Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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10
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of the following applications: Ser.
No.
60/XXX,XXX (converted to a provisional application from non-provisional
application Ser.
No. 08/786,161), filed January 21,1997; and Ser. No. 08/877,035, filed June
16,1997, which
is a continuation-in-part of Ser. No. 60/XXX,XXX (converted to a provisional
application
2 0 from non-provisional application Ser. No. 08/786,161), filed January
21,1997; all of which
are incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
2 5 such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
3 0 such as lymphokines, interferons, CSFs and interleukins) has matured
rapidly over the
past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA/amino acid sequence of
the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
3 5 cloning). More recent "indirect" cloning techniques such as signal
sequence cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA / amino acid sequences for proteins that are known to have biological
activity by
4 0 virtue of their secreted nature in the case of leader sequence cloning, or
by virtue of the
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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.
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 poiynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
{b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 101 to nucleotide 428;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 325 to nucleotide 461;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AA365_1 deposited under accession
number ATCC 98296;
{e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AA365_1 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AA365_1 deposited under accession number
2 0 ATCC 98296;
(g} a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AA365_1 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
2 5 (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
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
3 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
NO:1 from nucleotide 101 to nucleotide 428; the nucleotide sequence of SEQ ID
NU:1
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from nucleotide 325 to nucleotide 461; the nucleotide sequence of the full-
length protein
coding sequence of clone AA365_1 deposited under accession number ATCC 98296;
or the
nucleotide sequence of the mature protein coding sequence of clone AA365_1
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone AA365_1 deposited under accession number ATCC 98296. 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 76 to amino
acid
109.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 76 to
amino acid 109;
(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
2 0 AA365_1 deposited under accession number ATCC 98296;
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 76 to amino acid 109.
In one embodiment, the present invention provides a composition comprising an
2 5 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 245 to nucleotide 421;
3 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 413 to nucleotide 421;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BL67_2 deposited under accession
number ATCC 98296;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BL67 2 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BL67 2 deposited under accession number
ATCC 98296;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BL67 2 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(i} a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity;
{j) a polynucleotide which is an allelic variant of a polynucleotide of
(a~(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 245 to nucleotide 421; the nucleotide sequence of SEQ ID
N0:3 from
2 0 nucleotide 413 to nucleotide 421; the nucleotide sequence of the full-
length protein coding
sequence of clone BL67 2 deposited under accession number ATCC 98296; or the
nucleotide sequence of the mature protein coding sequence of clone BL67 2
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 5 clone BL67 2 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:4 from amino acid 1 to amino
acid 20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3 or SEQ ID N0:5.
3 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
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(b) the amino acid sequence of SEQ ID N0:4 from amino acid I to
amino acid 20;
(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
BL67_2 deposited under accession number ATCC 98296;
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 20.
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
N0:6;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:6 from nucleotide 292 to nucleotide 468;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BN130_1 deposited under accession
number ATCC 98296;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BN130_1 deposited under accession number ATCC 98296;
2 0 (e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BN130 1 deposited under accession number
ATCC 98296;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BN130_1 deposited under accession number ATCC 98296;
2 5 (g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:7;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:7 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
3 0 (a}-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ; and
(k) a polynudeotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(h).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:6 from nucleotide 292 to nucleotide 468; the nucleotide sequence of the
full-length
protein coding sequence of clone BN130_1 deposited under accession number ATCC
98296; or the nucleotide sequence of the mature protein coding sequence of
clone BN130_1
deposited under accession number ATCC 98296. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BN130_1 deposited under accession number ATCC 98296.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:6 or 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
consisting of:
(a) the amino acid sequence of SEQ ID N0:7;
(b) fragments of the amino acid sequence of SEQ ID N0:7; and
(c) the amino acid sequence encoded by the cDNA insert of clone
BN130_1 deposited under accession number ATCC 98296;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:7.
In one embodiment, the present invention provides a composition comprising an
2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 137 to nucleotide 2146;
2 5 {c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 218 to nucleotide 2146;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 189 to nucleotide 397;
(e) a polynucleotide comprising the nucleotide sequence of the full-
3 0 length protein coding sequence of clone CG99 2 deposited under accession
number ATCC 98296;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CG99_2 deposited under accession number ATCC 98296;
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(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CG99_2 deposited under accession number
ATCC 98296;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CG99 2 deposited under accession number ATCC 98296;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 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
N0:9 from nucleotide 137 to nucleotide 2146; the nucleotide sequence of SEQ ID
N0:9
from nucleotide 218 to nucleotide 2146; the nucleotide sequence of SEQ ID N0:9
from
nucleotide 189 to nucleotide 397; the nucleotide sequence of the full-length
protein coding
2 0 sequence of clone CG99_2 deposited under accession number ATCC 98296; or
the
nucleotide sequence of the mature protein coding sequence of clone CG99_2
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CG99_2 deposited under accession number ATCC 98296. In yet other
preferred
2 5 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:10 from amino acid 50 to amino
acid
87.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
3 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
. consisting of:
(a) the amino acid sequence of SEQ ID N0:10;
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(b) the amino acid sequence of SEQ ID N0:10 from amino acid 50 to
amino acid 87;
(c) fragments of the amino acid sequence of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CG99 2 deposited under accession number ATCC 98296;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10 or the amino acid
sequence
of SEQ ID N0:10 from amino acid 50 to amino acid 87.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 123 to nucleotide 1247;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 1 to nucleotide 419;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone C0618_1 deposited under accession
number ATCC 98296;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C0618_1 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequenre of the mature
protein coding sequence of clone C0618_1 deposited under accession number
ATCC 98296;
2 5 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C0618_1 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the
3 0 amino acid sequence of SEQ ID N0:12 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
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(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:11 from nucleotide 123 to nucleotide 1247; the nucleotide sequence of SEQ
ID N0:11
from nucleotide 1 to nucleotide 419; the nucleotide sequence of the full-
length protein
coding sequence of clone C0618_1 deposited under accession number ATCC 98296;
or the
nucleotide sequence of the mature protein coding sequence of clone C0618_1
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone C0618_1 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:12 from amino acid 1 to amino
acid
99.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:11.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:12;
2 0 (b) the amino acid sequence of SEQ ID N0:12 from amino acid 1 to
amino acid 99;
(c) fragments of the amino acid sequence of SEQ ID N0:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C0618_1 deposited under accession number ATCC 98296;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:12 or the amino acid
sequence
of SEQ ID N0:12 from amino acid 1 to amino acid 99.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
3 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 208 to nucleotide 402;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 184 to nucleotide 300;
(d) a poiynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone C0629_2 deposited under accession
number ATCC 98296;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C0629_2 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone C0629 2 deposited under accession number
ATCC 98296;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C0629 2 deposited under accession number ATCC 98296;
(h) a polynucleotfde encoding a protein comprising the amino acid
sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity;
(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 polynudeotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 208 to nucleotide 402; the nucleotide sequence of SEQ ID
N0:13
2 5 from nucleotide 184 to nucleotide 300; the nucleotide sequence of the full-
length protein
coding sequence of clone C0629_2 deposited under accession number ATCC 98296;
or the
nucleotide sequence of the mature protein coding sequence of clone C0629 2
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
3 0 clone C0629 2 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:14 from amino acid 1 to amino
acid
31.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) the amino acid sequence of SEQ ID N0:14 from amino acid 1 to
amino acid 31;
(c) fragments of the amino acid sequence of SEQ ID N0:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C0629 2 deposited under accession number ATCC 98296;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:14 or the amino acid
sequence
of SEQ ID N0:14 from amino acid 1 to amino acid 31.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:15 from nucleotide 94 to nucleotide 1059;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 1 to nucleotide 387;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CT645_1 deposited under accession
2 5 number ATCC 98296;
(e} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CT645_1 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of done CT645_1 deposited under accession number
3 0 ATCC 98296;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CT645_1 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:16;
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{i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:16 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(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
N0:15 from nucleotide 94 to nucleotide 1059; the nucleotide sequence of SEQ ID
N0:15
from nucleotide 1 to nucleotide 387; the nucleotide sequence of the full-
length protein
coding sequence of clone CT645_1 deposited under accession number ATCC 98296;
or the
nucleotide sequence of the mature protein coding sequence of clone CT645_1
deposited
under accession number ATCC 98296. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CT645_1 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID NOa6 from amino acid 1 to amino
acid
98.
2 0 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:15.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
2 5 (a) the amino acid sequence of SEQ ID N0:16;
(b) the amino acid sequence of SEQ ID N0:16 from amino acid 1 to
amino acid 98;
(c) fragments of the amino acid sequence of SEQ ID N0:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone
3 0 CT645_1 deposited under accession number ATCC 98296;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid
sequence
of SEQ ID N0:16 from amino acid 1 to amino acid 98.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 640 to nucleotide 1029;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 611 to nucleotide 814;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CW383_1 deposited under accession
number ATCC 98296;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CW383_1 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CW383_1 deposited under accession number
ATCC 98296;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CW383_1 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:18;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:18 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(g) above;
2 5 (k) a polynucleotide which encodes a species homologue of the protein
of (h) or {i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the poiynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
3 0 N0:17 from nucleotide 640 to nucleotide 1029; the nucleotide sequence of
SEQ ID N0:17
from nucleotide 611 to nucleotide 814; the nucleotide sequence of the full-
length protein
coding sequence of clone CW3$3_1 deposited under accession number ATCC 98296;
or
the nucleotide sequence of the mature protein coding sequence of clone CW383_1
deposited under accession number ATCC 98296. In other preferred embodiments,
the
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polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CW383_1 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:18 kom amino acid 1 to amino
acid
63.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected kom
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:18;
(b) the amino acid sequence of SEQ ID N0:18 kom amino acid 1 to
amino acid 63;
(c) kagments of the amino acid sequence of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CW383_1 deposited under accession number ATCC 98296;
the protein being substantially kee kom other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ iD N0:18 or the amino acid
sequence
of SEQ ID N0:18 kom amino acid 1 to amino acid 63.
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
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:19 kom nucleotide 509 to nucleotide 718;.
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DN167_1 deposited under accession
number ATCC 98296;
(d) a polynucleotide encoding the full-length protein encoded by the
3 0 cDNA insert of clone DN167_1 deposited under accession number ATCC 98296;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DN167_1 deposited under accession number
ATCC 98296;
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(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DN167_1 deposited under accession number ATCC 98296;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:20;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:20 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ; and
(k) a polynucleotide 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
N0:19 from nucleotide 509 to nucleotide 718; the nucleotide sequence of the
full-length
protein coding sequence of clone DN167_1 deposited under accession number ATCC
98296; or the nucleotide sequence of the mature protein coding sequence of
clone
DN167_1 deposited under accession number ATCC 98296. In other preferred
embodiments, the polynucleotide encodes the full-length or mature protein
encoded by
the cDNA insert of clone DN167_1 deposited under accession number ATCC 98296.
In
2 0 yet other preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising the amino acid sequence of SEQ ID N0:20 from
amino acid
1 to amino acid 65.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
2 5 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:20;
(b) the amino acid sequence of SEQ ID N0:20 from amino acid 1 to
3 0 amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID N0:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone
DN167 1 deposited under accession number ATCC 98296;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:20 or the amino acid
sequence
of SEQ ID N0:20 from amino acid 1 to amino acid 65.
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
N0:21;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:21 from nucleotide 177 to nucleotide 470;
(c) a polynucleoHde comprising the nucleotide sequence of SEQ ID
N0:21 from nucleotide 237 to nucleotide 470;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DN711 2 deposited under accession
number ATCC 98296;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone DN711 2 deposited under accession number ATCC 98296;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DN711 2 deposited under accession number
ATCC 98296;
2 0 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DN711 2 deposited under accession number ATCC 98296;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:22;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:22 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
3 0 (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
N0:21 from nucleotide 177 to nucleotide 470; the nucleotide sequence of SEQ ID
N0:21
from nucleotide 237 to nucleotide 470; the nucleotide sequence of the full-
length protein
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coding sequence of clone DN711 2 deposited under accession number ATCC 98296;
or
the nucleotide sequence of the mature protein coding sequence of clone DN711 2
deposited under accession number ATCC 98296. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone DN711 2 deposited under accession number ATCC 98296. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:22 from amino acid 1 to amino
acid
94.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:21.
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 N0:22;
(b) the amino acid sequence of SEQ ID N0:22 from amino acid 1 to
amino acid 94;
(c) fragments of the amino acid sequence of SEQ ID N0:22; and
(d) the amino acid sequence encoded by the cDNA insert of clone
DN711 2 deposited under accession number ATCC 98296;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequenre of SEQ ID N0:22 or the amino acid
sequence
of SEQ ID N0:22 from amino acid 1 to amino acid 94.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
2 5 yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
3 0 (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.
17
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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 Garner. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
2 0 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
2 5 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
3 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.
18
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Clone "AA365 1"
A polynucleotide of the present invention has been identified as clone "AA365
1".
AA365_1 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. AA365_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "AA365_1 protein').
The nucleotide sequence of AA365_1 as presently determined is reported in SEQ
ID N0:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AA365_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:2. Another possible reading frame
and
predicted amino acid sequence encoded by AA365_1 is encoded by base pairs 157-
610 of
SEQ ID NO:1 and is reported in SEQ ID N0:33. Amino acids 9 to 21 of SEQ ID
N0:33 are
a predicted leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 22 of SEQ ID N0:33, or are a transmernbrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AA365_1 should be approximately 700 bp.
The nucleotide sequence disclosed herein for AA365_1 was searched against the
2 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AA365_1 demonstrated at least some similarity with
sequences
identified as AA019239 (ze56c09.s1 Scares retina N2b4HR Homo sapiens cDNA
clone
362992 3'). Based upon sequence similarity, AA365_1 proteins and each similar
protein
or peptide may share at least some activity. The TopPredII computer program
predicts
2 5 two potential transmembrane domains within the AA365_1 protein sequence
centered
around amino acids 33 and 73 of SEQ ID N0:2, respectively; amino acids 14 to
26 are also
a possible leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 27 of SEQ ID N0:2.
3 0 Clone "BL67 2"
A polynucleotide of the present invention has been identified as clone "BL67
2".
BL67_2 was isolated from a human adult testes cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
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analysis of the amino acid sequence of the encoded protein. BL67 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"BL67_2 protein').
The nucleotide sequence of the 5' portion of BL67_2 as presently determined is
reported in SEQ ID N0:3. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:4. The predicted amino acid
sequence of
the BL67_2 protein corresponding to the foregoing nucleotide sequence is
reported in SEQ
ID N0:4. Amino acids 44 to 56 are a predicted leader/signal sequence, with the
predicted mature amino acid sequence beginning at amino acid 57, or are a
transmembrane domain. Additional nucleotide sequence from the 3' portion of
BL67_2,
including the polyA tail, is reported in SEQ ID N0:5.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BL67_2 should be approximately 970 bp.
The nucleotide sequence disclosed herein for BL67_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. No hits were found in the database.
Clone "BN130 1"
A polynucleotide of the present invention has been identified as clone
"BN130_1".
2 0 BN130_1 was isolated from a human adult placenta cDNA library using
methods which
are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BN130_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BN130_1 protein').
The nucleotide sequence of the 5' portion of BN130_1 as presently determined
is
reported in SEQ ID N0:6. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:7. The predicted amino acid
sequence of
the BN130_1 protein corresponding to the foregoing nucleotide sequence is
reported in
3 0 SEQ ID N0:7. Additional nucleotide sequence from the 3' portion of
BN130_1, including
the polyA tail, is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BN130_1 should be approximately 1200 bp.
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The nucleotide sequence disclosed herein for BN130_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BN130_1 demonstrated at least some similarity with
sequences
identified as AA023887 (mh93g07.r1 Soares mouse placenta 4NbMP13.5 14.5 Mus
musculus cDNA clone 458556 5'), T99892 (ye68f02.r1 Homo sapiens cDNA clone
122907
5'), U65410 (Human Mad2 (hsMAD2) mRNA, complete cds), and 223859 (H. Sapiens
partial cDNA sequence; clone 81C04; strand (-), single read). The predicted
amino acid
sequence disclosed herein for BN130_1 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
BN130_1 protein demonstrated at least some similarity to sequences identified
as D37934
(Rat mRNA for 5E5 antigen, complete cds). Based upon sequence similarity,
BN130_1
proteins and each similar protein or peptide may share at least some activity.
Clone "CG99 2"
A polynucleotide of the present invention has been identified as clone "CG99
2".
CG99 2 was isolated from a human adult testes cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CG99 2 is a full-
length clone,
2 0 including the entire coding sequence of a secreted protein (also referred
to herein as
"CG99_2 protein").
The nucleoiide sequence of CG99_2 as presently determined is reported in SEQ
ID N0:9. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CG99 2 protein corresponding to the
foregoing
2 5 nucleotide sequence is reported in SEQ ID NO:10. Amino acids 15 to 27 are
a predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 28, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CG99_2 should be approximately 2400 bp.
3 0 The nucleotide sequence disclosed herein for CG99_2 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CG99 2 demonstrated at least some similarity with
sequences
identified as L03813 Rattus norvegicus neurotrophin-3 receptor (trkC) mRNA,
complete
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cds). Based upon sequence similarity, CG99 2 proteins and each similar protein
or
peptide may share at least some activity.
Clone "C0618 1"
A polynucleotide of the present invention has been identified as clone
"C0618_1".
C0618_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. C0618_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "C0618_1 protein')
The nucleotide sequence of C0618_1 as presently determined is reported in SEQ
ID N0:11. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the C0618_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
C0618_1 should be approximately 1750 bp.
The nucleotide sequence disclosed herein for C0618_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. C0618_1 demonstrated at least some similarity with
sequences
identified as D87074 (Human male bone marrow myeloblast mRNA for KIAA0237
protein, complete cds), H34957 (EST110575 Rattus sp. cDNA), and T33172
(EST56933
Homo sapiens cDNA 3' end similar to None). The predicted amino acid sequence
disclosed herein for C0618_1 was searched against the GenPept and GeneSeq
amino acid
2 5 sequence databases using the BLASTX search protocol. The predicted C0618_I
protein
demonstrated at least some similarity to sequences identified as AF007836
(rab3 effector
[Rattus norvegicus]), D87074 (KIAA0237 protein; similar to a C.elegans protein
encoded
in cosmid T10A3(U41035) [Homo sapiens]), and D90195 (polyprotein [Japanese
encephalitis virus]). C0618_1 protein showed at least some similarity to rat
Rim, a
3 0 putative Rab3 effector. Rim is composed of an amino-terminal zinc-finger
motif and
carboxy-terminal PDZ and C2 domains. It binds only to GTP (but not to GDP)-
complexed
Rab3, and interacts with no other Rab protein. Rim is localized to presynaptic
active
zones in conventional synapses, and to presynaptic ribbons in ribbon synapses
(Wang et
al., 1997, Nature 388(6642): 593-598, incorporated by reference herein). Based
upon
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sequence similarity, C0618_1 proteins and each similar protein or peptide may
share at
least some activity. The TopPredII computer program predicts a potential
transmembrane
domain within the C0618_1 protein sequence centered around amino acid 350 of
SEQ ID
N0:12.
Clone "C0629 2"
A polynucleotide of the present invention has been identified as clone "C0629
2".
C0629_2 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. C0629_2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "C0629_2 protein").
The nucleotide sequence of C0629_2 as presently determined is reported in SEQ
ID N0:13. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the C0629_2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:14. Another possible reading
frame and
predicted amino acid sequence encoded by C0629_2 is encoded by basepairs 417
to 632
of SEQ ID N0:13 and is reported in SEQ ID N0:34; this alternative open reading
frame
2 0 could be joined to the reading frame reported in SEQ ID N0:14 if an
insertion or deletion
resulting in a frameshift was made in the sequence of SEQ ID N0:13. The
TopPredII
computer program predicts a potential transmembrane domains within the
alternative
C0629_2 protein sequence centered around amino acid 50 of SEQ ID N0:34.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 C0629_2 should be approximately 1800 bp.
The nucleotide sequence disclosed herein for C0629_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. C0629_2 demonstrated at least some similarity with
sequences
identified as U78857 (Rattus norvegicus calcium calmodulin dependent lcinase
CPG16
3 0 (cpgl6) mRNA, complete cds) and W77588 (me67b11.r1 Soares mouse embryo
NbME13.5
14.5 Mus musculus cDNA). The predicted amino acid sequence disclosed herein
for
C0629_2 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted C0629 2 protein demonstrated
at least
some similarity to sequences identified as U78857 (calcium calmodulin
dependent kinase
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CPG16 [Rattus norvegicus]). Based upon sequence similarity, C0629 2 proteins
and each
similar protein or peptide may share at least some activity.
Clone "CT645 1"
A polynucleotide of the present invention has been identified as clone
"CT645_1".
CT645_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. CT645_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CT645_1 protein").
The nucleotide sequence of CT645_1 as presently determined is reported in SEQ
ID N0:15. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CT645 1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CT645_1 should be approximately 2560 bp.
The nucleotide sequence disclosed herein for CT645_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. CT645_1 demonstrated at least some similarity with
sequences
identified as AA430737 (zw20g12.r1 Soares ovary tumor NbHOT Homo sapiens cDNA
clone 769894 5'), H34543 (EST111568 Rattus sp. cDNA 5' end), and N34881
(yy46g12.s1
Homo sapiens cDNA clone 276646 3' similar to contains element MER37 repetitive
element). Based upon sequenre similarity, CT645_1 proteins and each similar
protein or
2 5 peptide may share at least some activity. The TopPredII computer program
predicts two
potential transmembrane domains within the CT645_1 protein sequence centered
around
amino acids 75 and 275 of SEQ ID N0:16, respectively; amino acids 92 to 104 of
SEQ ID
N0:16 are a possible leader/signal sequence, with the predicted mature amino
acid
sequence beginning at amino and 105. The nucleotide sequence of CT645_1
indicates that
3 0 it may contain an Alu repetitive element.
Clone "CW383 1"
A polynucleotide of the present invention has been identified as clone
"CW383_1".
CW383_1 was isolated from a human fetal brain cDNA library using methods which
are
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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. CW383_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CW383_1 protein").
The nucleotide sequence of CW383_1 as presently determined is reported in SEQ
ID N0:17. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CW383_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:18.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CW383_1 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for CW383_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CW383_1 demonstrated at least some similarity with
sequences
identified as AA037470 (zk36g04.s1 Soares pregnant uterus NbHPU Homo Sapiens
cDNA
clone 484950 3'), AA037478 (zk36h05.s1 Soares pregnant uterus NbHPU Homo
sapiens
cDNA clone 484953 3'), AA088729 (z189c09.r1 Stratagene colon (#937204) Homo
Sapiens
cDNA clone 511792 5'), AA098551 (mk18f12.r1 Soares mouse p3NMF19.5 Mus
musculus
cDNA clone 493295 5'), 875968 (yi62d07.r1 Homo sapiens cDNA clone 143821 5'),
and
2 0 X85681 (H.sapiens mRNA for expressed sequence tag, clone CAM tESTIF6 (B)).
Based
upon sequence similarity, CW383_1 proteins and each similar protein or peptide
may
share at least some activity.
Clone "DN167 1"
2 5 A polynucleotide of the present invention has been identified as clone
"DN167 1".
DN167 1 was isolated from a human fetal brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. DN167_1 is a full-
length
3 0 clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "DN167_1 protein")
The nucleotide sequence of DN167_1 as presently determined is reported in SEQ
ID N0:19. What applicants presently believe to be the proper reading frame and
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predicted amino acid sequence of the DN167_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:20.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
done
DN167_1 should be approximately 850 bp.
The nucleotide sequence disclosed herein for DN167_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. DN167 1 demonstrated at least some similarity with
sequences
identified as AA405121 (zu65dOl.r1 Soares testis NHT Homo sapiens cDNA done
742849
5'), AC000045 (00(?64; HTGS phase 3, complete sequence), 851018 (yg71b02.s1
Homo
sapiens cDNA clone 38639 3' similar to contains Alu repetitive
element;contains MSR1
repetitive element), and 270233 (Human DNA sequence from cosmid V870H8,
between
markers DXS366 and DXS87 on dlromosome X contains ESTs). Based upon sequence
similarity, DN167_1 proteins and eadl similar protein or peptide may share at
least some
activity. The nucleotide sequence of DN167_1 indicates that it may contain an
Alu
repetitive element.
Clone "DN711 2"
A polynudeotide of the present invention has been identified as clone "DN711
2".
DN711_2 was isolated from a human fetal brain cDNA library using methods which
are
2 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. DN71I 2 is a full-
length
done, including the entire coding sequence of a secreted protein (also
referred to herein
as "DN711 2 protein").
2 5 The nucleotide sequence of DN711 2 as presently determined is reported in
SEQ
ID N0:21. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the DN711 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:22. Amino acids 8 to 20 are a.
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
3 0 amino acid 21, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
done
DN711 2 should be approximately 700 bp.
The nucleotide sequence disclosed herein for DN711 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
26
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FASTA search protocols. DN711 2 demonstrated at least some similarity with
sequences
identified as AA127018 (z122a03.s1 Scares pregnant uterus NbHPU Homo Sapiens
cDNA
clone 502636 3'), AA128221 (z122a03.r1 Scares pregnant uterus NbHPU Homo
sapiens
cDNA clone 502636 5'), AA444139 (zv51f01.r1 Scares testis NHT Homo sapiens
cDNA
clone 757177 5'), AA460975 (zx63c11.sI Scares total fetus Nb2HF8 9w Homo
Sapiens),
D62671 (Human aorta cDNA 5'-end GEN-312F07), D79837 (Human aorta cDNA 5'-end
GEN-335E12), D79860 (Human aorta cDNA 5'-end GEN-342C02), 824595 (yg36h06.r1
Homo sapiens cDNA clone 346315'), and 840285 (yf80h03.s1 Homo sapiens cDNA
clone).
Based upon sequence similarity, DN711 2 proteins and each similar protein or
peptide
may share at least some activity.
Deposit of Clones
Clones AA365_1, BL67_2, BN130_1, CG99_2, C0618_1, C0629_2, CT645_1,
CW383_1, DN167_l, and DN711 2 were deposited on January 17,1997 with the
American
Type Culture Collection as an original deposit under the Budapest Treaty and
were given
the accession number ATCC 98296, from which each clone comprising a particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
deposited material will be irrevocably removed upon the granting of the
patent, except
for the requirements specified in 37 C.F.R. ~ 1.808(b).
2 0 Each clone has been transfected into separate bacterial cells (E. coli) in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
2 5 pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning
(Kaufman et al.,
1991, Nucleic Acids Res. 29: 4485-4490); the pNOTs vector was derived from
pMT2
(Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR
sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
3 0 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.
27
CA 02278153 1999-07-20
wo 8oi rc'rwsssrom7
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
AA365_1 SEQ ID N0:23
BL67_2 SEQ ID N0:24
BN130_1 SEQ ID N0:25
CG99_2 SEQ ID N0:26
C0618_1 SEQ ID N0:27
C0629 2 SEQ ID N0:28
CT645_1 SEQ ID N0:29
CW383_1 SEQ ID N0:30
DN167_1 SEQ ID N0:31
DN711 2 SEQ ID N0:32
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-
2 5 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;
3 0 (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-32P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Uruncorporated
28
CA 02278153 1999-07-20
wo msoz rc~rrtrs~roioo7
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 lzl 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
pg / ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ~lg /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 0 a concentration greater than or equal to le+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
2 5 filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
3 0 hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, et al., Bio/Technology 10) 773-778 (1992) and
in R.S.
29
CA 02278153 1999-07-20
wo msoz rcr~s9sroioo~
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
(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
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
2 0 expression of such genes. Corresponding genes may therefore include but
are not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene '
is a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the genes)
3 0 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
ef al.,1997,
Biochem. Mol. Med. 62(1):11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol.
Biol. 58: 1
CA 02278153 1999-07-20
WO 98131802 PCTIUS98/01007
39; all of which are incorporated by reference herein). Transgenic arurnals
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 Bl, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
2 0 are mammals. Such organisms are useful for the development of non-human
models for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
2 5 the present invention also provides for soluble forms of such protein. In
such forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
3 0 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
31
CA 02278153 1999-07-20
WO 98131802 PC"TIUS98/01007
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
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.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
which also encode proteins which are identical, homologous, or related to that
encoded
by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
2 0 The present invention also includes polynucleotides capable of hybridizing
under
reduced stringency conditions, more preferably stringent conditions, and most
preferably
highly stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
2 5 stringent as, for example, conditions G-L; and reduced stringency
conditions are at least
as stringent as, for example, conditions M-R.
32
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WO 98/31802 PCT/US98/a1007
SMngency PolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
~P)t Buffer' and Buffers
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC Ta*; lxSSC
C DNA:RNA a 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 TD*; lxSSC Tp*; lxSSC
E RNA:RNA 2 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA s 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 TH*; 4xSSC T,,*; 4xSSC
I DNA:RNA Z 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
., K RNA:RNA Z 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- 50'C; 2xSSC
40C; 6xSSC, 50% fonmamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 T,.*; 6xSSC T,,*; 6xSSC
Q RNA:RNA s 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 0 R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
t: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
hybridizing a polynudeotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is O.15M NaCI) lOmM NaHzPO" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6{logo[Na'J) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'J is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
33
CA 02278153 1999-07-20
WO 98J31802 PCT/US98/01007
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce
the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 1_ 85, 537-566 (1990). As defined herein
"operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimlcrium,
or any bacterial
34
CA 02278153 1999-07-20
wo msoz rcrrtrs~oioo~
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 MaxBacO kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (198 incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
rell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffiruty
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(T1ZX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT}.
Finally, one or more reverse-phase high performance liquid chromatography
(lZl'-
HPLC) steps employing hydrophobic lZl'-HPLC media, e.g., silica gel having
pendant
CA 02278153 1999-07-20
wo msoZ rcr~rs9sioioo~
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgeruc
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
36
CA 02278153 1999-07-20
WO 98r31802 PCT/US98~1007
~JSES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
3 0 The proteins provided by the present invention can similarly be used in
assay to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
37
CA 02278153 1999-07-20
wo m8oz rcr~s9sroioo7
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein {such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. ICimmel
eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 0 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
2 5 can be added to the medium in or on which the microorganism is cultured.
C3rtokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation {either inducing or
inhibiting) activity or may
3 0 induce production of other cytokines in certain cell populations. Many
protein factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
38
CA 02278153 1999-07-20
wo soz rcrrtrs9sroioo7
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J,
Immunol. 152:1756-1761,1994.
Assays for cytokine production and/or proliferation of spleen cells, Iymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonai T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto.1994.
2 0 Assays for proliferation and differentiation of hematopoietic and
Iymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
2 5 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, j., Clark, S.C. and Turner, K. J. In
Current Protocols
3 0 in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto.1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
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CA 02278153 1999-07-20
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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 Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
{SLID)), e.g.,
in regulating (up or down) growth and proliferation of T and / or -B
lymphocytes, as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
2 0 infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
CA 02278153 1999-07-20
WO 98J31802 PCT/US98~11007
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B~), e.g.,
preventing
high level lymphokine synthesis by activated T rells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
toleranre by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
41
CA 02278153 1999-07-20
WO 98131802 PCTlUS98l01007
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
S humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11702-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T reps that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific toleranre of autoreactive T rells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL / Ipr/Ipr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
42
CA 02278153 1999-07-20
WO 98/31802 PCT/US98/01007
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
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
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
(3Z
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
43
CA 02278153 1999-07-20
WO 98131802 PCT/US98/01007
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience {Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974,1982; Handa
et al.,
J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-3500,1986;
Takai et al.,
J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol.153:3079-3092,
1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol.144:3028-3033,1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Imma~nology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
44
CA 02278153 1999-07-20
WO 98/31802 PGT/US98/01007
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
350(1,1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Re ating Activity
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
CA 02278153 1999-07-20
WO 98131802 PCT/US98/01007
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and / or in supporting the growth and proliferation
of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-
151,1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
2 5 Hematopoietic 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
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,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et a1. eds. Vol pp.1-21, Wiley-Liss, Inc.., New York,
NY.1994; Long
term bone marrow cultures in the presenre 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,
46
CA 02278153 1999-07-20
wo soz rcr~rsnroloo~
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, ef al. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY.1994.
Tissue Growth ActivitX
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and / or nerve tissue growth or
regeneration, as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and / or bone
growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and /or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue 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
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon / ligament-like
tissue inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
47
CA 02278153 1999-07-20
wo soz rcTrtrs9sroioo7
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to. attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and / or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson s disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
3 0 and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogeruc activity.
48
CA 02278153 1999-07-20
wo msoz rcrivs9sroioo~
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin; endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing pps. 71-112 (Maibach, HI and Rovee, DT, eds.),
Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activit~r
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
(3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
49
CA 02278153 1999-07-20
WO 98131802 PCTIUS98ro1007
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chemokinetic ActivitX
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast rells, eosinophils, epithelial and /
or endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by employing such protein or peptide in
any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146,1995; Muller et al Eur. j. Immunol. 25: 1744-1748; Gruber et
al. J. of
Immunol.152:5860-5867,1994; Johnston et a1. J. of Immunol. 153:1762-1768,1994.
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wo msoz rcTrtrs9sroioo7
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolyHc
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophiliac) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474,1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their ligands, rereptor phosphatases and their
ligands,
rereptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
ICruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
51
CA 02278153 1999-07-20
wo msoz rcTms9sroioo~
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-Inflamrnatorv Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
2 0 from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Su~pressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
52
CA 02278153 1999-07-20
wo mao2 rcT~rs~roioo~
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into canrer cells can reduce or eliminate the canrerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleotf des
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 S 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
rells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
53
CA 02278153 1999-07-20
WO 98131802 PGT/US98I01007
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-
1038,1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
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CA 02278153 1999-07-20
WO 98/31802 PGT/US98/01007
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); imrnunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a earner)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, ILr7, IL-8, IL-
9, IL-10, IL-11,
2 0 IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may further
contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
2 5 or to mininnize side effects. Conversely, protein of the present invention
may be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
3 0 A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
CA 02278153 1999-07-20
WO 98J31802 PCT/US98/01007
The pharmaceutical composition of the invention may be in the form of a
complex
of the protein{s) 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 MIiC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
2 0 and the like. Preparation of such liposomal formulations is within the
level of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
2 5 amount of each active component of the pharmaceutical composition or
method that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
3 0 a combination, the term refers to combined amounts of the active
ingredients that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
56
CA 02278153 1999-07-20
WO 98131802 PCT/(T598/Ol~07
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
compusitiou of the invention may additionally contain a solid Garner such as a
gelatin or
2 0 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
2 5 physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
3 0 When a therapeutically effective amount of protein of the present
invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
57
CA 02278153 1999-07-20
wo 9smao2 rc~rms9sroioo7
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringei s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 Ilg
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 Ilg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
2 0 present invention will vary, depending on the severity of the disease
being treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
2 5 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
3 0 carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. 85 2149-2154 (1963); J.L. Krstenansky, et
al., FEBS Lett.
211 ) 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
58
CA 02278153 1999-07-20
wo msoz rcr~s9sioioo~
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. in the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and / or cartilage formation,
the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
2 0 developing bone and cartilage and optimally capable of being resorbed into
the body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
2 5 application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrires are comprised of
pure proteins
3 0 or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
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CA 02278153 1999-07-20
WO 98/31802 PCT/US98/01007
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
carboxymethylrellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
2 0 In further compositions, proteins of the invention may be combined with
other
agents beneficial to the treatment of the bone and/or cartilage defect, wound)
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGF-(3), and insulin-like growth factor (IGF).
2 5 The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
3 0 various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
CA 02278153 1999-07-20
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the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
51
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 34
(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: (627) 876-5851
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 633 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
62
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
CTCTTACCCC TCCTCCTGTT TCATGGACAT TTCAGTGTTG CCAGTGAAAA AGTAATGGTT 60
TTAATTTGGT CCTTATTTTT AACCTGCCCC TGAGACTTAT ATGCTTGTTT ATACCATGTA 120
CGTAGTGTGT GATTGTATGT GTTTGTATTT GTCCACATGT CCCAAAACAT GGGCTGTTAC 180
TTCCTTTTTC TATCTTGGTT TCCTTATTCC CACCCTTCTT TTTCCACCCA GGTATCTGGA 240
CAGGAAGACT TCTCCCATCA GCTTTACCAG AGGAAGCTGC AGGCCCCACT GTGGCCCAGC 300
TCCCTGGGCA TCACTGATTG CTGTCAGTAT GTCACCTCCT GTCACCCCAA GAGATCAGAG 360
AGACGCAAGT ATGGCCGAGA CTTCCTGCTA CGTTTCCGCT TCTGCAGCAT CGCTTGTCAG 420
CGACCAGTAG GACTGGTCCT TATGGAAGGA GTGACAGATA CTAAGCCAGA GCGACCTGCG 480
GGTTGGGCTG AGTCTGTCCT TGAGGAAGAT GCATCGGAGC TTGAGCCTGC CTTCTCCAGG 540
ACTGTAGGTA CCATCCAGCA CTGCCTCCAC CTGACGTCAG TATATACCCC CTCTACCCCC 600
TCTAGAGCCA AAAAAAAAAA AAAAAAAA.AA AAA 633
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:2:
MetLeuVal TyrThr MetTyrValVal CysAsp CysMetCys LeuTyr
1 5 10 15
LeuSerThr CysPro LysThrTrpAla ValThr SerPhePhe TyrLeu
20 25 30
GlyPheLeu IlePro ThrLeuLeuPhe ProPro ArgTyrLeu AspArg
35 40 45
LysThrSer ProIle SerPheThrArg GlySer CysArgPro HisCys
50 55 60
63
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Gly Pro Ala Pro Trp Ala Ser Leu Ile Ala Val Ser Met Ser Pro Pro
65 70 75 80
Val Thr Pro Arg Asp Gln Arg Asp Ala Ser Met Ala Glu Thr Ser Cys
85 90 95
Tyr Val Ser Ala Ser Ala Ala Ser Leu Val Ser Asp Gln
100 105
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 421 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCGGCCGCAG GTCTAGAATT CAATCGGGGA AGTGGGCTGT CCAGCTGATG TGCAGCCTCA 60
AAAGAGACCC CAAGCCAGAC CCACCCACGA AAGGTACCCC TGAATTCTGG ACTCATAAAA 120
TCTCTTAGAT AATACATATT TGTTGTTTTA ACCCACTCAG TTTTGCATTG 3TTACACAGC 180
AATAGAAAAT AAAGGAGGCA GATGAAATTG ATGGAGAACA ATTGCAACGA AAACAGAATA 240
CACAATGCAC GAGCCTGTGT CAGGAATGAC AGTGCATTCC ACGGAAGAGT TGCACAGAGA 300
GAGACCAGTT ACAAGATGCC ATCAGGATAA CGGGAGCTGC GCTGGGCAGC TGGGCCTTCC 360
GACATCCTCC CTGGTTGCGG TGGCAGTGGT GGTGTCTGCT CCAAGTCAAG GCTGGCAGAG 420
A 421
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
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Met His Glu Pro Va1 Ser Gly Met Thr Val His Ser Thr Glu Glu Leu
1 5 10 15
His Arg Glu Arg Pro Val Thr Arg Cys His Gln Asp Asn Gly Ser Cys
20 25 30
' Ala Gly Gln Leu Gly Leu Pro Thr Ser Ser Leu Val Ala Val Ala Val
35 40 45
Val Val Ser Ala Pro Ser Gln Gly Trp Gln Arg
50 55
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TCTGGAAACA CAAACCTCAC TGAGTAACAA AGAAACAGTA TTTGAGGAAA GCCAATGTGA 60
TGAAAGAGAr ACGCCAGACT GGAAGGAGGG ACGGTCATCT GAGAACATAC AAAGCAAGTG 120
GCAAGCGTTA GTTTACACGT CCAAGGTGAC CGTCACATGC TCTGTCCAGG CAAGAGAGGC 180
AAGTGGACAC CCTGAGAAGG AAAAATGTTG TCAAAATAAA AACCTAATAT GTGAACCTAG 240
GAGTAAAATG GACACAGCTT GCGAGCAAAG AGACAGTGAG AAGATGAAGC CAAGGCACTC 300
TCCAGAATGC AGGTGACGGG AAAAAGAGAC AGTAACAGAT CCAAAGAGGT CCAGAAGGAA 360
TGACCATGGA GCGGAAAGAA AGF~AAAAAAA AAA 393
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
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GCTCCGGTGG CTGCGCAGCG TCGGCGGGGAGCGCGGGCTGCGGAGAGGCG GGCCGGGCCA60
AGCGGAGCCG AGCGAGCGGG AGCGCGGCGTCCGGGAGGCGGCGGAGACGC GGGGCTCGGA120
GGGTCAGCCT CTTATCGTAG CAGGTCTCCTCGGCACGCCCCCCTTGTTTC GCCCCACGGC180
CAAGCCCGCC GCGGGCCGGC GTGCGCTGGTCACTGAGGCCCAGGTCGCCG CCGCGGCGCG240
TTTTTGAAAT CATGAATCCT GTTTATAGTCCTGGATCTTCTGGGGTTCCC TATGCAAATG300
CCAAAGGAAT TGGTTATCCA GCTGGTTTTCCCATGGGCTATGCAGCAGCA GCTCCCTGCC360
TATTCTCCTA ACATGTATCC TGGAGCGAATCCTACCTTCCAAACAGGTTA CACTCCTGGC420
ACACCTTACA AAGTGTCCTG TTCCCCCACCAGCGGGGCTGTGCCACCG 468
(2) INFORMATION FOR SEQ
ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID. N0:7:
Met Pro Lys Glu Leu Val Ile Gln Leu Val Phe Pro Trp Ala Met Gln
1 5 10 15
Gln Gln Leu Pro Ala Tyr Ser Pro Asn Met Tyr Pro Gly Ala Asn Pro
20 25 30
Thr Phe Gln Thr Gly Tyr Thr Pro Gly Thr Pro Tyr Lys Val Ser Cys
35 40 45
Ser Pro Thr Ser Gly Ala Val Pro Pro
50 55
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 366 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
AAGTACCGGG CCCCAGGAAC GCCCACTTAC AGCTATGTGC CCCCTCAGTG GTGATCACCT 60
GCAAATGTTT GAGGACGGAG CTGTGCAGTC ACATTATTGG GGATTCCACA GCTGGTGCTG 120
CAGGCCTTGC GCCTCCAACC AGGACTTTCT TCTTAATGCT CTCGACACTT AGCTAAACAC 180
GACTATATCC CGGCCCAGCA GGCCCCAGCG CCGTTAGTCT CCAGCTGACT CTGTGGGTTG 240
GTCTTAAAGC AAATTCTGTT TTGTGGACTG CCTGGCAATT TTTTAGCTAA CTGTAATGAT 300
AAAAAGGGAG TATTAATCTA TTCTGAATCA TATCTAGTTG AATGCATGTT TAA,AAAP.AAA 360
A~ 366
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2423 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NC:9:
AGGGCCCGCC GCTGGGATGC CGAGCGCCCG CGCCGCCGCT GCCTCTGTCC60
TCCGCGCGCT
GCTCAGCTGA AGGCGCACAG GATTCAATTA CTGGACTTGT CAACTCTGCC120
AGTGTACGTG
CCATTTCTCT TCCACTATGA GAGGACCGAT TGTATTGCAC ATTTGTCTGG180
CTTTCTGTAG
CCTTCTGCTT TTCAGCGTTG CCACACAATG TCTGGCCTTC CCCAAAATAG240
AAAGGAGGAG
GGAGATAGCA CATGTTCATG CGGAAAAAGG GCAGTCCGAT AAGATGAACA300
CCGATGACCT
AGAAAATAGC TCTGTTACCT CAAAGCAGAC TCCCCAACTG GTGGTCTCTG360
AAGATCCAAT
GATGATGTCA GCAGTACCAT CGGCAACATC ATTAAATAAA GCATTCTCGA420
TTAACAAAGA
AATCCAGCCT GGACAAGCTG GGCTCATGCA AACAGAACGC CCTGGTGTTT480
CCACACCTAC
TGAGTCAGGT GTCCCCTCAG CTGAAGAAGT ATTTGGTTCC AGCCAGCCAG540
AGAGAATATC
TCCTGAAAGT GGACTTGCCA AGGCCATGTT AACCATTGCT ATCACTGCGA600
CTCCTTCTCT
GACTGTTGAT GAAAAGGAGG AACTCCTTAC AAGCACTAAC TTTCAGCCCA660
TTGTAGAAGA
GATCACAGAA ACCACAAAAG GTTTTCTGAA GTATATGGAT AATCAATCAT720
TTGCAACTGA
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AAGTCAGGAA GGAGTTGGTT TGGGACATTC ACCTTCATCC TATGTGAATA 780
CTAAGGAAAT
GCTAACCACC AATCCAAAGA CTGAGAAATT TGAAGCAGAC ACAGACCACA 840
GGACAACTTC
TTTTCCTGGT GCTGAGTCCA CAGCAGGCAG TGAGCCTGGA AGCCTCACCC 900
CTGATAAGGA
GAAGCCTTCG CAGATGACAG CTGATAACAC CCAGGCTGCT GCCACCAAGC 960
AACCACTCGA
AACTTCCGAG TACACCCTGA GTGTTGAGCC AGAAACTGAT AGTCTGCTGG 1020
GAGCCCCAGA
AGTCACAGTG AGTGTCAGCA CAGCTGTTCC AGCTGCCTCT GCCTTAAGTG 1080
ATGAGTGGGA
TGACACCAAA TTAGAGAGTG TAAGCCGGAT AAGGACCCCC AAGCTTGGAG 1140
ACAATGAAGA
GACTCAGGTG AGAACGGAGA TGTCTCAGAC AGCACAAGTA AGCCATGAGG 1200
GTATGGAAGG
AGGCCAGCCT TGGACAGAGG CTGCACAGGT GGCTCTGGGG CTGCCTGAAG 1260
GGGAAACACA
CACGGGCACA GCCCTGCTAA TAGCGCATGG GAATGAGAGA TCACCTGCTT 1320
TCACTGATCA
AAGTTCCTTT ACCCCCACAA GTCTGATGGA AGACATGAAA GTTTCCATTG 1380
TGAACTTGCT
CCAAAGTACG GGAGACTTCA CGGAATCCAC CAAGGAAAAC GATGCCCTGT 1440
TTTTCTTAGA
AACCACTGTT TCTGTCTCTG TATATGAGTC TGAGGCAGAC CAACTGTTGG 1500
GAAATACAAT
GAAAGACATC ATCACTCAAG AGATGACAAC AGCTGTTCAA GAGCCAGATG 1560
CCACTTTATC
CATGGTGACA CAAGAGCAGG TTGCTACCCT CGAGCTTATC AGAGACAGTG 1620
GCAAGACTGA
GGAAGAAAAG GAGGACCCCT CTCCTGTGTC TGACGTTCCT GGTGTTACTC 1680
AGCTGTCAAG
AAGATGGGAG CCTCTGGCCA CTACAATTTC AACTACAGTC GTCCCTTTGT 1740
CTTTTGAAGT
TACTCCCACT GTGGAAGAAC AAATGGACAC AGTCACAGGG CCAAATGAGG 1800
AGTTCACACC
AGTTCTGGGA TCTCCAGTGA CACCTCCTGG AATAATGGTG GGGGAACCCA 1860
GCATTTCCCC
TGCACTTCCT GCTTTGGAGG CATCCTCTGA GAGAAGAACT GTTGTTCCAT 1920
CTATTACTCG
TGTTAATACA GCTGCCTCAT ATGGCCTGGA CCAACTTGAA TCTGAAGAGA 1980
CGGGGTTTCA
CCATGTTGCC CAGGCTCGTC TCAAACTCCT GGGCTTAAGA AGTCTGCCTG 2040
CCTCGGCCTC
CCAAAGTGTT GGAATTACAA GTGTGAACTC CTGCACCCAG CCAAGAAAAT 2100
ATTTAAACAG
CTGCCTGAAA TGGAAATTGA ATCCAAAACA TTTTGCCACT GGTGTTTGAA 2160
GTGGACTATC
TTGTCTCAAG CCTTTTTATT TTCTCATGAA TGTACCTTCT AAATATTGTT 2220
AAGGCTACAA
TGGAGGAAAT TTTAAATTAT TATATTTACT ATTTTACAGT GAGTGGAAAA 2280
TTTGAAATTT
AGTAAAAAGT TTTCAAGAAA TTTTTATAGA TTAATAATGG AGTTTAAAAA 2340
GTCATGGCGA
TGAGGCATTT AACAAGGAGA GAACTGAATA AATTAACAGA CATGTTTAAG 2400
GTF~P.AAAAAA
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2423
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 670 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Arg Gly Pro Ile Val Leu His Ile Cys Leu Ala Phe Cys Ser Leu
1 5 10 15
Leu Leu Phe Ser Val Ala Thr Gln Cys Leu Ala Phe Pro Lys Ile Glu
20 25 30
Arg Arg Arg Glu Ile Ala His Val His Ala Glu Lys Gly Gln Ser Asp
35 40 45
Lys Met Asn Thr Asp Asp Leu Glu Asn Ser Ser Val Thr Ser Lys Gln
50 55 60
Thr Pro Gln Leu Val Val Ser Glu Asp Pro Met Met Met. Ser Ala Val
65 70 75 80
Pro Ser Ala Thr Ser Leu Asn Lys Ala Phe Ser Ile Asn Lys Glu Ile
85 90 95
Gln Pro Gly Gln Ala Gly Leu Met Gln Thr Glu Arg Pro Gly Val Ser
100 105 110
Thr Pro Thr Glu Ser Gly Val Pro Ser Ala Glu Glu Val Phe Gly Ser
115 120 125
Ser Gln Pro Glu Arg Ile Ser Pro Glu Ser Gly Leu Ala Lys Ala Met
130 135 140
Leu Thr Ile Ala Ile Thr Ala Thr Pro Ser Leu Thr Val Asp Glu Lys
145 150 155 160
Glu Glu Leu Leu Thr Ser Thr Asn Phe Gln Pro Ile Val Glu Glu Ile
165 170 175
Thr Glu Thr Thr Lys Gly Phe Leu Lys Tyr Met Asp Asn Gln Ser Phe
180 185 190
Ala Thr Glu Ser Gln Glu Gly Val Gly Leu Gly His Ser Pro Ser Ser
195 200 205
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Tyr Val Asn Thr Lys GIu Met Leu Thr Thr Asn Pro Lys Thr Glu Lys
210 215 220
Phe Glu Ala Asp Thr Asp His Arg Thr Thr Ser Phe Pro Gly Ala Glu
225 230 235 240
Ser Thr Ala Gly Ser Glu Pro Gly Ser Leu Thr Pro Asp Lys Glu Lys
245 250 255
Pro Ser Gln Met Thr Ala Asp Asn Thr Gln Ala Ala Ala Thr Lys Gln
260 265 270
Pro Leu Glu Thr Ser Glu Tyr Thr Leu Ser Val Glu Pro Glu Thr Asp
275 280 285
Ser Leu Leu Gly Ala Pro Glu Val Thr Val Ser Val Ser Thr Ala Val
290 295 300
Pro Ala Ala Ser Ala Leu Ser Asp Glu Trp Asp Asp Thr Lys Leu Glu
305 310 315 320
Ser Val Ser Arg Ile Arg Thr Pro Lys Leu Gly Asp Asn Glu Glu Thr
325 330 335
Gln Val Arg Thr Glu Met Ser Gln Thr Ala Gln Val Ser His Glu Gly
340 345 350
Met Glu Gly Gly Gln Pro Trp Thr Glu Ala Ala Gln Val Ala Leu C;ly
355 360 365
Leu Pro Glu Gly Glu Thr His Thr Gly Thr Ala Leu Leu Ile Ala His
370 375 380
a Gly Asn Glu Arg Ser Pro Ala Phe Thr Asp Gln Ser Ser Phe Thr Pro
385 390 395 400
Thr Ser Leu Met GIu Asp Met Lys Val Ser Ile Val Asn Leu Leu Gln
405 410 415
Ser Thr Gly Asp Phe Thr Glu Ser Thr Lys Glu Asn Asp Ala Leu Phe
420 425 430
Phe Leu Glu Thr Thr Val Ser Val Ser Val Tyr Glu Ser Glu Ala Asp
435 440 445
GIn Leu Leu Gly Asn Thr Met Lys Asp Ile Ile Thr Gln Glu Met Thr
450 455 460
Thr Ala Val Gln Glu Pro Asp Ala Thr Leu Ser Met Val Thr Gln Glu
465 470 475 480
Gln Val Ala Thr Leu Glu Leu Ile Arg Asp Ser Gly Lys Thr Glu Glu
485 490 495
Glu Lys Glu Asp Pro Ser Pro Val Ser Asp Val Pro Gly Val Thr Gln
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500 505 510
Leu Ser Arg Arg Trp Glu Pro Leu Ala Thr Thr Ile Ser Thr Thr Val
515 520 525
Val Pro Leu Ser Phe Glu Val Thr Pro Thr Val Glu Glu Gln Met Asp
530 535 540
Thr Val Thr Gly Pro Asn Glu Glu Phe Thr Pro Val Leu Gly Ser Pro
545 550 555 560
Val Thr Pro Pro Gly Ile Met Val Gly Glu Pro Ser Ile Ser Pro Ala
565 570 575
Leu Pro Ala Leu Glu Ala Ser Ser Glu Arg Arg Thr Val Val Pro Ser
580 585 590
Ile Thr Arg Val Asn Thr Ala Ala Ser Tyr Gly Leu Asp Gln Leu Glu
595 600 605
Ser Glu Glu Thr Gly Phe His His Val Ala Gln Ala Arg Leu Lys Leu
610 615 620
Leu Gly Leu Arg Ser Leu Pro A1a Ser Ala Ser Gln Ser Val Gly Ile
625 630 635 640
Thr Ser Val Asn Ser Cys Thr Gln Pro Arg Lys Tyr Leu Asn Ser Cys
645 650 655
Leu Lys Trp Lys Leu Asn Pro Lys His Phe Ala Thr Gly Val
660 665 670
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1302 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
CAGGATGGGA TCCTCATAGA GGGGCAGATA ATGTTTCTAC TAAATCTTCG GACAGTGATG 60
TAAGTGATAT ATCTGCGGTT TCAAGGACTA GTAGTGCTTC TCGTTTCAGC AGCACAAGCT 120
ACATGTCTGT CCAATCAGAA CGCCCAGGAG GAAACAAGAA AATCAGTGTC TTTACATCCA 180
AAATGCAAAG CAGACAAATG GGCATATCAG GGAAGAACAT GACAAAAAGC ACCAGCATCA 240
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GTGGAGACATGTGCTCACTG GAGAAGAATG ATGGCAGCCAGTCTGACACT GCAGTGGGCA300
CCTTGGGCACCAGTGGCAAA AAGCGGCGCT CTAGCCTTGGTGCCAAAATG GTAGCTATCG360
TTGGTCTGTCACGGAAAAGT CGCAGTGCTT CTCAGCTCAGCCAAACGGAA GCAGGAGGTW420
AAAAACTAAGGAGCACTGTC CAAAGAAGTA CAGAAACAGGCCTGGCCGTG GAAATGAGGA480
ACTGGATGACTCGACAGGCA AGCCGAGAGT CTACAGATGGTAGCATGAAC AGCTACAGCT540
CAGAAGGAAATCTGATTTTC CCTGGTGTTC GCTTGGCCTCTGATAGCCAG TTCAGTGATT600
TCCTGGATGGCCTTGGCCCT GCTCAGCTAG TGGGACGCCAGACTCTGGCA ACACCTGCAA660
TGGGTGACATTCAGGTAGGA ATGATGGACA AAAAGGGACAGCTGGAGGTA GAAATCATCC720
GGGCCCGTGGCCTTGTTGTA AAACCAGGTT CCAAGACACTGCCAGCACCG TATGTAAAAG780
TGTATCTATTAGATAACGGA GTCTGCATAG CCAAAAAGGGACAGCTGGAG GTAGAAATCA840.
TCCGGGCCCGTGGCCTTGTT GTAAAACCAG GTTCCAAGACACTGCCAGCA CCGTATGTAA900
AAGTGTATCTATTAGATAAC GGAGTCTGSA TAGCCAAAAAGAAAACAAAA GTGGCAAGAA960
AAACGCTGGAACCCCTTTAC CAGCAGYTAT TATCTTTCGAAGAGAGTCCA CAAGGAAAAG2020
TTTTACAGATCATCGTCTGG GGAGATTATG GCCGCATGGATCACAAATCT TTTATGGGAG1080
TGGrCCAGATACTTTTAGAT GAACTAGAGC TATCCAATATGGTGATCGGA TGGTTCAAAC114C
TTTTCCCACCTTCCTCCCTA GTAGATCCAA CCTTGGCCCCTCTGACAAGA AGAGCTTCCC1200
AATCATCTCTGGAAAGTTCA ACTGGACCTT CTTACTYTCGTTCATAGCAG CTGTAAAAAA1260
ATTGTTGTCACAGCAACCAG CGTTACAAAA P~~i~AP.AAAAAAA 1302
(2) INFORMATION
FOR
SEQ
ID N0:12:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 374 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Ser Val Gln Ser Glu Arg Pro Gly Gly Asn Lys Lys Ile Ser Val
1 5 10 15
Phe Thr Ser Lys Met Gln Ser Arg Gln Met Gly Ile Ser Gly Lys Asn
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20 25 30
Met Thr Lys Ser Thr Ser Ile Ser Gly Asp Met Cys Ser Leu Glu Lys
35 40 45
Asn Asp Gly Ser Gln Ser Asp Thr Ala Val Gly Thr Leu Gly Thr Ser
50 55 60
Gly Lys Lys Arg Arg Ser Ser Leu Gly Ala Lys Met Val Ala Ile Val
65 70 75 80
Gly Leu Ser Arg Lys Ser Arg Ser Ala Ser Gln Leu Ser Gln Thr Glu
85 90 95
Ala Gly Gly Xaa Lys Leu Arg Ser Thr Val Gln Arg Ser Thr Glu Thr
100 105 110
Gly Leu Ala Val Glu Met Arg Asn Trp Met Thr Arg Gln Ala Ser Arg
115 120 125
Glu Ser Thr Asp Gly Ser Met Asn Ser Tyr Ser Ser Glu Gly Asn Leu
130 135 140
Ile Phe Pro Gly Val Arg Leu Ala Ser Asp Ser Gln Phe Ser Asp Phe
145 150 155 160
Leu Asp Gly Leu Gly Pro Ala Gln Leu Val Gly Arg Gln Thr Leu Ala
165 170 175
Thr Pro Ala Met Gly Asp Ile Gln Val Gly Met Met Asp Lys Lys Gly
180 185 190
Gln Leu Glu Val Glu Ile Ile Arg Ala Arg Gly Leu Val Val Lys Pro
195 200 205
Gly Ser Lys Thr Leu Pro Ala Pro Tyr Val Lys Val Tyr Leu Leu Asp
210 215 220
Asn Gly Val Cys Ile Ala Lys Lys Gly Gln Leu Glu Val Glu Ile Ile
225 230 235 240
Arg Ala Arg Gly Leu Val Val Lys Pro Gly Ser Lys Thr Leu Pro Ala
245 250 255
Pro Tyr Val Lys Val Tyr Leu Leu Asp Asn Gly Val Xaa Ile Ala Lys
260 265 270
Lys Lys Thr Lys Val Ala Arg Lys Thr Leu Glu Pro Leu Tyr Gln Gln
275 280 285
Leu Leu Ser Phe Glu Glu Ser Pro Gln Gly Lys Val Leu Gln Ile Ile
290 295 300
Val Trp Gly Asp Tyr Gly Arg Met Asp His Lys Ser Phe Met Gly Val
305 310 315 320
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Ala Gln Ile Leu Leu Asp Glu Leu Glu Leu Ser Asn Met Val Ile Gly
325 330 335
Trp Phe Lys Leu Phe Pro Pro Ser Ser Leu Val Asp Pro Thr Leu Ala
340 345 350
Pro Leu Thr Arg Arg Ala Ser Gln Ser Ser Leu Glu Ser Ser Thr Gly
355 360 365
Pro Ser Tyr Xaa Arg Ser
370
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1627 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:13:
GGCCTACTGAATAACAATAGTTATTTTTGAAAGATCATTGTTTCCCTAATTCTGTGGCTA60
AGAGGGGCCCATTTAGGAAGAGGTCAGGAGGGTGGTTGAGGCGGGGAGGGGGACTCTTAA120
GGAAAAGACAACTCAAACGGGAGGATGAAAAAGCATTGTATTTGATTGTTTTCTTTCCTG180
TAGGATGATGCCTCCCAGGAGAATAACATGCAAGCTGAGGTGACAGGTAAACTAAAACAG240
CACTTTAATAATGCGCTCCCCAAACAGAACAGCACTACCACCGGGGTCTCCGTCATCATG300
GTGAGTGGAAGGCGGCAGGTCTGGCCTGACTGCGGAGCCGGCCTTGAAGTTTTTGAATTA360
GGTAGCCGGGAGCTGCCCTCACATGGAAGTTGGTGCCTTCCGTAGTCCTATTTCATATGA420
AGATTGGCTTGGCATGTGGAGGGCACTCATTCGGCAACTCCCAGGCTTTGGGCACTGTGT480
GGAGGGGCTTGTGTAGGGACCAGCAGGCCTGGTGTGAGGGGTCCAGGCGTCAAGGAGCTC540
CTGGCTGGGCCCTCTGGGCAGCTGCTTCCACTCTTGTCTCTGCCTTCTCATCTAGAGAGA600
CTCCCAAGCCCTGGAGGGGTGTGTTGTGTTAGGAATTAACTCCCTGCCTACCCCAAGGCC560
TCAGAAATAGATTATTAGAGATGTGAATTATTCTTTGAGACTTGGGATAAGAAACAGCCA720
AAGCTAAACATATTTCAGTTTTAAAAAATCAGTGTTTTATAAAACACAGTTTGGGGCTTT780
TAAAGGTACATAATCAAGGAAAi~AAATATATATTCATTTTTCAGGGTTGGTAACATTTTA840
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TGAGATGTCA GTGACAACGA TGGCCTTATT TTTTTCAGCC TTTTCTTCTT 900
CCAAAATGTT
TCTTAAGGCA ACTCTCCTAA ATACATAAAC ACAACAAATT AAAATGAAAA GTGACATGAG960
AGTAAATGAA TCAAAAGGAA AAAACATTGA ACCAGAGGTG AGGGCAGCAC ACCCGCAGCA1020
GCTGTCCAGG CCTGAGCCAA TGCAACCCTG GGCGGGAAGG CCAGCTCACC GTGAGCAGGT1080
AGAAGCCAGC CAGCCACCCA GGCAGGGACC TTGGTTCTCC CCACACACTC CCAGGAGCAG1140
GGAACAGGGG TGGAGTGGCC TTTCCCAGAG CTGGAGTTGG CTGCAGCAGC TTTCGAATCA1200
GACCTGCCAA GGTGATGGGC GTCTGAGTTT CACATCTGGG CCCCCCGTGA CCCCACTGAG1260
TCCTGACAGC TAAGGATGGG CCACCTCCAC AGCTCCGTCA CTCGTACTTG GGACAGGCCT1320
CTCATCCTCT GGGAAGGTCC TCCTTGTTTC CTACCCAACT AGAAGGGAAA CAGTGGCATA1380
TTCTCATGGT ACATGGTTGT CTGAAAGCCT TACCTAGGAA GACGCAGGGT CTAGATAGAA1440
GCTATAAGGA AGCCACACAC ATAACCCACA TCCCCACACC CCCAACATCC CCCACACTCC1500
CCACACCCCC CACACCCCCC ACATCCCCAC CATAATTACC CCCACCTCCA AATATCTCAT1560
AAAAAAAAAA AAAAAAAAAA P,F~~~.AAAAAA AAAAAAAAAA AAAAAAP,AAA P,~~i~AAAAAAA16
2
0
AAAAAAA
162.
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Gln Ala Glu Val Thr Gly Lys Leu Lys Gln His Phe Asn Asn Ala
1 5 10 15
Leu Pro Lys Gln Asn Ser Thr Thr Thr Gly Val Ser Val Ile Met Val
20 25 30
Ser Gly Arg Arg Gln Val Trp Pro Asp Cys Gly Ala Gly Leu Glu Val
35 40 45
Phe Glu Leu Gly Ser Arg Glu Leu Pro Ser His Gly Ser Trp Cys Leu
50 55 60
CA 02278153 1999-07-20
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Pro
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2468 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
AAATTTTCAT CTCTAAATCA AAAAGTGAAG CAGACCAAAT CCAATGTAAA TATTGGCAAC60
CTCCGAAAGC TAGGAAACTT TACCAAACCT GAAATGAAAG TTAACTTTCT AAAACCAAAC120
TTAAAAGTAA ATCTTTGGAA ATCAGATAGT AGTCTTGAAA CTATGGAAAA CACAGGAGTG180
ATGGATAAGG TTCAGGCAGA GTCTGATGGG GACATGTCTT CAGATAATGA CTCATACCAC240
TCTGATGAAT TCCTTACAAA TTCTAAGTCT GATGAAGACA GGCAGCTAGC TAACTCATTA300
GAGAGTGTAG GGCCAATAGA TTACGTTCTT CCTAGTTGTG GTATTATTGC CTCAGCGCCT360
CGATTGGGCA GTCGGTCCCA GTCTCTTAGC AGCACAGATA GTAGCGTTCA TGCTCCTTCA420
GAGATTACTG TTGCTCATGG GAGTGGGCTT GGAAAAGGCC AGGAGTCTCC TTTGAAGAAA480
AGTCCTTCTG CTGGCGACGT ACACATATTG ACTGGCTTTG CCAAGCCTAT GGATATTTAC540
TGCCACAGAT TTGTGCAAGA TGCACAGAAC AAAGTGACCC ACCTATCAGA GACCAGATCT600
GTGTCTCAGC AGGCTAGTCA GGAAAGAAAT CAAATGACCA ATCAAGTTTC AAATGAAACC660
CAATCAGAAT CAACAGAACA GACACCTTCT CGGCCATCGC AATTAGATGT CTCTCTTTCT720
GCAACAGGCC CACAGTTTTT GTCAGTTGAG CCAGCGCATT CAGTTGCATC TCAAAAAACC780
CCCACCTCCG CTTCCAGCAT GCTTGAACTT GAGACAGGGC TTCATGTAAC TCCTTCTCCT840
TCAGAGAGCA GTAGCAGCAG AGCAGTCTCT CCCTTTGCCA AGATTCGAAG TTCCATGGTC900
CAGGTTGCTA GTATTACCCA AGCTGGATTA ACCCATGGGA TAAACTTTGC AGTGTCAAAA960
GTTCAGAAGA GTCCTCCAGA ACCTGAAATC ATTAATCAAG TCCAGCAAAA TGAACTTAAA1020
AAGATGTTTA TACAATGCCA GACACGGATA ATTCAGATTT AGCTTTTAGC CATAAGAATC1080
CTTCCATGGC TTTTATTTAA AAATATGAAA TTTTCACCTC TTGGGGTATT TTAATTGTAC1140
76
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TGTCTGAACC CAGGGATCAC AAATTCTGTT CATTGGAAAG GGTTTTAAAC1200
GGAGTCGGAA
CCTGAGTAGA TTTCCAAATT TTACAGCCAG GACTACAGAA GTGCATCATT1260
CTAGAATGTG
TAGACCTGAG TAGCTTATAC ACTACAGAGC ACTTTGCTTA TTTGAAAGTA1320
ATTCAGCAAC
AGGTCACTTT GGGATATAAC CTGAACCTTT TTTTGGAGTG GGGTGGGTAG1380
ACTACAGTAG
ACACAAGGGC TGGACATGCA GATGCTTAGG GGATTAGCGT TTTTCATAAT1440
TTGTTCTGTT
TGTCAGTTCA TTCCTGTGTG TTCTTACCTC TACAAAGTAA ATTACACATT1500
TAGTTTTTAG
TGACTTTAAC ATGTTACTGA AGCATTTGAA TATAAAGCTA TTTTAGTTTT1560
GATGGCTTAA
CTGTTCCCTG AGGAGTTGAG GGTTATTGAC ACTAAAAAAA TGAATTCTCA1620
TTTGATCCTA
ATTTTCCCCG TATTCTACTT GAACACATTA AAAATACTCT GCTGCCTATA1680
CAATGTAAAC
CTAGGAGCAT TAAGACTTGT CACACAGTAA ACCTGATACA TCAGAGGTGA1740.
ATACCAGCAC
CTATTAGGTT TCATTTTGCT GTTTTCAGGA ATGTAAGAAC ACCCATATTG1800
GCTACTGGAA
ATTCTAGCAG TCAGTCAGGT TTTAATTTAT TCCAGGAGGG GCATCCTCGA1860
CATCTTATGT
AGATGATCCA CAACTTCAAA ATTTAGTCTG GGCCTAGTGC AGTGGCTCAC1920
ACCTATAATC
CCAACACTTT GGGAGGCCAG GAGTTTTGAG ACCAGCCTGG GAAACATCTG1980
CCTCTACAAA
AAAATACAAA AATTAGCTGG GCATAGTGGT GCATGCCTGT GTTTCTAGCT2040
ACGCAGGAGG
ATTGCTTGAG CCCATGAGAT TGAGGCTGCA GTGAGCTGTG ATCGTGCCAC2100
TGACCTCCAG
CCTGGGGGAC AGAGCAAGAC CGTGTCTCAA AAACAATTTA GTCTGAAACA2160
CAATTGTGCT
GAATCTGTCT GACTATAACT CTGACCACAC AGAACCAGGG CTGCCCCTGT2220
AATCCCCACA
GTAAGAAAGT TGTATGGCAT ATTCCAACAA GTATTGGTTC GTCTGGTGTC2280
TTTAGAGCTT
TACTCTGTTG AAGTGACTGA TTCTCAACTG AACATTATGT CGTTACTTTG2340
ATAAGCATTC
CACTTTTGTT ATTTATTAGT GCTATCTTTT TTTTTTTACG TGTTAAATCT2400
TGTGATTATT
AAAATAAAGT ACCATTGTAA TTTAAAGTGA P,~~~,p.AAAAp,A AAAAAAAAAA2 4
AA~p,~A 6
0
AAAAAAAA
2468
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Lys Val Asn Phe Leu Lys Pro Asn Leu Lys Val Asn Leu Trp Lys
1 5 10 15
Ser Asp Ser Ser Leu Glu Thr Met Glu Asn Thr Gly Val Met Asp Lys
20 25 30
Val Gln Ala Glu Ser Asp Gly Asp Met Ser Ser Asp Asn Asp Ser Tyr
35 40 45
His Ser Asp Glu Phe Leu Thr Asn Ser Lys Ser Asp Glu Asp Arg Gln
50 55 60
Leu Ala Asn Ser Leu Glu Ser Val Gly Pro Ile Asp Tyr Val Leu Pro
65 70 75 80
Ser Cys Gly Ile Ile Ala Ser Ala Pro Arg Leu Gly Ser Arg Ser Gln
85 90 95
Ser Leu Ser Ser Thr Asp Ser Ser Val His Ala Pro Ser Glu Ile Thr
100 105 110
Val Ala His Gly Ser Gly Leu Gly Lys Gly Gln Glu Ser Pro Leu Lys
115 120 125
Lys Ser Pro Ser Ala Gly Asp Val His Ile Leu Thr Gly Phe Ala Lys
130 135 140
Pro Met Asp Ile Tyr Cys His Arg Phe Val Gln Asp Ala Gln Asn Lys
145 150 155 160
Val Thr His Leu Ser Glu Thr Arg Ser Val Ser Gln Gln Ala Ser Gln
165 170 175
Glu Arg Asn Gln Met Thr Asn Gln Val Ser Asn Glu Thr Gln Ser Glu
180 185 190
Ser Thr Glu Gln Thr Pro Ser Arg Pro Ser Gln Leu Asp Val Ser Leu
195 200 205
Ser Ala Thr Gly Pro Gln Phe Leu Ser Val Glu Pro Ala His Ser Val
210 215 220
Ala Ser Gln Lys Thr Pro Thr Ser Ala Ser Ser Met Leu Glu Leu Glu
225 230 235 240
Thr Gly Leu His Val Thr Pro Ser Pro Ser Glu Ser Ser Ser Ser Arg
245 250 255
Ala Val Ser Pro Phe Ala Lys Ile Arg Ser Ser Met Val Gln Val Ala
260 265 270
78
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Ser Ile Thr Gln Ala Gly Leu Thr His Gly Ile Asn Phe Ala Val Ser
275 280 285
Lys Val Gln Lys Ser Pro Pro Glu Pro Glu Ile Ile Asn Gln Val Gln
290 295 300
Gln Asn Glu Leu Lys Lys Met Phe Ile Gln Cys Gln Thr Arg Ile Ile
305 310 315 320
Gln Ile
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 1044 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:17:
GCGGCCGCGGTGGTCCGGGC CGCCGTGTCT GCTTCCAGAG CCAAATCTGC o0
CGAGGCTGGA
ATTGCCGGGGAGGCCCAAAG CAAGAAGCCA GTGTCCAGGC CGGCCACCGC 120
TGCCGCTGCC
GCTGCCGGCTCCAGGGAGCC CCGTGTCAAG CAAGGTCCAA AAATTTATAG 180
TTTTAATTCT
ACAAATGATTCTAGTGGTCC TGCAAATCTG GATAAATCTA TTTTGAAAGT 240
GGTAATTAAT
AACAAACTAGAGCAAAGAAT TATTGGAGTG ATCAATGAGC ATAAAAAGCA 300
AAATAATGAC
AAAGGAATGATTTCTGGAAG ACTTACTGCC AAAAAATTGC AGGATTTATA 360
CATGGCTTTA
CAAGCATTTTCATTTAAGAC AAAGGACATT GAAGATGCCA TGACCAATAC 420
ACTCTTATAT
GGAGGTGACCTTCATTCTGC CTTGGATTGG CTCTGTTTAA ACCTTTCAGA 480
TGATGCACTT
CCTGAAGGATTCAGTYAGGA ATTWGAAGAG CAGAAACCTA AAAGTAGGCA 540
TAAATTTCAG
TCTCCTCAAATATAAGTTAC TATTTCACCT CCATTGCAAC CTAAAACAAA 600
AACATATGAA
GAGGACCCTAAGAGTAAGCC AAAAAAGGAA GAAAAAAATA TGGAAGTAAA 660
TATGAAAGAG
TGGATTTTACGATATGCTGA ACAACAAAAT GAAGAAGAAA AGAATGAGAA 720
TTCTAAAAGT
TTAGAAGAGGAGGAAAAATT TGACCCTAAT GAAAGGTACT TACATCTTGC 780
AGCAAAACTG
CTGGATGCAAAAGAACAAGC AGCTACCTTT AAACTAGAAA AAAACAAGCA 840
AGGCCAAAAA
79
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GAGGCTCAGG AAAAAATAAG GAAATTTCAA AGAGAAATGG AAACTTTAGA AGACCATCCA 900
GTATTTAACC CAGCCATGAA GATTTCACAT CAACAAAATG AAAGGAAAAA GCCTCCTGTA 960
GCCACAGAAG GAGAAAGTGC ATTGAATTTT AATTTATTTG AAAAATCTGC AGCTGCTACT 1020
GAAGAAGAGA AAAAAAAAAA AAAA 1044
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:18:
Met Glu Asn Lys Glu IleLeu Arg Tyr GluGln
Val Met Trp Ala Gln
1 5 10 15
Asn Glu Glu Asn Glu SerLys Ser Leu GluG1u
Glu Lys Asn Glu Glu
20 25 30
Lys Phe Pro Glu Arg LeuHis Leu Ala LysLeu
Asp Asn Tyr Ala Leu
35 40 45
Asp Ala Lys Glu Gln Ala Ala Thr Phe Lys Leu Glu Lys Asn Lys Gln
50 55 60
Gly Gln Lys Glu Ala Gln Glu Lys Ile Arg Lys Phe Gln Arg Glu Met
65 70 75 80
Glu Thr Leu Glu Asp His Pro Val Phe Asn Pro Ala Met Lys Ile Ser
85 90 95
His Gln Gln Asn Glu Arg Lys Lys Pro Pro Val Ala Thr Glu Gly Glu
100 105 110
Ser Ala Leu Asn Phe Asn Leu Phe Glu Lys Ser Ala Ala Ala Thr Glu
115 120 125
Glu Glu
130
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 764 base pairs
(B) TYPE: nucleic acid
8~
CA 02278153 1999-07-20
wo msoz rcrms9sroioo~
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
AGGTCTAGAA TTTGTTATTT TAAGTTGAAA TTTGAAGATT GGCTGAGTTG GTTTCATGAT60
ATTGTTGCCT ACTGATATCT TACACAGAGT ACGAGTACAT CTGGTTGAAA GAGAAAAGGC120
AGAGAGAAAA GCAGACTTTT TGGAGTTGTT GTTCTCAGGC ACTAACAGAT TCATCCCTTA180
TATGCATATG AATGGACAAT AAGATTTCTT GGATATTTTC ATGGCAAAGG TGAGGAGAAA240
TTTTTATTGC TTTTTGAAGA AACATGATTT TTATTACCTT GGGTTATTCC AGGAGATAAT300
TGATAAATGT TGAGTAGTTT GTTGGTCTTC TTCTTAAAGG AGGATCAAGA GAAGCAGTAC360
AGCCTGGGTG ACAGAGCGAG ACTCTGTCTC AAATAAAAAA AAAGAAGGAA ACATACTGTA420
GCCAGACGCC ACCTATAGTC CCAAGTAGCT GGGATTACAG GACAAAACAA TTGAAGAGCT480
CAGGCAATCT TTAGCAAATG TTGAAAGGAT GAAAGAGAAG GCAAATGTTG AAACGATGAA540
AGAGAAGGCA GTTGTGAAAA CAGAAAACTT GAAAACTACA TTAGACTCTG CAGAGCAAAA600
GGCAAGATCA GACAAAGAGA AGACCCAGCA GATGTTAGAT GCTGTCACTT CTGAGCCCCC650
AACAGCAAAG AGCGCACCTG AAGAAGTATC AGGACAAGAA CAAGAGGTTT TTTCAAAATA720
GTAAAATTAA AATTAATTTA GTTGAAGTGA T~!~~AAAAP,AA AAAA 7
6
4
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Met Lys Glu Lys Ala Asn Val Glu Thr Met Lys Glu Lys Ala Val Val
10 15
Lys Thr Glu Asn Leu Lys Thr Thr Leu Asp Ser Ala Glu Gln Lys Ala
81
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20 25 30
Arg Ser Asp Lys Glu Lys Thr G1n Gln Met Leu Asp Ala Val Thr Ser
35 40 45
Glu Pro Pro Thr Ala Lys Ser Ala Pro Glu Glu Val Ser Gly Gln Glu
50 55 60
Gln Glu Val Phe Ser Lys
65 70
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 666 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GCGAGTCTGT CGATCCCAGG CCAGAGACAA GGCAGACTAA TAAAGAAGCT60
GGTTCATTTG
CCTTCCAGCA CCTCCTCTCT TCTCCTTTTG CCCAAACTCA 3TGAGCATTT120
CCCAGTGAGT
AAGAAGCATC CTCTGCCAAG ACCAAAAGGA AAGAAGAAAA GCCAAAATGA180
AGGGCCAAAA
AACTGATGGT ACTTGTTTTC ACCATTGGGC TAACTTTGCT CAAGCCATGC240
GCTAGGAGTT
CTGCAAATCG CCTCTCTTGC TACAGAAAGA TACTAAAAGA CACAACCTTC300
TCACAACTGT
CGGAAGGAGT AGCTGACCTG ACACAGATTG ATGTCAATGT TTCTGGGATG360
CCAGGATCAT
GGAAGGGATG TGAGATGATC TGTTACTGCA ACTTCAGCGA TGCCCAAAAG420
ATTGCTCTGC
ACGTTTTCTT TGGACCAAAG ATCTCTTTCG TGATTCCTTG TGAGAATCTT480
CAACAATCAA
CATGTATTCT GGAGAACACC ATTCCTGATT TCCCACAAAC CAGTATAACT540
TGCACTACAT
GCATTTCTAG TTTCTATATA GTGCAATAGA GCATAGATTC TACTTGTCTA600
TATAAATTCT
AGACAAGTAA ATCTGTGTTA AACAAGTAGT AATAAAAGTT p,F~e~AAAAAAA660
AATTCAATCT
666
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 amino acids
(B) TYPE: amino acid
82
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Met Lys Leu Met Val Leu Val Phe Thr Ile Gly Leu Thr Leu Leu Leu
1 5 10 15
Gly Val Gln Ala Met Pro Ala Asn Arg Leu Ser Cys Tyr Arg Lys Ile
20 25 30
Leu Lys Asp His Asn Cys His Asn Leu Pro Glu Gly Val Ala Asp Leu
35 40 45
Thr Gln Ile Asp Val Asn Val Gln Asp His Phe Trp Asp Gly Lys Gly
0 5'5 6 0
Cys Glu Met Ile Cys Tyr Cys Asn Phe Ser Glu Leu Leu Cys Cys Pro
65 70 75 80
Lys Asp Val Phe Phe Gly Pro Lys Ile Ser Phe Val Ile Pro Cys Asn
85 90 95
Asn Gln
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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 N0:23:
CNAAGCATATA AGTCTCAGGG GCAGGTT 28
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CNATTGCTGTG TAACCAATGC AAAACTGA 29
(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:
CNCAGAAGAT CCAGGACTAT AAACAGGAT 2g
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
CNGACAAATGT GCAATACAAT CGGTCCTC 2g
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
84
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(ii} MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GNCCATTTGTC TGCTTTGCAT TTTGGATG 2g
(2) INFORMATION FOR SEQ ID N0:28:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
CNGCTTGCATG TTATTCTCCT GGGAGGCA 2g
(2) INFORMATION FOR SEQ ID N0:29:
(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 N0:29:
CNATCACTCCT GTGTTTTCCA TAGTTTCA 2g
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
CA 02278153 1999-07-20
wo mso2 pcT~s98~oioo7
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GNTCTTTTGCA TCCAGCAGTT TTGCTGCA 29
(2) INFORMATION FOR SEQ ID N0:31:
(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 N0:31:
CNACATTTGCC TTCTCTTTCA TCCTTTCA 29
(2) INFORMATION FOR SEQ ID N0:32:
(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 N0:32:
TNAACTCCTAG CAGCAAAGTT AGCCCAAT 29
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 151 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
86
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Met Ser Gln Asn Met Giy Cys Tyr Phe Leu Phe Leu Ser Trp Phe Pro
1 5 10 15
Tyr Ser His Pro Ser Phe Ser Thr Gln Val Ser Gly Gln Glu Asp Phe
20 25 30
Ser His Gln Leu Tyr Gln Arg Lys Leu Gln Ala Pro Leu Trp Pro Ser
35 40 45
Ser Leu G1y Ile Thr Asp Cys Cys Gln Tyr Val Thr Ser Cys His Pro
50 55 60
Lys Arg Ser Glu Arg Arg Lys Tyr Gly Arg Asp Phe Leu Leu Arg Phe
65 70 75 80
Arg Phe Cys Ser Ile Ala Cys Gln Arg Pro Val Gly Leu Val Leu Met
85 90 95
Glu Gly Val Thr Asp Thr Lys Pro Glu Arg Pro Ala Gly Trp Ala Glu
100 105 110
Ser Val Leu Glu Glu Asp Ala Ser Glu Leu Glu Pro Ala Phe Ser Arg
115 120 125
Thr Val Gly Thr Ile Gln His Cys Leu His Leu Thr Ser Val Tyr Thr
130 135 140
Pro Ser Thr Pro Ser Arg Ala
145 150
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Met Lys Ile Gly Leu Ala Cys Gly Gly His Ser Phe Gly Asn Ser Gln
1 5 10 15
Ala Leu Gly Thr Val Trp Arg Gly Leu Cys Arg Asp Gln Gln Ala Trp
20 25 30
87
CA 02278153 1999-07-20
WO 98J31802 PCT/US98/01007
Cys Glu Gly Ser Arg Arg Gln Gly Ala Pro Gly Trp Ala Leu Trp Ala
35 40 45
Ala Ala Ser Thr Leu Val Ser Ala Phe Ser Ser Arg Glu Thr Pro Lys
50 55 60
Pro Trp Arg Gly Val Leu Cys
88
