGCATGTTCCTCTTCTCATCAATATCCTAAAAGACATTTATTTTTTACACACT600
CCTTGGGAAAAATTAACTTTTTTTCACTGAAAATATTTCCTTTTTTGGTTATCTTGATCT660
CAGATTATTTTGTGAAAGAATTTTACTGTACTTAGTTCAAAAGAGTAGAAAGAATGATTT720
ACTATTGCAGACATATGTAGGGTAAAATCATAATTTATTTAAACTGACTGTACAACACCA780
TTTAGAGTTGATATTGACATAAATGTTATTAGCCTACTAATTTGGAACTGCATTTCTCAA840
CAATGCTGGCAAGCATCTTCCGTACTTAGCATACCAAGTTGTAGGGGAGAGACTGTGTAT900
ATATTTTTTAAAAGCAATCCAATGGATTTGTTTTTGTTCATATTTTGAAAACAACTCGAA960
GGATTTTTCTTATTTTAGAAGGTAGAAAATATGTGGACCTGTGTTTTAAATTAATATGTA1020
TTTATAAATTATAACATTGAATGATACAACAGAGCTCTAATATTGATGTTCTCACTTTCT1080
GATATTTAATTTTTATAAATGTTTTTGTAAGTAACTAATTGTAGTATTGCTTTTAAATAG1140
TTTTAAAATCTATCTCAAATAAGTTCTGCCCAGACCTATTTCCTTAGGACAGTATTCTAA1200
71
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AGTTCAGTAG TCCAGTGTGA GCTTGAAATACTCCTAGATCCCGTCAGTGA TTTTTATATA1260
TACCAATACA CAAGCAAATT ATTAATATACCAAAAACCTAATTTCTGCAT TGCATTAATA1320
AAATACTTGT TATTTTGCCT TTATCCAATTCAGTTGATTAGGTGAAATAG AAAATTAATC1380
TTTTCAAATG TGCATATGAT CATAATTTTTCAAAATGTTACTGTGGTCAT TTTTGGTGCA1440
TGATGCCAAG TTTATCTTTA GTTAGCCATTGCCACCTGATATGTAATGAC AAATGTTTTA1500
CTATCTGATC ATTGGGTTGT AGATTAAACTATTTTTTTTCTCTGTAGATT CTCACTACAT1560
TTTCAAATAT GTACTTGGAA AAAGCTGCAAAGTGCTACCTATAAGCAAAT TAGTAACTTT1620
GGCCTTATCT ATGCATAGTG TTCGCTCAGTTTGGTTTTGATAAATTGATT GACTTGCAGT1680
TCCTTTGTAG TATTTTAACT TATTGTGATGTAATGAATTTTTTGAAATGT TTATTTGATT1740
AGCTGTTAAA ATATAGGAAA GAATGTAGCTTTGCATGAAATAAATTACAT TTCTACAAGT1800
Tp~AA.A.AAAA F~~ AAAAA.AP'AA 18
p~~AAAAAP AA 31
(2) INFORMATION FOR SEQ :
ID N0:15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino a cids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Val Asp Ile Asp Ile Asn Val I1e Ser Leu Leu Ile Trp Asn Cys Ile
1 5 10 15
Ser Gln Gln Cys Trp Gln Ala Ser Ser Val Leu Ser Ile Pro Ser Cys
20 2~5 30
Arg Gly Glu Thr Val Tyr Ile Phe Phe Lys Ser Asn Pro Met Asp Leu
35 40 45
Phe Leu Phe Ile Phe
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 980 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
72
,. ,
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:16:
CTAAAGTTTA CTGGTGAGGA GCTTGAACTG AAGAATCATA CTCCAGCCTG60
AGTAGAAGCA
GGTGACAGAG CAAGACTCTG TCTCAAAAAA TAAACAATAG TATCCATCTT120
AAAAATTATT
CCCACAATTA CTATTAAAGA TTCTTAAAAT ATTGAAGGAA ACGAATTATG180
TTCTGGAAAT
CTTTAAGATT TTAAACCTAC AGAGTAAGTT TTGCCATTTA CTAGCTGTGT240
AATTCTGTCT
AAACTTGGAG AGATCAGCTT GAGACTCTCT TTTTTAATCT GTCAAATGGG300
GAGCCTCATT
TCTCAGAAAA TTGTGAGGAT TAAATGAAAT AGTTGTTTAG AACAGTTCTT360
ATTACCTGTA
GGAACATAGT AAAAACTCAG AAAGTGTTCA GAGTATATTA TTGTGATGTG420
GTAAAATGTT
GGAATTGCTG ATCCAGTTGA CTCAATTCAC ACTGGATTTG AATAAGGAGA480
ACTGCAAATT
TGAAGATTGA GTCACATTGT TCTCTTTGAT TTAGACAACA TCACTCAAAC540
CCATTTTCTT
AACCCTATTA CTTCAGTAGC AATGAACTGT TATTAAGCPT CTCACTAGTT600
CTCTGGATTT
CCTTTTCTTC AGCTGTATGG CACCCTGTCT CAGAGGCTCC TCAGCTGGGT660
TCCTGTACAC
AAGGTGAGCC AACGTTACCA GGAGTATATG ATTTCAAAGT CTTTCATAGA720
CTGAGAGGCC
AGGCTGTGCT TGGGCAAGTA GAACTTTTCA CCAGAGATGT GAAGTTATCA780
TCATACAGTC
AGGTCAGAGA AGAGGAAAAG AGACTCAGAG CTCTAGTCTT TCATTCGGGA840
AAACTGTGTT
ACAAATGGTG TCGTACCAAA TGGCTGAAAA TTACTCTGAG AAACATCTCT900
GATCCCAGGC
CTCTTCTGAA TTAAACTTGC ACAGTGGCAA P~~.AAA.AAAAA AAAAAAAAAA9
P.,~~ 6
0
p,~~~AAAAAAA 1'~~i~AAAP.AAA ~ 9
8
0
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Asn Cys Leu Trp Ile Leu Leu Ser Ile Ser Leu Val Pro Phe Leu
1 5 10 15
Gln Leu Tyr Gly Thr Leu Ser Ser Cys Thr Pro Glu Ala Pro Gln Leu
20 25 30
Gly Lys Val Ser Gln Arg Tyr Gln Glu Tyr Met Leu Arg Gly His Phe
35 40 45
Lys Val Phe His Arg Arg Leu Cys Leu Gly Lys
50 55
(2) INFORMATION FOR SEQ ID N0:18:
(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 N0:18:
TNTCTGTCATG GAATCGCCTT GCAGAGAA 2g
(2) INFORMATION FOR SEQ ID N0:19:
(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:
CNGCCACAGAA GCCTGAGACA GAACAAAC 2g
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
TNAAATGCCCG GAGCACTTCA GGGCTCTT 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:
TNTTATAGTAC AGCCAAGAGA TGCATACA 29
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ANCAGAAGATG GTGACTGAAA TGGATACC 2g
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02280968 1999-08-10
WO 98136067 PCT/US98102'767
(D) TOPOLOGY: linear
(ii) MOLECULE TxPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GNAGACAACAC AAAGACAATC ACTAAAGC 2g
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CNTACAACTTG GTATGCTAAG TACGGAAG 2g
(2) INFORMATION FOR SEQ ID N0:25:
(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 N0:25:
GNAGTAATAGG GTTGTTTGAG TGATGTTG 2g
76
