Note: Descriptions are shown in the official language in which they were submitted.
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
5
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of Ser. No. 60/XXX,XXX (converted
to
a provisional application from non-provisional application 08/805,819), filed
February 26,
1997, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
2 0 polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
2 5 past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA/amino acid sequence of
the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
3 0 isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
3 5 cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
SUBSTITUTE SHEET (RULE 2B)
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 73 to nucleotide 954;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 208 to nucleotide 954;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1 to nucleotide 630;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BD380_1 deposited under accession
number ATCC 98337;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA _insert of clone BD380 1 deposited under accession number ATCC 98337;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BD380_1 deposited under accession number
ATCC 98337;
2 0 (h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BD380_1 deposited under accession number ATCC 98337;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(j) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:2 having biological activity, the
fragment
comprising the amino acid sequence from amino acid 142 to amino acid 151 of
SEQ ID N0:2;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
3 0 (1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
2
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 73 to nucleotide 954; the nucleotide sequence of SEQ_ID
NO:1 from
nucleotide 208 to nucleotide 954; the nucleotide sequence of SEQ ID NO:1 from
nucleotide 1 to.nucleotide 630; the nucleotide sequence of the full-length
protein coding
sequence of clone BD380_1 deposited under accession number ATCC 98337; or the
nucleotide sequence of the mature protein coding sequence of clone BD380_1
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BD380 1 deposited under accession number ATCC 98337. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 1 to amino
acid 186.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 1 to
amino acid 186;
2 0 (c) fragments of the amino acid sequence of SEQ ID N0:2 comprising
the amino acid sequence from amino acid 142 to amino acid 151 of SEQ ID N0:2;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
BD380_1 deposited under accession number ATCC 98337;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 1 to amino acid 186.
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
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 45 to nucleotide 281;
3
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 61 to nucleotide 419; .
(d) a polynucieotide comprising the nucleotide sequence of the full
length'protein -coding sequence of clone BQ115 2 deposited under accession
number ATCC 98337;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA _insert of clone BQ115 2 deposited under accession number ATCC 98337;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BQ115 2 deposited under accession number
ATCC 98337;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA _insert of clone BQ115 2 deposited under accession number ATCC 98337;
(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, the fragment
comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ
ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
2 0 (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).
2 5 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:3 from nucleotide 45 to nucleotide 281; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 61 to nucleotide 419; the nucleotide sequence of the full-length
protein coding
sequence of clone BQ115_2 deposited under accession number ATCC 98337; or the
nucleotide sequence of the mature protein coding sequence of clone BQ115 2
deposited
3 0 under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BQ115 2 deposited under accession number ATCC 98337. 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 7 to amino
acid 79.
4
CA 02281015 1999-08-13
. WO 98/38209 PCT/US98/03697
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
In other embodiments, the present invention provides a composition comprising
a protein, whefein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 7 to
amino acid 79;
(c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID N0:4;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
BQ115 2 deposited under accession number ATCC 98337;
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 7 to amino acid 79.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
2 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 158 to nucleotide 985;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 497 to nucleotide 985;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:5 from nucleotide 319 to nucleotide 923;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CC198_1 deposited under accession
number ATCC 98337;
(f) a polynucleotide encoding the full-length protein encoded by the
3 0 cDNA insert of clone CC198 1 deposited under accession number ATCC 98337;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CC198_1 deposited under accession number
ATCC 98337;
5
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CC198_1 deposited under accession number_ATCC 98337;
(i) a polynucleotide encoding a protein comprising the amino acid
sequeriee of SEQ ID N0:6;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
comprising the amino acid sequence from amino acid 133 to amino acid 142 of
SEQ ID N0:6;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleoddes specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 158 to nucleotide 985; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 497 to nucleotide 985; the nucleotide sequence of SEQ ID N0:5 from
nucleotide 319 to nucleotide 923; the nucleotide sequence of the full-length
protein coding
sequence of clone CC198_1 deposited under accession number ATCC 98337; or the
2 0 nucleotide sequence of the mature protein coding sequence of clone CC198_1
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CC198_1 deposited under accession number ATCC 98337. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
2 5 comprising the amino acid sequence of SEQ ID N0:6 from amino acid 61 to
amino acid
256.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
In other embodiments, the present invention provides a composition comprising
3 0 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ ID N0:6 from amino acid 61 to
amino acid 256;
. 6
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
the amino acid sequence from amino acid 133 to amino acid 142 of.SEQ ID N0:6;
and
-(d) the amino acid sequence encoded by the cDNA insert of clone
CCi98_1 deposited under accession number ATCC 98337;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6 or the amino acid
sequence
of SEQ ID N0:6 from amino acid 61 to amino acid 256.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 21 to nucleotide 674;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1164 to nucleotide 1465;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CJ317_4 deposited under accession
number ATCC 98337;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CJ317_4 deposited under accession number ATCC 98337;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CJ317_4 deposited under accession number
ATCC 98337;
2 5 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CJ317_4 deposited under accession number ATCC 98337;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(i) a polynucleotide encoding a protein comprising a fragment of the
3 0 amino acid sequence of SEQ ID N0:8 having biological activity, the
fragment
comprising the amino acid sequence from amino acid 104 to amino acid 113 of
SEQ ID N0:8;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(g) above;
7
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(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
N0:7 from nucleotide 21 to nucleotide 674; the nucleotide sequence of SEQ ID
N0:7 from ,
nucleotide 1164 to nucleotide 1465; the nucleotide sequence of the full-length
protein
coding sequence of clone CJ317_4 deposited under accession number ATCC 98337;
or the
nucleotide sequence of the mature protein coding sequence of clone CJ317_4
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CJ317_4 deposited under accession number ATCC 98337.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a} the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8 comprising
2 0 the amino acid sequence from amino acid 104 to amino acid 113 of SEQ ID
N0:8;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
CJ317_4 deposited under accession number ATCC 98337;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 protein comprises the amino acid sequence of. SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 951 to nucleotide 1037;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CS319_1 deposited under accession
number ATCC 98337;
8
CA 02281015 1999-08-13
WO 98/38209 PCT/CJS98/03697
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CS319_1 deposited under accession number ATCC 98337;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein-coding sequence of clone CS319_l deposited under accession number
ATCC 98337;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CS319_1 deposited under accession number ATCC 98337;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:10 having biological activity, the fragment
comprising the amino acid sequence from amino acid 9 to amino acid 18 of SEQ
ID NO:10;
(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).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:9 from nucleotide 951 to nucleotide 1037; the nucleotide sequence of the
full-length
protein coding sequence of clone CS319_1 deposited under accession number ATCC
98337; or the nucleotide sequence of the mature protein coding sequence of
clone CS319_l
deposited under accession number ATCC 98337. In other preferred embodiments,
the
2 5 polynucleotide encodes the full-length or mature protein encoded by the
cDNA insert of
clone CS319_1 deposited under accession number ATCC 98337.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9 or SEQ ID N0:11.
In other embodiments, the present invention provides a composition comprising
3 0 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b} fragments of the amino acid sequence of SEQ ID N0:10 comprising
the amino acid sequence from amino acid 9 to amino acid 18 of SEQ ID NO:10;
and
9
CA 02281015 1999-08-13
W O 98/38209 PCT/US98103697
(c) the amino acid sequence encoded by the cDNA insert of clone
CS319_1 deposited under accession number ATCC 98337;
the protein being substantially free from other-m-ammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:10.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 79 to nucleotide 1134;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 692 to nucleotide 1008;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DL504_3 deposited under accession
number ATCC 98337;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA _insert of clone DL504_3 deposited under accession number ATCC 98337;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DL504_3 deposited under accession number
2 0 ATCC 98337;
(g) a polynucieotide encoding the mature protein encoded by the
cDNA _insert of clone DL504 3 deposited under accession number ATCC 98337;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:13;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:13 having biological activity, the fragment
comprising the amino acid sequence from amino acid 171 to amino acid 180 of
SEQ ID N0:13;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:12 from nucleotide 79 to nucleotide 1134; the nucleotide sequence of SEQ ID
N0:12
from nucleotide 692 to nucleotide 1008; the nucleotide sequence of the full-
length protein
coding sequence of clone DL504_3 deposited under accession number ATCC 98337;
or the
nucleotide sequence of the mature protein coding sequence of clone DL504_3
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone DL504_3 deposited under accession number ATCC 98337.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:12.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:13;
(b) fragments of the amino acid sequence of SEQ ID N0:13 comprising
the amino acid sequence from amino acid 171 to amino acid 180 of SEQ ID N0:13;
and
{c) the amino acid sequence encoded by the cDNA insert of clone
DL504_3 deposited under accession number ATCC 98337;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:13.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 1599 to nucleotide 2543;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 174 to nucleotide 396;
3 0 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DN747_7 deposited under accession
number ATCC 98337;
(e} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone DN747_7 deposited under accession number ATCC 98337;
11
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DN747_ .7 deposited under accession number
ATCC 98337;
fig) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DN747 .7 deposited under accession number ATCC 98337;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:15;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 having biological activity, the fragment
comprising the amino acid sequence from amino acid 152 to amino acid 161 of
SEQ ID N0:15;
(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
N0:14 from nucleotide 1599 to nucleotide 2543; the nucleotide sequence of SEQ
ID N0:14
2 0 from nucleotide 174 to nucleotide 396; the nucleotide sequence of the full-
length protein
coding sequence of clone DN747_7 deposited under accession number ATCC 98337;
or
the nucleotide sequence of the mature protein coding sequence of clone DN747_7
deposited under accession number ATCC 98337. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 5 clone DN747_7 deposited under accession number ATCC 98337.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:14.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:15;
(b) fragments of the amino acid sequence of SEQ ID N0:15 comprising
the amino acid sequence from amino acid 152 to amino acid 161 of SEQ ID N0:15;
and
12
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(c) the amino acid sequence encoded by the cDNA insert of clone
DN747_7 deposited under accession number ATCC 98337; _
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:15.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 4 to nucleotide 1224;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 1 to nucleotide 336;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone DU123_1 deposited under accession
1 S number ATCC 98337;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone DU123_1 deposited under accession number ATCC 98337;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone DU123_1 deposited under accession number
2 0 ATCC 98337;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone DU123_1 deposited under accession number ATCC 98337;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:17;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:17 having biological activity, the fragment
comprising the amino acid sequence from amino acid 198 to amino acid 207 of
SEQ ID N0:17;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
3 0 (a}-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i} above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
13
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:16 from nucleotide 4 to nucleotide 1224; the nucleotide sequence of SEQ ID
N0:16
from nucleotide 1 to nucleotide 336; the nucleotide sequence of the full-
length protein
coding sequence of clone DU123_1 deposited under accession number ATCC 98337;
or the
nucleotide sequence of the mature protein coding sequence of clone DU123_1
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone DU123_1 deposited under accession number ATCC 98337. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:17 from amino acid 1 to amino
acid
111.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:16.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:17;
(b) the amino acid sequence of SEQ ID N0:17 from amino acid 1 to
amino acid 111;
2 0 (c) fragments of the amino acid sequence of SEQ ID N0:17 comprising
the amino acid sequence from amino acid 198 to amino acid 207 of SEQ ID N0:17;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
DU123_1 deposited under accession number ATCC 98337;
2 S the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:17 or the amino acid
sequence
of SEQ ID N0:17 from amino acid 1 to amino acid lll.
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
N0:18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 233 to nucleotide 370;
14
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 293 to nucleotide 370;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 1 to nucleotide 361;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone FB78 1 deposited under accession
number ATCC 98337;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone FB78_1 deposited under accession number ATCC 98337;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone FB78_I deposited under accession number
ATCC 98337;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone FB78_1 deposited under accession number ATCC 98337;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:19;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:19 having biological activity, the fragment
comprising the amino acid sequence from amino acid 18 to amino acid 27 of SEQ
2 0 ID N0:19;
(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
2 5 (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
N0:18 from nucleotide 233 to nucleotide 370; the nucleotide sequence of SEQ ID
N0:18
from nucleotide 293 to nucleotide 370; the nucleotide sequence of SEQ ID N0:18
from
- 3 0 nucleotide 1 to nucleotide 361; the nucleotide sequence of the full-
length protein coding
sequence of clone FB78_1 deposited under accession number ATCC 98337; or the
nucleotide sequence of the mature protein coding sequence of clone FB78_1
deposited
under accession number ATCC 98337. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
CA 02281015 1999-08-13
WO 98/38209 PCT/US98103697
clone FB78_1 deposited under accession number ATCC 98337. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:19-from amino acid 1 to amino
acid
43. -
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:18.
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 N0:19;
(b) the amino acid sequence of SEQ ID N0:19 from amino acid 1 to
amino acid 43;
(c) fragments of the amino acid sequence of SEQ ID N0:19 comprising
the amino acid sequence from amino acid 18 to amino acid 27 of SEQ ID N0:19;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
FB78_1 deposited under accession number ATCC 98337;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:19 or the amino acid
sequence
2 0 of SEQ ID N0:19 from amino acid 1 to amino acid 43.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
2 5 modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
3 0 (b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
16
CA 02281015 1999-08-13
WO 98/38209 PCTNS98/03697
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided-by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature) can then be determined from such nucleotide sequence. The amino
acid
2 0 sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 5 As used herein a "secreted" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
3 0 which are transported across the membrane of the endoplasmic reticulum.
Clone "BD380 1"
A polynucleotide of the present invention has been identified as clone
"BD380_1 ".
BD380_1 was isolated from a human fetal kidney cDNA library using methods
which are
17
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
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. BD380_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BD380 1 protein").
The nucleotide sequence of BD380_1 as presently determined is reported in SEQ
ID NO:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BD380_l protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:2. Amino acids 33 to 45 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 46, or are a transmembrane domain.
The EcoRI /NotI restriction fragment obtainable from the deposit containing
clone
BD380_1 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for BD380_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA _search protocols. BD380 1 demonstrated at least some simuariry wmn
seduenee~
identified as AA112524 (zm28e05.r1 Stratagene pancreas (#937208) Homo sapiens
cDNA
clone 526976 5' _similar to SW:CD63 HUMAN P08962 CD63 ANTIGEN), AA113814
(zm29b04.r1 Stratagene pancreas (#937208) Homo Sapiens cDNA clone 527023 5'),
M79068
(EST01216 Homo sapiens cDNA clone HHCPJ65), N72328 (yv31f12.r1 Homo Sapiens
cDNA clone), and Q61176 (Human brain Expressed Sequence Tag EST01216). The
predicted amino acid sequence disclosed herein for BD380_1 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted BD380_1 protein demonstrated at least some similarity to
sequences
2 5 identified as M37033 (CD53 glycoprotein [Homo sapiens]), 891446 (Human
CD53
antigen), 892313 (Retinal degradation slow protein), 593788 (ocular melanoma-
associated
antigen, OMA81H [human,uvealJ), X97227 (CD53 gene product [Mus musculus]), and
268880 (T14G10.6 [Caenorhabditis elegans]). Based upon sequence similarity,
BD380_1
proteins and each similar protein or peptide may share at least some activity.
The
3 0 TopPredII computer program predicts four potential transmembrane domains
within the
BD380_1 protein sequence, centered around amino acids 33, 73,104, and 242 of
SEQ ID
N0:2, respectively.
18
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Clone "BO115 2"
A polynucleotide of the present invention has been identified as clone
"BQ115_2".
BQ115_2 was isolated from a human adult colon (adenocarcinoma Caco2) 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.
BQ115_2
is a full-length clone, including the entire coding sequence of a secreted
protein (also
referred to herein as "BQ115_2 protein")
The nucleotide sequence of BQ115_2 as presently determined is reported in SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BQ115 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BQ115_2 should be approximately 400 bp.
The nucleotide sequence disclosed herein for BQ115_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BQ115_2 demonstrated at least some similarity with
sequences
identified as AA130380 (z130d03.r1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 503429 5'), N99899 (zb87e10.s1 Homo Sapiens cDNA clone 310602 3'),
T85228
2 0 (yd33e08.r1 Homo Sapiens cDNA clone 110054 5'), T97822 (ye54f02.r1 Homo
Sapiens
cDNA, and U73629 (Human chromosome 11 114g8 cosmid, complete sequence). The
similarity of BQ115 2 to that of the U73629 (Human chromosome 11 114g8 cosmid)
sequence, the gene corresponding to BQ115_2 is likely located on human
chromosome 11.
Based upon sequence similarity, BQ115 2 proteins and each similar protein or
peptide
2 5 may share at least some activity. The TopPredII computer program predicts
two potential
transmembrane domains within the BQ115_2 protein sequence centered around
amino
acids 29 and 67 of SEQ ID N0:4, respectively; amino acids 55 to 67 of SEQ ID
N0:4are also
a possible leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 68.
Clone "CC198 1"
A polynucleotide of the present invention has been identified as clone
"CC198_1".
CC198_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
19
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CC198_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CC198_1 protein").
The nucleotide sequence of CC198_1 as presently determined is reported in SEQ
ID N0:5. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CC198_l protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6. Amino acids 101 to 113 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 114, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CC198_1 should be approximately 1120 bp.
The nucleotide sequence disclosed herein for CC198_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CC198_1 demonstrated at least some similarity with
sequences
identified as AA037314 (zc52h04.r1 Soares senescent fibroblasts NbHSF Homo
sapiens
cDNA clone 325975 5' similar to WP:T07A5.2 CE01647 YK10 HOMOLOG), AA102090
(zk87c12.s1 Soares pregnant uterus NbHPU Homo Sapiens cDNA clone 489814 3'
similar
to WP T07A5.2 CE01647 YK10 HOMOLOG), H94093 (yw58d12.r1 Soares placenta
2 0 8to9weeks -2NbHP8to9W Homo Sapiens cDNA clone 256439 5' similar to SP:YK10
YEAST
P36I25 HYPOTHETICAL 32.0 KD PROTEIN IN GCN3-DAL80 INTERGENIC), N33417
(yy09a04.s1 Homo sapiens cDNA clone 270702 3' similar to WP:T07A5.2 CE01647
YK10
HOMOLOG), T87106 (yd88e12.r1 Homo Sapiens cDNA clone 115342 5' similar to SP
YK10 YEAST P36125 HYPOTHETICAL 32.0 KD PROTEIN IN GCN3-DAL80
2 S INTERGENIC), and 248055 (Caenorhabditis elegans cosmid T07A5). The
predicted
amino acid sequence disclosed herein for CC198 1 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted CC198_1 protein demonstrated at least some similarity to sequences
identified
as U96638 (unc-50 related protein; URP (Rattus norvegicus]), 248055 (T07A5.2
3 0 (Caenorhabditis elegans]), and 295397 (unknown [Schizosaccharomyces
pombe]). Based
upon sequence similarity, CC198_1 proteins and each similar protein or peptide
may
share at least some activity. The TopPredII computer program predicts five
potential
transmembrane domains within the CC198_1 protein sequence, centered around
amino
acids 110, 145, 190, 223, and 251 of SEQ ID N0:6, respectively.
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Clone "CT317 4"
A polynucleotide of the present invention has been identified as clone
"CJ317_4".
CJ317_4 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. CJ317_4 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"CJ317 4 protein").
The nucleotide sequence of CJ317_4 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading~frame and
the
predicted amino acid sequence of the CJ317_4 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CJ317_4 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for CJ317 4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CJ317_4 demonstrated at least some similarity with
sequences
identified as AA212333 (mu78d07.r1 Stratagene mouse melanoma (#937312) Mus
musculus cDNA clone 651661 5' similar to TR:C~927407 6927407 ACTIN BINDING
2 0 PROTEIN), AA251247 (zs03h05.r1 NCI CGAP_GCB1 Homo sapiens cDNA clone),
D20285 (Human HL60 3'directed MboI cDNA, HUMGS01259, clone pm1854), 816712
(yf33h12.s1 Homo sapiens cDNA clone 128711 3'), 840626 (yf72g12.s1 Homo
sapiens
cDNA clone 280513'), and T20115 (Human gene signature HUMGS01259). The
predicted
amino acid sequence disclosed herein for CJ317_4 was searched against the
GenPept and
2 5 GeneSeq amino acid sequence databases using the BLASTX search protocol.
The
predicted CJ317_4 protein demonstrated at least some similarity to sequences
identified
as U18795 (Saccharomyces cerevisiae chromosome V cosmids 9669, 8334, 8199, and
lambda clone 1160 [Saccharomyces cerevisiae]), U18795 (Ye1057cp [Saccharomyces
cerevisiae]), and X89858 (Anillin [Drosophila melanogaster]). Based upon
sequence
3 0 similarity, CJ317_4 proteins and each similar protein or peptide may share
at least some
activity. The TopPredII computer program predicts a potential transmembrane
domain
within the CJ317_4 protein sequence centered around amino acid 20 of SEQ ID
N0:8.
21
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Clone "CS319 1"
A polynucleotide -of the present invention has been identified as clone
"CS319_l".
Clones were first 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 were
identified as encoding a secreted or- transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. Probes derived
from these
cDNAs were then used to isolate CS319_1 from a human adult testes cDNA
library.
CS319_l is a full-length clone, including the entire coding sequence of a
secreted protein
(also referred to herein as "CS319_1 protein").
The nucleotide sequence of the 5' portion of CS319_1 as presently determined
is
reported in SEQ ID N0:9. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID NO:10. The predicted amino acid
sequence
of the CS319_1 protein corresponding to the foregoing nucleotide sequence is
reported in
SEQ -ID NO:10. Additional nucleotide sequence from the 3' portion of CS319 1,
including the polyA tail, is reported in SEQ ID NO:11.
The EcoRI /Notl restriction fragment obtainable from the deposit containing
clone
CS319_1 should be approximately 1600 bp.
The nucleotide sequence disclosed herein for CS319_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. CS319_1 demonstrated at least some similarity with
sequences
identified as AA062561 (zf68c09.r1 Soares pineal gland N3HPG Homo Sapiens cDNA
clone 382096 5' similar to contains Ll.t2 L1 repetitive element), AC000378
(***
SEQUENCING IN PROGRESS *** Human Chromosome 11p15.5 pac pDJ1173a5; HTGS
phase 1, 4 unordered pieces), H04709 (ph2f06u_19/1TV Homo Sapiens cDNA clone
ph2f06u_19/1TV), L10574 (Human Chromosome 7 STS sWSS208; single read), 839402
(yh95e03.r1 Homo sapiens cDNA clone 137500 5'), 281310 (Human DNA sequence
from
cosmid 019A on chromosome 6 Contains HLA DNA gene and STS), 282217 (***
SEQUENCING IN PROGRESS *** Human DNA sequence *** SEQUENCING IN
PROGRESS *** from clone 78B3; HTGS phase 1), and 295704 (Human DNA sequence
from
3 0 4PTEL, Huntington's Disease Region, chromosome 4p16.3). Based upon
sequence
similarity, CS319_1 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of CS319 1 indicates that it may contain one
or more
Ll repeat elements.
22
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Clone "DL504 3"
A polynucleotide of the present invention has been identified as clone
"DL504_3".
DL504_3 was isolated from a human adult brain cDNA-library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or -transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. DL504_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "DL504_3 protein").
The nucleotide sequence of DL504_3 as presently determined is reported in SEQ
ID N0:12. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the DL504_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:13.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
DL504_3 should be approximately 2400 bp.
The nucleotide sequence disclosed herein for DL504_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. DL504_3 demonstrated at least some similarity with
sequences
identified as D81501 (Human fetal brain cDNA 5'-end GEN-168H07.1), H24009
(ym49e05.s1 Homo Sapiens cDNA clone 51376 3'), H50251 (yo28h02.s1 Homo Sapiens
2 0 cDNA clone 179283 3'), 812817 (yf57b10.r1 Homo Sapiens cDNA clone 26249
5'), U58886
(Mus musculus SH3-containing protein SH3P4 mRNA, complete cds), and X99657
(H.sapiens mRNA for protein containing SH3 domain). The predicted amino acid
sequence disclosed herein for DL504 3 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
2 5 DL504_3 protein demonstrated at least some similarity to sequences
identified as 871943
(Grb3-3 protein), U58886 (SH3P4 [Mus musculus]), and X99657 (SH3-containing
Grb-2-like
2 [Homo sapiens]). Based upon sequence similarity, DL504 3 proteins and each
similar
protein or peptide may share at least some activity.
3 0 Clone "DN747 7"
A polynucleotide of the present invention has been identified as clone
"DN747_7".
DN747_7 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
23
CA 02281015 1999-08-13
WO 98/38209 PCTIUS98/03697
analysis of the amino acid sequence of the encoded protein. DN747_7 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "DN747_7 protein").
The -nucleotide sequence of DN747_7 as presently determined is reported in SEQ
ID N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the DN747_7 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15.
The EcoRI/Notl restriction fragment obtainable from the deposit containing
clone
DN747_7 should be approximately 3450 bp.
The nucleotide sequence disclosed herein for DN747_7 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA _search protocols. DN747_7 demonstrated at least some similarity with
sequences
identified as AA195883 (zp92f09.r1 Stratagene HeLa cell s3 937216 Homo Sapiens
cDNA
clone 627689 5' similar to WP:D2045.8 CE00608 TNF-ALPHA INDUCED PROTEIN B12),
AA199716 (zq74h11.r1 Stratagene hNT neuron (#937233) Homo Sapiens cDNA clone
647397 5'), U55984 (Human chromosome 18q12.1-q12.2 clone 36 mRNA sequence),
W67965 (zd39f04.s1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone 343039
3'),
W68044 (zd39f04.r1 Soares fetal heart NbHHI9W Homo Sapiens cDNA clone 343039
5'),
and X79321 (R. norvegicus (Wistar) mRNA for tau microtubule-associated
protein). The
2 0 predicted amino acid sequence disclosed herein for DN747_7 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted DN747_7 protein demonstrated at least some similarity to
sequences
identified as U80842 (similar to human tumor necrosis factor-alpha-induced
protein B12
(GI 179304) and several potassium channel proteins (see, U42975)
[Caenorhabditis
2 5 elegans -]). Based upon sequence similarity, DN747_7 proteins and each
similar protein or
peptide may share at least some activity. The TopPredII computer program
predicts a
potential transmembrane _domain within the DN747_7 protein sequence around
amino
acid 120 of SEQ -ID N0:15. The nucleotide sequence of DN747_7 indicates that
it may
contain one or more of the following nucleotide repeat regions: simple CCA
repeat,
3 0 simple AT repeat, and L1PA2.
Clone "DU123 1"
A polynucleotide of the present invention has been identified as clone
"DU123_1".
DU123_1 was isolated from a human fetal brain cDNA library using methods which
are
24
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
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. DU123_l is a full-
length
clone, includingthe entire coding sequence of a secreted protein (also
referred to herein
as "DU123_1 protein").
The nucleotide sequence of DU123_1 as presently determined is reported in SEQ
ID N0:16. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the DU123_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:17. If a frameshift were
introduced into
the nucleotide sequence of SEQ ID N0:16 at position 1224, the predicted amino
acid
sequence for the DU123_1 protein would be extended by 96 amino acids.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
DU123_1 should be approximately 1450 bp.
The predicted amino acid sequence disclosed herein for DU123_1 was searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted DU123_1 protein demonstrated at least some
similarity
to sequences identified as L35240 (enigma protein (Homo sapiens]), U23769
(CLP36
[Rattus norvegicus]), and U48247 (protein kinase C-binding protein Enigma
[Rattus
norvegicus]). These protein sequences contain LIM domains which bind two zinc
atoms
2 0 and have been implicated in the recognition of tyrosine residues in tight
turns. The
predicted DU123_1 protein also shows some homology to human protein tyrosine
phosphatase. Based upon sequence similarity, DU123_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 20 amino terminal amino acids of
the
2 S DU123_1 protein sequence of SEQ ID N0:17.
Clone "FB78 1"
A polynucleotide of the present invention has been identified as clone
"FB78_1".
FB78_1 was isolated from a human adult placenta cDNA library using methods
which are
3 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637}, or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. FB78_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"FB78_1 protein").
CA 02281015 1999-08-13
WO 98/38209 PCT/US98103697
The nucleotide sequence of FB78_1 as presently determined is reported in SEQ
ID
N0:18. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the FB78 1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:19. Amino acids 8 to 20 are a
predicted
S leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 21, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
FB78_1 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for FB78_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. FB78_1 demonstrated at least some similarity with
sequences
identified as N50885 (yy92e05.s1 Homo sapiens cDNA clone 281024 3'), Q03823
(Poly GT
enhancer element), T08769 (EST06661 Homo Sapiens cDNA clone HIBBJ45 5' end),
and
U12968 (Human clone S3/1 dinucleotide repeat-rich region). Based upon sequence
similarity, FB78_1 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of FB78_1 indicates that it may contain a
simple CA
repeat sequence.
Deposit of Clones
2 0 Clones BD380_l, BQ115_ _ - -2, CC198_1, CJ317_4, CS319_l, DL504_3,
DN747_7,
DU123_l, and FB78_1 were deposited on February 26, 1997 with the American Type
Culture Collection as an original deposit under the Budapest Treaty and were
given the
accession number ATCC 98337, from which each clone comprising a particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
2 5 deposited material will be irrevocably removed upon the granting of the
patent, except
for the requirements specified in 37 C.F.R. ~ 1.808(b).
Each clone has been transfected into separate bacterial cells {E. coli) in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
3 0 appropriate fragment for such clone. Each clone was deposited in either
the pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman
et al.,
1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from
pMT2
(Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR
sequences,
26
CA 02281015 1999-08-13
WO 98/38209 PCT/LTS98/03697
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
orientation) in the deposited isolate. In such instances, the cDNA insert can
still be
isolated by digestion with EcoRI and NotI. However, NotI will then produce the
5' site
and EcoRI will produce the 3' site for- placement of the cDNA in proper
orientation for
expression in a suitable vector. The cDNA may also be expressed from the
vectors in
which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of the
oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
BD380_1 ' SEQ ID N0:20
BQ115_2 ~ SEQ ID N0:21
2 0 CC198_1 SEQ ID N0:22
CJ317 4 SEQ ID N0:23
CS319_1 SEQ ID N0:24
DL504 3 SEQ ID N0:25
DN747_7 SEQ ID N0:26
DU123_l SEQ ID N0:27
FB78_1 SEQ ID N0:28
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
3 0 nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
27
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's'"), if any; _
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
- A or T and 4 degrees for each G or C).
S The oligonucleotide should preferably be labeled with g--''P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
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
lZg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
2 0 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
2 5 NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
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
3 0 by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for
15 minutes.
A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
28
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then_be verified by restriction
analysis,
hybridization analysis, or DNA sequencing
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature form
of such protein may be obtained by expression of the disclosed full-length
polynucleotide
2 0 (preferably those deposited with ATCC) in a suitable mammalian cell or
other host cell.
The sequence of the mature form of the protein may also be determinable from
the amino
acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
2 5 are transcribed to produce the mRNAs from which cDNA polynucleotide
sequences are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
3 0 be isolated in accordance with known methods using the sequence
information disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
29
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
Organisms that have enhanced, reduced-, or modified expression of the genes)
corresponding to the polynucleotide sequences disclosed herein are provided.
The
desired change in gene expression . can be achieved through the use of
antisense
polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from
the
gene (Albert and Morris,1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky
et al.,1997,
Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res.
Mol. Biol. 58: 1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 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
2 0 can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. NatI. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
2 5 Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
3 0 assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains-from sequence information.
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
Species homologs of the disclosed polynucleotides and proteins are also
provided
by the present invention. As used herein, a "species homologue" is a protein
or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide, as determined by those of skill in the art. Species homologs
may be
2 0 isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source from the desired species.
Preferably,
species homologs are those isolated from mammalian species.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
2 5 which also encode proteins which are identical, homologous, or related to
that encoded
by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
under
3 0 reduced stringency conditions, more preferably stringent conditions, and
most preferably
highly stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown 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
31
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
- ~p)t Buffer' and Buffer'
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
~ 42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA s 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 T~*; lxSSC Tfl*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; IxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 T"*; 4xSSC T"*; 4xSSC
I DNA:RNA ~ 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
SOC; 4xSSC, 50% formamide
L RNA:RNA <50 T, *; 2xSSC T, *; 2xSSC
M DNA:DNA s 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
p DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
2 P DNA:RNA <50 T,,*; 6xSSC T,.*; 6xSSC
0
RNA:RNA z 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
t~ The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
2 5 hybridizing a polynucleotide to a target polynucleotide of unknown
sequence, the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaH,_PO" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
3 0 (lxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and
wash buffers; washes are
performed for 15 minutes after hybridization is complete.
*TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
32
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,o[Na']) +0.41(%G+C) -
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
hybridization buffer ([Na'] for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
~ where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
2 0 Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to
produce the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185 537-566 (1990). As defined herein "operably
linked" means that the isolated polynucleotide of the invention and an
expression control
2 5 sequence are situated within a vector or cell in such a way that the
protein is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
protein. Mammalian host cells include, for example, monkey COS cells, Chinese
Hamster
3 0 Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells,
human Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
33
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Alternatively, it may be possible to produce the protein in lower eukaryotes
such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987), incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
2 0 The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 5 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
3 0 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharinacia (Piscataway, NJ)
and
34
CA 02281015 1999-08-13
WO 9$/3$209 PCT/US98/03697
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,~ne or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
2 0 including protein activity. Thus, they may be employed as biologically
active or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 5 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
3 0 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
CA 02281015 1999-08-13
WO 9$/3$209 PCT/US98/03697
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be
useful_for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleoddes provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
2 0 constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
2 5 information to derive PCR primers for genetic fingerprinting; as a probe
to "subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
3 0 immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
36
CA 02281015 1999-08-13
WO 98/3$209 PCT/US98/03697
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
s throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
2 0 Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory
Press, Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel
eds.,1987.
Nutritional Uses
2 5 Polynucleotides and proteins of the, present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
or amino acid supplement, use as a carbon source, use as a nitrogen source and
use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
3 0 or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
can be added to the medium in or on which the microorganism is cultured.
37
CA 02281015 1999-08-13
WO 98/8209 PCT/US98/03697
Cytokine and Cell Proliferadon/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
{either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and
CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
2 0 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
2 5 Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994;
and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
3 0 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky,
P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
38
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C.-and Turner, K. J. In
Current Protocols
in Immunology: J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. j.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating: or Suppressing Activity
2 0 A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
2 5 as effecting the cytolytic activity of NK cells and other cell
populations. These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
3 0 herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various
fungal infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
39
CA 02281015 1999-08-13
WO 98/38209 PCT/IJS98/03697
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
2 0 the tolerizing agent has ceased. Operationally, tolerance can be
demonstrated by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
2 5 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
3 0 the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992} and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
2 0 Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 5 Administration of reagents which block costimulation of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
3 0 the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr mice or NZ8 hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
41
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means o'f- up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
2 0 costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 5 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
3 0 transfected tumor cells are returned to the patient to result in
expression of the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
42
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, cair 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
~i2
microglobulin protein or an MHC class II a chain protein and an MHC class II p
chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
2 0 limitation, those described in: Current Protocols in Immunology, Ed by J.
E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492, 1981; Hemnann et al., J. Immunol. 128:1968-1974, 1982; Handa
et al.,
2 5 J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-
3500,1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
3 0 Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Th1/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
43
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
j.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
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; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
2 0 proteins that prevent apoptosis after superantigen induction and proteins
that regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 5 Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation, those described in: Antica et al.,
Blood
84:111-117, 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 Regulatinn Activity
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
' 44
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating-various anemias or for
use in
conjunctiomvixh irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
2 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 5 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
3 0 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freslzney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et aI. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et a1. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
2 0 well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 5 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
3 0 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
46
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may hare prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
2 0 peripheral nervous system diseases and neuropathies, as well as mechanical
and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 5 Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
3 0 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
47
CA 02281015 1999-08-13
WO 98/38209 PCTlUS98/03697
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such. as organs (including,- for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/1t;035 (bone, cartilage,
tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
2 0 Assays for wound healing activity include, without limitation, those
described in:
Winter, ~idermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.),
Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, j.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
activities. Inhibins are characterized by their ability to inhibit the release
of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
3 0 invention, alone or in heterodimers with a member of the inhibin a family,
may be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
48
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
~3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
United States Patent 4,798,885. A protein of the invention may also be useful
for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
2 0 treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 5 population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
of cells can be readily determined by employing such protein or peptide in any
known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
3 0 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
49
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, _ Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter- 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,
1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
2 0 described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick
et al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Ligand Activity
2 5 A protein of the present invention may also demonstrate activity as
receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
3 0 cellular adhesion molecules (such as selectins, integrins and their
ligands) and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
development of cellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
The activity of a protein of the invention may, a-mong other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
2 0 suppressing production of other factors which more directly inhibit or
promote an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
2 5 lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
30 Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss
or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
51
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
2 0 suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
2 5 Cancer cells have also been shown to express cadherins of a different
tissue type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
3 0 reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherir<s, preventing
the cells from
52
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
cancer, the less cadherin expression there will be; and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
2 0 A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
3 0 agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
53
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
2 0 salts, buffers, stabilizers, solubilizers, and other materials well known
in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
2 5 such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-
8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may further
contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
3 0 pharmaceutical composition to produce a synergistic effect with protein of
the invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
54
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
S compositions of the invention may comprise a protein of the invention in
such multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
2 0 The pharmaceutical composition.of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
2 5 monoglycerides, diglycerides, sulfatides, Iysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
3 0 As used herein, the term "therapeutically effective amount" means the
total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
2 0 cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
2 5 composition of the invention may additionally contain a solid carrier such
as a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
3 0 may be added. The liquid form of the pharmaceutical composition may
further contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
. 56
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringei s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
2 0 response. Larger doses of protein of the present invention may be
administered until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 ug to
about 1 mg)
2 5 of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
3 0 will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
5?
CA 02281015 1999-08-13
WO 98/38209 PCT/LJS98/03697
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Arner.Chem.Soc. 85 2149-2154 (1963); J.L. Krstenansky, et
al., FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
2 0 tissue damage. Topical administration may be suitable for wound healing
and tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
2 5 composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
3 0 The choice of matrix material is based on biocompatfbility,
biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
58
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
2 0 acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
2 5 are prevented from infiltrating the matrix, thereby providing the protein
the opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
3 0 (EGF), platelet derived growth factor (PDGF), transforming growth factors
(TGF-a and
TGF-~3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
59
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be Formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also' be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
2 0 Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPIrICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v} COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1694 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
61
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(ii) MOLECULE TYPE: cDNA
(xi) SEQE1ENCE DESCRIPTION: SEQ ID N0:1:
CCACGAGCGC TGGCTGAGGG ACCGAGCCGG AGAGCCCCGG AGCCCCCGTA 60
ACCCGCGCGG
GGAGCGCCCA GGATGCCGCG CGGGGACTCG GAGCAGGTGC GCTACTGCGC 120
GCGCTTCTCC
TACCTCTGGC TCAAGTTTTC ACTTATCATC TATTCCACCG TGTTCTGGCT 180
GATTGGGGCC
CTGGTCCTGT CTGTGGGCAT CTATGCAGAG GTTGAGCGGC AGAAATATAA 240
AACCCTTGAA
AGTGCCTTCC TGGCTCCAGC CATCATCCTC ATCCTCCTGG GCGTCGTCAT 300
GTTCATGGTC
TCCTTCATTG GTGTGCTGGC GTCCCTCCGT GACAACCTGT ACCTTCTCCA 360
AGCATTCATG
TACATCCTTG GGATCTGCCT CATCATGGAG CTCATTGGTG GCGTGGTGGC 420
CTTGACCTTC
CGGAACCAGA CCATTGACTT CCTGAACGAC AACATTCGAA GAGGAATTGA 480
GAACTACTAT
GATGATCTGG ACTTCAAAAA CATCATGGAC TTTGTTCAGA AAAAGTTCAA 540
GTGCTGTGGC
GGGGAGGACT ACCGAGATTG GAGCAAGAAT CAGTACCACG ACTGCAGTGC 600
CCCTGGACCC
CTGGCCTGTG GGGTGCCCTA CACCTGCTGC ATCAGGAACA CGACAGAAGT 660
TGTCAACACC
ATGTGTGGCT ACAAAACTAT CGACAAGGAG CGTTTCAGTG TGCAGGATGT 720
CATCTACGTG
CGGGGCTGCA CCAACGCCGT GATCATCTGG TTCATGGACA ACTACACCAT 780
CATGGCGGGC
ATCCTCCTGG GCATCCTGCT TCCCCAGTTC CTGGGGGTGC TGCTGACGCT 840
GCTGTACATC
ACCCGGGTGG AGGACATCAT CATGGAGCAC TCTGTCACTG ATGGGCTCCT 900
GGGGCCCGGT
GCCAAGCCCA GCGTGGAGGC GGCAGGCACG GGATGCTGCT TGTGCTACCC 960
CAATTAGGGC
CCAGCCTGCC ATGGCAGCTC CAACAAGGAC CGTCTGGGAT AGCACCTCTC 1020
AGTCAACATC
GTGGGGCTGG ACAGGGCTGC GGCCCCTCTG CCCACACTCA GTACTGACCA 1080
AAGCCAGGGC
TGTGTGTGCC TGTGTGTAGG TCCCACGGCC TCTGCCTCCC CAGGGAGCAG 1140
AGCCTGGGCC
TCCCCTAAGA GGCTTTCCCC GAGGCAGCTC TGGAATCTGT GCCCACCTGG 1200
GGCCTGGGGA
ACAAGGCCCT CCTTTCTCCA GGCCTGGGCT ACGGGGGAGG GAGAGCCTGA 1260
GGCTCTGCTC
AGGGCCCATT TCATCTCTGG CAGTGCCTTG GCGGTGGTAT TCAAGGCAGT 1320
TTTGTAGCAC
CTGTAATTGG GGAGAGGGAG TGTGCCCCTC GGGGCAGGAG GGAAGGGCAT 1380
CTGGGGAAGG
GCAGGAGGGA AGAGCTGTCC ATGCAGCCAC GCCCATGGCC AGGTTGGCCT 1440
CTTCTCAGCC
62
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
TCCCAGGTGC CTTGAGCCCT CTTGCAAGGG CGGCTGCTTC CTTGAGCCTA GTTTTTTTAC 1500
GTGATTTTTG TAACATTCAT TTTTTTGTAC AGATAACAGG AGTTTCTGAC TAATCAAAGC 1560
TGGTATTTCC CCGCATGTCT TATTCTTGCC CTTCCCCCAA CCAGTTTGTT AATCAAACAA 1620
TAAAAACATG TTTTTTTTAA F,~~,AAAAAAAA A.F~~AAAAAAA p~~.AAAAAAAA p,~~AAAAAAAA 16
8 0
1694
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Pro Arg Gly Asp Ser Glu Gln Val Arg Tyr Cys Ala Arg Phe Ser
1 5 10 15
Tyr Leu Trp Leu Lys Phe Ser Leu Ile Ile Tyr Ser Thr Val Phe Trp
20 25 30
Leu Ile Gly Ala Leu Val Leu Ser Val Gly Ile Tyr Ala Glu Val Glu
35 40 45
Arg Gln Lys Tyr Lys Thr Leu Glu Ser Ala Phe Leu Ala Pro Ala Ile
50 55 60
Ile Leu Ile Leu Leu Gly Val Val Met Phe Met Val Ser Phe Ile Gly
65 70 75 80
Val Leu Ala Ser Leu Arg Asp Asn Leu Tyr Leu Leu Gln Ala Phe Met
85 90 95
Tyr Ile Leu Gly Ile Cys Leu Ile Met Glu Leu Ile Gly Gly Val Val
100 105 110
Ala Leu Thr Phe Arg Asn Gln Thr Ile Asp Phe Leu Asn Asp Asn Ile
115 120 125
Arg Arg Gly Ile Glu Asn Tyr Tyr Asp Asp Leu Asp Phe Lys Asn Ile
130 135 140
Met Asp Phe Val Gln Lys Lys Phe Lys Cys Cys Gly Gly Glu Asp Tyr
145 150 155 160
63
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Arg Asp Trp Ser Lys Asn Gln Tyr His Asp Cys Ser Ala Pro Gly Pro
165 170 175
Leu Ala Cys Gly Val Pro Tyr Thr Cys Cys Ile Arg Asn Thr Thr Glu
180 185 190
Val Val Asn Thr Met Cys Gly Tyr Lys Thr Ile Asp Lys Glu Arg Phe
195 200 205
Ser Val Gln Asp Val Ile Tyr Val Arg Gly Cys Thr Asn Ala Val Ile
210 215 220
Ile Trp Phe Met Asp Asn Tyr Thr Ile Met Ala Gly Ile Leu Leu Gly
225 230 235 240
Ile Leu Leu Pro Gln Phe Leu Gly Val Leu Leu Thr Leu Leu Tyr Ile
245 250 255
Thr Arg Val Glu Asp Ile Ile Met Glu His Ser Val Thr Asp Gly Leu
260 265 270
Leu Gly Pro Gly Ala Lys Pro Ser Val Glu Ala Ala Gly Thr Gly Cys
275 280 285
Cys Leu Cys Tyr Pro Asn
290
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
GTTCTTCCGGTGGCGGAGCG GCGGATTAGCCTTCGCGGGGCAAAATGGAG CTCGAGGCCA60
TGAGCAGATATACCAGCCCA GTGAACCCAGCTGTCTTCCCCCATCTGACC GTGGTGCTTT120
TGGCCATTGGCATGTTCTTC ACCGCCTGGTTCTTCGTTTACGAGGTCACC TCTACCAAGT180
ACACTCGTGATATCTATAAA GAGCTCCTCATCTCCTTAGTGGCCTCACTC TTCATGGGCT240
TTGGAGTCCTCTTCCTGCTG CTCTGGGTTGGCATCTACGTGTGAGCACCC AAGGGTAACA300
ACCAGATGGCTTCACTGAAA CCTGCTTTTGTAAATTACTTTTTTTTACTG TTGCTGGAAG360
TGTCCCACCTGCTGCTCATA ATAAATGCAGATGTATAGCAF,~~AAAAAAAA p~A 419
64
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids
(B) TYPE: amino acid
(C~ STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Glu Leu Glu Ala Met Ser Arg Tyr Thr Ser Pro Val Asn Pro Ala
1 5 10 15
Val Phe Pro His Leu Thr Val Val Leu Leu Ala Ile Gly Met Phe Phe
20 25 30
Thr Ala Trp Phe Phe Val Tyr Glu Val Thr Ser Thr Lys Tyr Thr Arg
35 40 45
Asp Ile Tyr Lys Glu Leu Leu Ile Ser Leu Val Ala Ser Leu Phe Met
50 55 60
Gly Phe Gly Val Leu Phe Leu Leu Leu Trp Val Gly Ile Tyr Val
65 ' 70 75
(2) INFORMATION FOR SEQ ID NO: S:
(i! SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1214 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGGAAGCCCG CCCGGTGGCG GCTGGGGTCG GCTGCTGGGA GGAGGTGGTG GGCTGGTTCG 60
GACGTGGGTCGAGGCTGTAGCAGGACTCCAGGGTTGGGAA GAACATGGAA AGTGACCTCC120
CTGCCAAATAACTCAGAAGAGGAGTGTCGGTAGCCAAATG TTTCTTCAGA ATACGTGTAA180
AAGAAATGTTTTTCTTCCATCTAGGAAGATGTTACCGAGT ACTTCAGTGA ATTCCTTAGT240
GCAGGGGAAC GGAGTCTTGA ATTCCAGGGA TGCGGCAAGA CACACAGCCG GAGCGAAACG 300
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/0369'7
CTACAAATAT CTGAGAAGGCTTTTCCGCTT TCGGCAAATGGACTTTGAAT TTGCTGCCTG360
GCAGATGCTC TACCTGTTCACATCCCCACA GAGAGTTTAC-AGAAATTTTC ATTATCGAAA420
ACAGACGAAG GACCAGTGGGCCAGAGATGA CCCTGCTTTCTTGGTCCTGT TAAGTATCTG480
GCTCTGTGTG TCCACTATAGGATTTGGCTT TGTGCTGGACATGGGATTCT TTGAGACAAT540
AAAGCTTCTC CTTTGGGTTGTACTCATAGA TTGTGTAGGCGTTGGTCTTC TGATAGCAAC600
TTTAATGTGG TTCATCTCTAACAAGTATTT AGTGAAACGACAGAGCAGAG ACTATGATGT660
GGAATGGGGC TATGCTTTTGATGTGCATCT CAATGCTTTTTATCCACTCC TGGTCATTTT720
GCATTTTATC CAGCTTTTTTTCATCAACCA TGTTATCCTGACAGACACAT TTATTGGATA780
TTTAGTTGGA AATACCTTATGGTTGGTTGC AGTTGGCTATTATATCTATG TAACTTTCCT840
GGGATACAGT GCATTGCCATTTTTGAAAAA TACAGTAATTCTTCTGTATC CATTTGCACC900
TCTGATTCTG CTCTACGGGCTTTCCCTGGC ACTGGGATGGAACTTCACCC ATACTCTCTG960
TTCTTTCTAT AAGTACAGAGTGAAATAAAA AGTGAGAAGAAGATTCAATC GTAACTGTGT1020
CAACAGTATT GTGAAGTGATCATTTCTTGT AAAACTTGTAAATAAACTAT CATCTTTGTA1080
GATATCTTAA AGGTGTAAAGTTTGCAAATT TGAAGAAATATATATTAACA CTGTGGTCAG1140
GTACATTCCT TAAAACTAATTAAATGTACA TTTCTATAATAAATATTTTT TAAACTAAAA1200
1214
(2) INFORMATION
FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH : 276 amino acids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
linear
(ii) MOLECULE PE: protein
TY
(xi)SEQUENCEDESCRIPTION: N0:6:
SEQ
ID
MetPhe GlnAsn Thr Cys ArgAsnVal Phe ProSer
Leu Lys Leu Arg
1 5 10 15
LysMet ProSer Thr Ser AsnSerLeu Val GlyAsn
Leu Val Gln Gly
20 25 30
ValLeu SerArg Asp Ala ArgHisThr Ala AlaLys
Asn Ala Gly Arg
35 40 45
66
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Tyr Lys Tyr Leu Arg Arg Leu Phe Arg Phe Arg Gln Met Asp Phe Glu
50 55 60
Phe Ala Ala Trp Gln Met Leu Tyr Leu-Phe_ Thr Ser Pro Gln Arg Val
65 70 75 80
Tyr Arg Asn Phe His Tyr Arg Lys Gln Thr Lys Asp Gln Trp Ala Arg
85 90 95
Asp Asp Pro Ala Phe Leu Val Leu Leu Ser Ile Trp Leu Cys Val Ser
100 105 110
Thr Ile Gly Phe Gly Phe Val Leu Asp Met Gly Phe Phe Glu Thr Ile
125 120 125
Lys Leu Leu Leu Trp Val Val Leu Ile Asp Cys Val Gly Val Gly Leu
130 135 140
Leu Ile Ala Thr Leu Met Trp Phe Ile Ser Asn Lys Tyr Leu Val Lys
145 150 155 160
Arg Gln Ser Arg Asp Tyr Asp Val Glu Trp Gly Tyr Ala Phe Asp Val
165 170 175
His Leu Asn Ala Phe Tyr Pro Leu Leu Val Ile Leu His Phe Ile Gln
180 185 190
Leu Phe Phe Ile Asn His Val Ile Leu Thr Asp Thr Phe Ile Gly Tyr
195 200 205
Leu Val Gly Asn Thr Leu Trp Leu Val Ala Val Gly Tyr Tyr Ile Tyr
210 215 220
Val Thr Phe Leu Gly Tyr Ser Ala Leu Pro Phe Leu Lys Asn Thr Val
225 230 235 240
Ile Leu Leu Tyr Pro Phe Ala Pro Leu Ile Leu Leu Tyr Gly Leu Ser
245 250 255
Leu Ala Leu Gly Trp Asn Phe Thr His Thr Leu Cys Ser Phe Tyr Lys
260 265 270
Tyr Arg Val Lys
275
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1762 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: cDNA
67
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(xi1 SEQUENCE DESCRIPTION:
SEQ ID N0:7:
GCAACATTCA TTCTTCAGTC ATGGCCAGTCCAGGAGGTCTTAGTGCTGTG CGAACCARCA60
ACTTCGCCCT TGTTGGATCT TACACATTATCATTGTCTTC 120
AGTAGGAAAT
ACTAAGTTTG
TTCTGGACAA GATAAATTAT GATGTAAGAGAGCGAGAGCT 180
ACTGGGCTAT
TTGTTCCAGG
AAAAGGTCCC CTTTTTATCT TCTTTGGAAGGTCATATTTATTTAAAAATA AAATGTCAAG240
TGAATTCCAG TGTTGAAGAA AGAGGTTTTCTAACCATATTTGAAGATGTT AGTGGTTTTG300
GTGCCTGGCA TCGAAGATGG TGTGTTCTTTCTGGAAACTGTATATCTTAT TGGACTTATC360
CAGATGATGA GAAACGCAAG AATCCCATAGGAAGGATAAATCTGGCTAAT TGTACCAGTC420
GTCAGATAGA ACCAGCCAAC AGAGAATTTTGTGCAAGACGCAACACTTTT GAATTAATTA480
CTGTCCGACC ACAAAGAGAA GATGACCGAGAGACTCTTGTCAGCCAATGC AGGGACACAC540
TCTGTGTTAC CAAGAACTGG CTGTCTGCAGATACTAAAGAAGAGCGGGAT CTCTGGATGC600
AAAAACTCAA TCAAGTTCTT GTTGATATTCGCCTCTGGCAACCTGATGCT TGCTACGAAC660
CTATTGGAAA GCCTTAAACC GGGAAATTTCCATGCTATCTAGAGGTTTTT GATGTCATCT720
TAAGAAACAC ACTTAAGAGC ATCAGATTTACTGATTGCATTTTATGCTTT AAGTACGAAA780
GGGTTTGTGC CAATATTCAC TACGTATTATGCAGTATTTATATCTTTTGT ATGTAAAACT840
TTAACTGATT TCTGTCATTC ATCAATGAGTAGAAGTAAATACATTATAGT TGATTTTGCT900
AAATCTTAAT TTAAAAGCCT CATTTTCCTAGAAATCTAATTATTCAGTTA TTCATGACAA960
TATTTTTTTA AAAGTAAGAA ATTCTGAGTTGTCTTCTTGGAGCTGTAGGT CTTGAAGCAG1020
CAACGTCTTT CAGGGGTTGG AGACAGAAACCCATTCTCCAATCTCAGTAG TTTTTTCGAA1080
AGGCTGTGAT CATTTATTGA TCGTGATATGACTTGTTACTAGGGTACTGA AAAAAATGTC1140
TAAGGCCTTT ACAGAAACAT TTTTAGTAATGAGGATGAGAACTTTTTCAA ATAGCAAATA1200
TATATTGGCT TAAAGCATGA GGCTGTCTTCAGAAAAGTGATGTGGACATA GGAGGCAATG1260
TGTGAGACTT GGGGGTTCAA TATTTTATATAGAAGAGTTAATAAGCACAT GGTTTACATT1320
TACTCAGCTA CTATATATGC AGTGTGGTGCACATTTTCACAGAATTCTGG CTTCATTAAG1380
ATCATTATTT TTGCTGCGTA GCTTACAGACTTAGCATATTAGTTTTTTCT ACTCCTACAA1440
GTGTAAATTG AAAAATCTTT ATATTAAAAAAAGTAAACTGTTATGAAGCT GCTATGTACT1500
AATAATACTT TGCTTGCCAA AGTGTTTGGGTTTTGTTGTTGTTTGTTTGT TTGTTTGTTT1560
68
CA 02281015 1999-08-13
WO 98/38209 PCT/IJS98/03697
TTGGTTCATG AACAACAGTG TCTAGAAACC CATTTTGAAA GTGGAAAATT ATTAAGTCAC 1620
CTATCACCTT TAAACGCCTT TTTTTAAAAT TATAAAATAT TGTAAAGCAG GGTSTC-AACT 1680
TTTAAATACA CTTTGAACTT CTTCTCTGAA TTATTAAAGT TCCTTTATGA CCTCATTTAT 1740
AAACAYTAAA AAAAAAAAAA AA 1?62
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Ala Ser Pro Gly Gly Leu Ser Ala Val Arg Thr Xaa Asn Phe Ala
1 5 10 15
Leu Val Gly Ser Tyr Thr Leu Ser Leu Ser Ser Val Gly Asn Thr Lys
20 25 30
Phe Val Leu Asp Lys Ile Asn Tyr Asp Val Arg Glu Arg Glu Leu Leu
35 40 45
Gly Tyr Leu Phe Gln Glu Lys Val Pro Phe Leu Ser Ser Leu Glu Gly
50 55 60
His Ile Tyr Leu Lys Ile Lys Cys Gln Val Asn Ser Ser Val Glu Glu
65 70 75 80
Arg Gly Phe Leu Thr Ile Phe Glu Asp Val Ser Gly Phe Gly Ala Trp
85 90 95
His Arg Arg Trp Cys Val Leu Ser Gly Asn Cys Ile Ser Tyr Trp Thr
100 105 110
Tyr Pro Asp Asp Glu Lys Arg Lys Asn Pro Ile Gly Arg Ile Asn Leu
115 120 125
Ala Asn Cys Thr Ser Arg Gln Ile Glu Pro Ala Asn Arg Glu Phe Cys
130 135 140
Ala Arg Arg Asn Thr Phe Glu Leu Ile Thr Val Arg Pro Gln Arg Glu
145 150 155 160
Asp Asp Arg Glu Thr Leu Val Ser Gln Cys Arg Asp Thr Leu Cys Val
165 170 ~ 175
69
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Thr Lys Asn Trp Leu Ser Ala Asp Thr Lys Glu Glu Arg Asp Leu Trp
180 185 190
Met Gln Lys Leu Asn Gln Val Leu Val Asp Ile Arg Leu Trp Gln Pro
195 200 205
Asp Ala Cys Tyr Glu Pro Ile Gly Lys Pro
210 215
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1037 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:9:
TCGGGAGTTG GTTGCCTTTT CACTTTATTGTTTCCTTTGCTGCACAAAATATTTTAAGTT60
TAATGTAGTA TAATTTATTT TAGTCTTTTTCACCTGTGCTTTTGGTTTCATTGGACCTTC120
TAATGCCCTG AAGCTTTTCT CTTATGTTTTCTTCTAGGAGTTTTATGGTTTTAGGCCTTA180
TGCATAAATC TGTGAGTTAA TGTATATATATGTTGTAAAATAAGACCCATCTCCATATTT240
GGCATGTGGA TATTCAGTTT CCCAAACACCACTTGTTGTTGTTTTATGTTTAACTTTACT300
TTCACCAGTT TAGTATAAAG GATACAATTCAGGAACAGCCAAGAGGAAGAGATGTAAATG360
GCAAGGAAAG ATGGAGCGAG TAGGTAACCCTGCTAAATAGCCATGATTGAGAAAATCCCC420
CTTCAGTTGT GCTCTCCAAG AGCAGCTTACTGGCAAAGAGACATGCTTACCATTATGACT480
TATATGACTT ATATGATGCC TCACTTTGTAACTATCACAAAGCCAGACACAGACTCTGCA540
AATTCCCCTT TTTCTTCCAC AATCAAATGAACAATTTTATCTTCAGTGGTCAGAACAAAA600
NACTTGTTAA ACAAATTTNG CTTAAGATTCTCTCCTTCCCACAGGCCTTGAACTTTGATT660
CACCCTCAGT CTGAGCTTAC ATACAACTTATCTTTATGCCTCCTCCTAAGAATAGGCTAA720
CTTCAGGGTG AAATATCCTC TGATCTGGGATCTAATTTTGCCACACTCCATACTGCCCTC780
CAGCTTTCAT CTTTTTTCAA ATCTTTTTTGNTCCTCCCTGTAAAAGGAACCCCTTATCTT840
CTTAACCTTT CAAATACTTG CAGATATTTTGCTTGGTGCTTCCCATCTATTGCAATACCC900
CTTTAGATAA AGTCAATTCT TATCTAAAATCAAATTCATTTTATTTGACAATGTTTACAA960
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
ACAACCCCAG GACGATAACA ATTACACTCT CAATACTGGC ATCACACCTT CACAATTACA 1020
CTAACCCCAA CCTAGGC - 1037
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Phe Thr Asn Asn Pro Arg Thr Ile Thr Ile Thr Leu Ser Ile Leu
1 5 10 15
Ala Ser His Leu His Asn Tyr Thr Asn Pro Asn Leu Gly
20 25
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TTCAGTGATC TTTTGCAAGC TCAAGATTGG CTTCAGCTGG GAGTCATTGG GCTGATGTNT 60
NTGATAACTG GTCAAGGTTA CCCACTTAGT TTTAAATGAG AAAATATCCA GGGTCTGAAG 120
AATCAAGTAA AACCACTCAA ATTCAGTTTT TATGTTTANT AAGAGGGAAG GAAATTGTAG 180
AACACTTCCA TTTNTTACCT CAACAGGAAA AAAAAAAGTA TCTATTTACT TAAGACATGT 240
AATTCAATTN TTATTTCCAG NGTAACAAAT CATTTAGTAA ACAATTATGT NTGAACACTT 300
NTAGGAGTTA CCAAATCTCA TGACATAAAA TTTAGCATTA AACTCTGACA TTCAGATGAC 360
AAAATACAGA ACAGATTTTC CACTGTCATA AATTCTCCAA TCTGAAAAAA AP.AAAAAAA 419
(2) INFORMATION FOR SEQ ID N0:12:
71
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2536 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:12:
GGCCGCCCCGCTCGGCCCTCCAGTCCCGCTCCGCCGCCTCCCTCCCGCACAGCAGCCGCC60
AGCGCGGCCTCCTGCACCATGTCGGTGGCCGGCCTCAAGAAGCAGTTCCATAAAGCCACT120
CAGAAAGTGAGTGAGAAGGTTGGAGGAGCTGAAGGAACCAAGCTAGATGATGACTTCAAA180
GAGATGGAAAGGAAAGTGGATGTCACCAGCAGGGCTGTGATGGAAATAATGACTAAAACA240
ATTGAATACCTTCAACCCAATCCAGCTTCCAGAGCTAAGCTCAGCATGATCAACACCATG300
TCAAAAATCCGTGGCCAGGAGAAGGGGCCAGGCTATCCTCAGGCAGAGGCGCTGCTGGCA360
GAGGCCATGCTCAAATTTGGAAGAGAGCTTGGAGATGATTGCAACTTTGGCCCAGCACTT420
GGTGAGGTCGGGGAGGCCATGCGGGAACTGTCGGAGGTCAAAGACTCTTTGGACATAGAA480
GTGAAGCAGAACTTCATTGACCCTCTTCAGAATCTTCATGACAAAGATCTTAGGGAAATT540
CAACATCATCTAAAGAAGTTGGAGGGTCGACGCCTGGATTTTGATTATAAGAAGAAACGA600
CAAGGCAAGATTCCGGATGAAGAGCTTCGTCAAGCTCTAGAGAAATTTGATGAGTCTAAG660
GAAATTGCTGAGTCAAGCATGTTCAATCTCTTGGAGATGGATATTGAACAAGTGAGCCAG720
CTCTCTGCACTTGTGCAAGCTCAGCTGGAGTACCACAAGCAGGCAGTCCAGATCCTGCAG780
CAAGTCACGGTCAGACTGGAAGAAAGAATAAGACAGGCTTCATCTCAGCCTAGAAGGGAA840
TATCAACCTAAACCACGAATGAGCCTGGAGTTTCCAACTGGAGACAGTACTCAGCCCAAT900
GGGGGTCTCTCCCACACAGGCACTCCCAAACCTTCAGGTGTCCAAATGGATCAGCCCTGC960
TGCCGAGCTCTGTACGACTTTGAACCTGAAAATGAAGGGGAGTTGGGATTTAAAGAGGGC1020
GATATCATCACACTCACTAACCAAATTGATGAGAACTGGTATGAGGGGATGCTGCATGGC1080
CATTCAGGCTTCTTCCCCATCAATTATGTGGAAATTCTGGTTGCCCTGCCCCATTAGGAT1140
GTTATGCTGGCTGGCTCGCCTCCTCTTGACCCAGATAGTTACGGTTAACCACTGCTTTGG1200
CAATGCTGCTTATAACACATCCCAAGTGCAGGCCGCAGTGGTCCACGTCATCCAGCCCCA1260
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
CCAAGTGACT TTGGTTGACTTGTGGGCTCCCACAGGAGTCATGGTGATGG ATGATATCCT1320
CTTAGCCTGG TGGGCGTGGCATGTGCTTTTTAAAACATCATCTGAGACCA GCCAGTAGTC1380
ACAGAACTGC TGTTTACACAGTTCTCAGGAGGCTGTGGTTTCTTAGAATA TGACCATGAG1440
CCATTTCACA GAAAAACCATCCCACCGAAGATATTGTCTATCACCCCAGG GGCCATCTGA1500
AGGTCTCTTT GCATTTCTCCATGCAAAGAGGAGAAAGCTTTTGCTTTCAC ACTGTCCCTT1560
CCCAAATATG TGAGTCATGGAATTGTCAAAGTAAGCCTTCCCTCACCAGC AAATTGTCTC1620
CTGATCTGAA TGAATTTGTCTCTTAATGCATCCATAGAAAAGTGTTAATT GTGGGTTCAA1680
AGCATTCTCT GCAAATAGGCATCTCAGCTCCTCACACTTATGGCTATTTC TGACGTATAG1740
CCAGTTTTCT TCCCTCCTTGCTATTAAAGCCAGAGCGGTAATTCCAAATT ATTTTTCAGT1800
AAGACAGTTA ATCAGCATTATTGTGAGAGGGACTGAAAAGAAATTCTCCA TTATGAGGAA1860
TTGGGAAGAA ATCTGGTATCCAAGCTTAAATTTCTTGCTATACAGAAACT ATGTATGTAT1920
TTAGGCTATT TCTGAAGGGCACAGGGAAGGGGGAACAAATATCTTCACTT CAGTTTTATT1980
TGTGAATTAC ATGTTTCATGAATCCATTTGGCACAGAGACACAAGGAAGA AAACACTAGT2040
AACCATCTTT CCACTAGTTCATATACTGAGAAACAGTAAATACCTTTCCT TTCCACTTTT2100
ACCCTGTGTT CTTTGAACATCATTTGTGCAGATTCTGCCCTCAATGAGGA CCAAATAAAG2160
ATGATTTTTG TGCTTAGCAGTTTAAGGTATATGGCTGCATATGCAAAACT CTTTCCCAAT2220
TCAGTCGCTA CTTTTACTTCTGCCCTTTCTATCCATCGTCTTCATTTTGT GTGTACAGTG2280
CTGTGTGTAA GCTTATCAGTGTGTTTTTTTATTTGTATCAGTCATGAAAG TCCTGTTAGG2340
TATCCAGAGT TCTATTTATCTAGCTGTACAGACTCTTTCAGAGGTTTAAC GTGCTGCTTC2400
CGATGTGCCA CCTGCAGTAGTGGATCATGTGGAGTGAAAGGCAAATCTTA CTGCTTAATG2460
TATAAACTCT CACCACAGGAAGCATCGCTGTTTCCAATAAATATTGCTGA AGACAGAAAA2520
2536
(2) INFORMATION :
FOR SEQ ID N0:13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
protein
73
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Met Ser Val Ala Gly Leu Lys Lys Gln Phe His Lys Ala Thr Gln Lys
1 5 10 15
Val Ser Glu Lys Val Gly Gly Ala Glu Gly Thr Lys Leu Asp Asp Asp
20 25 30
Phe Lys Glu Met Glu Arg Lys Val Asp Val Thr Ser Arg Ala Val Met
35 40 45
Glu Ile Met Thr Lys Thr Ile Glu Tyr Leu Gln Pro Asn Pro Ala Ser
50 55 60
Arg Ala Lys Leu Ser Met Ile Asn Thr Met Ser Lys Ile Arg Gly Gln
65 70 75 80
Glu Lys Gly Pro Gly Tyr Pro Gln Ala Glu Ala Leu Leu Ala Glu Ala
85 9p 95
Met Leu Lys Phe Gly Arg Glu Leu Gly Asp Asp Cys Asn Phe Gly Pro
100 105 110
Ala Leu Gly Glu Val Gly Glu Ala Met Arg Glu Leu Ser Glu Val Lys
115 120 125
Asp Ser Leu Asp Ile Glu Val Lys Gln Asn Phe Ile Asp Pro Leu Gln
130 135 140
Asn Leu His Asp Lys Asp Leu Arg Glu Ile Gln His His Leu Lys Lys
145 150 155 160
Leu Glu Gly Arg Arg Leu Asp Phe Asp Tyr Lys Lys Lys Arg Gln Gly
165 170 175
Lys Ile Pro Asp Glu Glu Leu Arg Gln Ala Leu Glu Lys Phe Asp Glu
180 185 190
Ser Lys Glu Ile Ala Glu Ser Ser Met Phe Asn Leu Leu Glu Met Asp
195 200. 205
Ile Glu Gln Val Ser Gln Leu Ser Ala Leu Val Gln Ala Gln Leu Glu
210 215 220
Tyr His Lys Gln Ala Val Gln Ile Leu Gln Gln Val Thr Val Arg Leu
225 230 235 240
Glu Glu Arg Ile Arg Gln Ala Ser Ser Gln Pro Arg Arg Glu Tyr Gln
245 250 255
Pro Lys Pro Arg Met Ser Leu Glu Phe Pro Thr Gly Asp Ser Thr Gln
260 265 270
Pro Asn Gly Gly Leu Ser His Thr Gly Thr Pro Lys Pro Ser Gly Val
74
CA 02281015 1999-08-13
WO 9$/3209 PCT/US98/03697
275 280 285
Gln Met Asp Gln Pro Cys Cys Arg Ala -Leu Tyr Asp Phe Glu Pro Glu
290 295 300
Asn Glu-Gly Glu Leu Gly Phe Lys Glu Gly Asp Ile Ile Thr Leu Thr
305 310 315 320
Asn Gln Ile Asp Glu Asn Trp Tyr Glu Gly Met Leu His Gly His Ser
325 330 335
Gly Phe Phe Pro Ile Asn Tyr Val Glu Ile Leu Val Ala Leu Pro His
340 345 350
(2} INFORMATION FOR SEQ ID N0:14:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3443 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ACATTGCTGC CTAAAGAAAC TCAGCAGCCT CAGGCCCAAT TCTGCCACTT CTGGTTTGGG 60
TACAGTTAAA GGCAACCCTG AGGGACTTGG CAGTARAAAT CCAGGGCCTC CCCTGGGGCT 120
GGCARCTTCG TGTGCAGCTA GAGCTTTACC TGAAAGGAAG TCTCTGGGCC CAGAACTCTC 180
CACCAAGAGC CTCCCTGCCG TTCGCTGAGT CCCAGCAATT CTCCTAAGTT GAAGGGATCT 240
GAGAAGGAGA AGGAAATGTG GGGTAGATTT GGTGGTGGTT AGAGATATGC CCCCCTCATT 300
ACTGCCAACA GTTTCGGCTG CATTTCTTCA CGCACCTCGG TTCCTCTTCC TGAAGTTCTT 360
GTGCCCTGCTCTTCAGCACCATGGGCCTTCTTATACGGAA TCTCCCCCTT420
GGCTCTGGGA
GTGGGGCAGGCTCTTGGGGCCAGCCTAAGATCATGGTTTAGGGTGATCAGTGCTGGCAGA480
TAAATTGAAAAGGCACGCTGGCTTGTGATCTTAAATGAGGACAATCCCCCCAGGGCTGGG540
CACTCCTCCCCTCCCCTCACTTCTCCCACCTGCAGAGCCAGTGTCCTTGGGTGGGCTAAA600
TAGGATATACTGTATGCCGGCTCCTTCAAGCTGCTGACTCACTTTATCAATAGTTCCATT660
TAAATTGACT TCAGTGGTGA GACTGTATCC TGTTTGCTAT TGCTTGTTGT GCTATGGGGG 720
GAGGGGGGAG GAATGTGTAA GATAGTTAAC ATGGGCAAAG GGAGATCTTG GGGTGCAGCA 780
CA 02281015 1999-08-13
WO 98_/3$209 PCT/US98/03697
CTTAAACTGC CTCGTAACCC TTTTCATGAT TTCAACCACA TTTGCTAGAG GGAGGGAGCA 840
GCCACGGAGT TAGAGGCCCT TGGGGTTTCT CTTTTCCACT GACAGGCTTT CCCAGGCAGC 900
TGGCTAGTTC ATTCCCTCCC CAGCCAGGTG CAGGCGTAGG AATATGGACA TCTGGTTGCT 960
TTGGCCTGCT GCCCTCTTTC AGGGGTCCTA AGCCCACAAT CATGCCTCCC TAAGACCTTG 1020
GCATCCTTCC CTCTAAGCCG TTGGCACCTC TGTGCCACCT CTCACACTGG CTCCAGACAC 1080
ACAGCCTGTG CTTTTGGAGC TGAGATCACT CGCTTCACCC TCCTCATCTT TGTTCTCCAA 1140
GTAAAGCCAC GAGGTCGGGG CGAGGGCAGA GGTGATCACC TGCGTGTCCC ATCTACAGAC 1200
CTGCAGCTTC ATAAAACTTC TGATTTCTCT TCAGCTTTGA AAAGGGTTAC CCTGGGCACT 2260
GGCCTAGAGC CTCACCTCCT AATAGACTTA GCCCCATGAG TTTGCCATGT TGAGCAGGAC 1320
TATTTCTGGC ACTTGCAAGT CCCATGATTT CTTCGGTAAT TCTGAGGGTG GGGGGAGGGA 1380
CATGAAATCA TCTTAGCTTA GCTTTCTGTC TGTGAATGTC TATATAGTGT ATTGTGTGTT 1440
TTAACAAATG ATTTACACTG ACTGTTGCTG TAAAAGTGAA TTTGGAAATA AAGTTATTAC 1500
TCTGATTAAA P,~~.~AAAAAAA P,~1~~ AAAA~AA~ ~GC GGCCGCGAGA 1560
ACCAGCGTGA GTTGGAGGAG GACTCTTTTG GGCTGGCCAT GGACGAGGAC GGTCGCAAGT 1620
TCGTCTACTT CAAGTCCCTC GGGCCCTACC ACAAGTCGCG CTCGTCGTCG TGGAGCAAGA 1680
AGCGCGCCGA GAGCAGCGAC GAGGAGAACT TGCCCCGCAT GTATGAGACG GGCACCGAGT 1740
TCTGCCCCTA CGCCAGCTTC GTCAAGTACC TGTCGAAACG CAACCCTCTC TGCAAGGCGT 1800
TCTTCCAGCG GCCCCGGGAC CACTGCAGCG AGGGCGATGT GACCTGGTAC GAGAACAAAG 1860
CCATCGGCAA GAACTTGCTA GGCACTCGGA TGCAGATGCT CTCCAAGGCG GCCAAGCTCT 1920
CCAAGACCTA CACCAACCAC TGCATCGGCG CCGTCTCCAT CGCCACGCTC AACAGCATCG 1980
CGGGCATTGG CACCAAGCTG GGCTCGCCCG CCCCGCAGGG CTGCTACGCC GAGGCTCTGA 2040
ACGGGGCGGC ACGGCACAAC TCCCACCACC CCCCCACCCA TCCCTCCCAC CACCACCGCC 2100
CCCAGCCGCC CTCGCTGGGG AACACTTACA TCCTCCCCAA AGACAGCCAG GTCGGGCCCG 2160
ACGTGAAATC CGAGGCTGCG CCCAAGCGCG CCCTGTACGA GTCTGTGTTC GGGTCGGGGG 2220
AAATCTGCGG CCCCACTTCC CCCAAAAGAC TTTGTATCCG CCCCTCGGAG CCTGTGGATG 2280
CGGTGGTGGT GGTTTCCGTG AAACACGACC CCCTGCCTCT TCTTCCAGAA GCCAATGGGC 2340
ACAGAAGCAC CAATTCTCCC ACAATAGTTT CACCTGCTAT TGTTTCCCCC ACCCAGGACA 2400
GTCGGCCCAA TATGTCAAGA CCTCTGATCA CTAGATCCCC TGCATCTCCA CTGAACAACC 2460
76
CA 02281015 1999-08-13
WO 98/3$209 PCT/US98/03697
AAGGCATCCCTACTCCAGCA CAACTCACAA GCCTGTCCAC ATTGATGTGG2520
AATCCAATGC
GCGGCCACATGTACACCAGC AGCSTGGCCACCCTCACCAP.ATACCCTGAA TCCAGAATCG2580
GAAGACTTTTTGATGGTACA GAGCCCATTGTTTTGGACAGTCTCARACAG CACTATTTCA2640
TTGACAGAGATGGACAGATG TTCAGATATATCTTGAATTTTCTACGAACA TCCAAACTCC2700
TCATTCCTGATGATTTCAAG GACTACACTTTGTTATATGAAGAGGCAAAA TATTTTCAGC2760
TTCAGCCCATGTTGTTGGAG ATGGAAAGATGGAAGCAGGACAGAGAAACT GGTCGATTTT2820
CAAGGCCCTGTGAGTGCCTC GTCGTGCGTGTGGCCCCAGACCTCGGAGAA AGGATCACGC2880
TAAGCGGTGACAAATCCTTG ATAGAAGAAGTATTTCCAGAGATCGGCGAC GTGATGTGTA2940
ACTCTGTCAATGCAGGCTGG AATCACGACTCGACGCACGTCATCAGGTTT CCACTAAATG3000
GCTACTGTCACCTCAACTCA GTCCAGGTCCTCGAGAGGTTGCAGCAAAGA GGATTTGAAA3060
TCGTGGGCTCCTGTGGGGGA GGAGTAGACTCGTCCCAGTTCAGCGAATAC GTCCTTCGGC3120
GGGAACTGAGGCGGACGCCC CGTGTACCCTCCGTCATCCGGATAAAGCAA GAGCCTCTGG3180
ACTAAATGGACATATTTCTT ATGCAAAAAGGAAAACACACACAACCAATA ACTCAAACAA3240
AAAAGGGACATTTATGTGCA GTTGGGACAGCAAACCAAGTCCTGGACGTA AAATCGAATA3300
AAAGACACATTTATATCCAA TAGAGACCACACCTGTATTCATATGGGAAC AATTGGAATA3360
GTGATATCCTCAAGGTGTAA AAAATATATAAATATATATATATATGTCAA AAGGTAGGAA3420
ATGCAAAAAAP,~~,~AAAAAA~1 3443
AAA
(2) INFORMATION
FOR
SEQ
ID N0:15:
(i} SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 315 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Asp Glu Asp Gly Arg Lys Phe Val Tyr Phe Lys Ser Leu Gly Pro
1 5 10 15
Tyr His Lys Ser Arg Ser Ser Ser Trp Ser Lys Lys Arg Ala Glu Ser
20 25 30
77
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
Ser Asp Glu Glu Asn Leu Pro Arg Met Tyr Glu Thr Gly Thr Glu Phe
35 40 45
Cys Pro Tyr Ala Ser Phe Val Lys Tyr LeuSer Lys Arg Asn Pro Leu
50 55 60
Cys Lys Ala Phe Phe Gln Arg Pro Arg Asp His Cys Ser Glu Gly Asp
65 70 75 BO
Val Thr Trp Tyr Glu Asn Lys Ala Ile Gly Lys Asn Leu Leu Gly Thr
85 90 95
Arg Met Gln Met Leu Ser Lys Ala Ala Lys Leu Ser Lys Thr Tyr Thr
100 105 110
Asn His Cys Ile Gly Ala Val Ser Ile Ala Thr Leu Asn Ser Ile Ala
115 120 125
Gly Ile Gly Thr Lys Leu Gly Ser Pro Ala Pro Gln Gly Cys Tyr Ala
130 135 140
Glu Ala Leu Asn Gly Ala Ala Arg His Asn Ser His His Pro Pro Thr
145 150 155 160
His Pro Ser His His His Arg Pro Gln Pro Pro Ser Leu Gly Asn Thr
165 170 175
Tyr Ile Leu Pro Lys Asp Ser Gln Val Gly Pro Asp Val Lys Ser Glu
180 185 190
Ala Ala Pro Lys Arg Ala Leu Tyr Glu Ser Val Phe Gly Ser Gly Glu
195 200 205
Ile Cys Gly Pro Thr Ser Pro Lys Arg Leu Cys Ile Arg Pro Ser Glu
210 215 220
Pro Val Asp Ala Val Val Val Val Ser Val Lys His Asp Pro Leu Pro
225 230 235 240
Leu Leu Pro Glu Ala Asn Gly His Arg Ser Thr Asn Ser Pro Thr Ile
245 250 255
Val Ser Pro Ala Ile Val Ser Pro Thr Gln Asp Ser Arg Pro Asn Met
260 265 270
Ser Arg Pro Leu Ile Thr Arg Ser Pro Ala Ser Pro Leu Asn Asn Gln
275 280 285
Gly Ile Pro Thr Pro Ala Gln Leu Thr Lys Ser Asn Ala Pro Val His
290 295 300
Ile Asp Val Gly Gly His Met Tyr Thr Ser Ser
305 310 315
(2) INFORMATION FOR SEQ ID N0:16:
7g
CA 02281015 1999-08-13
WO 98/38209 PCTNS98/03697
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1511 base pairs
(B) TYPE: nucleic acid -
(C) STRANDEDNESS: double _
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:16:
AACATGGGCACAGGGGATTTTATCTGCATT TCCATGACTGGAGGGGCGCCCTGGGGGTTC60
AGATTGCAAGGTGGCAAGGAGCAGAAGCAG CCCTTACAAGTTGCAAAGATTCGAAATCAG120
AGCAAAGCCTCTGGGTCTGGGCTCTGTGAG GGAGATGAAGTGGTTTCCATCAATGGCAAC180
CCTTGTGCAGATCTCACCTACCCTGAAGTC ATCAAGCTCATGGAAAGCATAACAGACTCT240
CTCCAAATGCTCATCAAAAGACCATCCAGT GGAATAAGTGAGGCTTTGATATCTGAAAAT300
GAAAACAAAAACCTCGAGCATCTCACACAT GGGGGTTATGTGGAAAGTACCACCCTGCAG360
ATTCGACCGGCCACAAAGACCCAGTGCACA GAATTCTTCCTCGCCCCTGTCAAGACTGAA420
GTTCCCCTAGCTGAGAACCAAAGAAGTGGT CCCGACTGTGCAGGCAGCTTGAAAGAAGAA480
ACAGGCCCGAGCTACCAAAGGGCTCCCCAA ATGCCTGACTCCCAAAGAGGACGCGTGGCA540
GAAGAGCTGATCTTAAGGGAGAAGGTAGAA GCGGTACAGCCTGGGCCTGTGGTTGAGCTG600
CAACTGTCCCTTTCACAGGAGAGACATAAG GGCGCTAGTGGCCCTTTAGTGGCTCTCCCG660
GGAGCTGAAAAATCTAAGTCTCCTGACCCA GACCCTAACTTGTCACATGACAGGATTGTC720
CACATAAATTCGATCCCTACTAATGAGAAA GCAGACCCTTTCCTGAGGTCCAGCAAGATA780
ATCCAGATCTCCAGTGGCAGAGAGTTGAGA GTGATCCAGGAAAGTGAAGCAGGAGATGCG840
GGACTGCCCCGGGTGGAAGTGATCCTCGAC TGCTCTGACAGGCAGAAGACAGAAGGGTGC900
AGGCTTCAGGCAGGAAAGGAGTGTGTGGAT TCTCCAGTGGAAGGAGGGCAGTCAGAAGCA960
CCTCCTTCTCTGGTATCCTTTGCCGTCTCA TCAGAAGGCACAGAGCAGGGAGAAGATCCA1020
CGCTCGGAAAAAGATCACAGCAGACCTCAC AAGCACCGAGCGCGGCATGCACGGCTCAGG1080
AGGAGTGAAAGCCTGTCAGAAAAACAAGTG AAGGAAGCAAAATCTAAATGCAAAAGCATT1140
GCCCTTCTTCTAACGGATGCTCCCAACCCC AAYTCCAAGGGGGTGTTGATGTTTAAGAAG1200
CGACGTCGGAGGGCCAGGAAATAACACCCT AGTTAGCTACGGTACTGGCGAGCTTGAGCG1260
' 79
CA 02281015 1999-08-13
WO 98_/3$209 PCT/US98/03697
AGAGGCGGAC GAGGAGGAAG AAGGTGACAA GGAGGATACA TGTGAAGTAG CATTTCTTGG 1320
TGCAAGCGAA TCAGAGGTGG ATGAAGAGTT ATTGTCTGAC GTTGACGACA ACACACAAGT 1380
TGTGAACTTT GACTGGGATT CTGGACTGGT GGACATTGAA AAGAAACTGA ACAGAGGGGA 1440
CAAGATGGAG ATGTTACCAG ACACCACAGG CAAGGGAGAC ACTGACTCCA GCCAAGAAAA 1500
1511
A.F~ A
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE DESCRIPTION:
SEQ
ID
N0:17:
Met GlyThr GlyAspPhe IleCysIle SerMetThrGly GlyAlaPro
1 5 10 15
Trp GlyPhe ArgLeuGln GlyGlyLys GluGlnLysG1n ProLeuGln
20 25 30
Val AlaLys IleArgAsn GlnSerLys AlaSerGlySer GlyLeuCys
35 40 45
Glu GlyAsp GluValVal SerIleAsn GlyAsnProCys AlaAspLeu
50 55 60
Thr TyrPro GluValIle LysLeuMet GluSerIleThr AspSerLeu
65 70 75 80
Gln MetLeu IleLysArg ProSerSer GlyIleSerGlu AlaLeuIle
85 90 95
SerGlu AsnGluAsn LysAsnLeu GluHisLeuThr HisGly Tyr
Gly
100 105 110
ValGlu SerThrThr LeuGlnIle ArgProAlaThr LysThr Cys
Gln
115 120 125
ThrGlu PhePheLeu AlaProVal LysThrGluVal ProLeu Glu
Ala
130 135 140
AsnGln ArgSerGly ProAspCys AlaGlySerLeu LysGlu Thr
Glu
145 150 155 160
CA 02281015 1999-08-13
WO 98J3$209 PCT/US98/03697
Gly Pro Ser Tyr Gln Arg Ala Pro Gln Met Pro Asp Ser Gln Arg Gly
165 170 175
Arg Val Ala Glu Glu Leu Ile Leu Arg.Glu Lys Val Glu Ala Val Gln
180 185 190
Pro Gly Pro Val Val Glu Leu Gln Leu Ser Leu Ser Gln Glu Arg His
195 200 205
Lys Gly Ala Ser Gly Pro Leu Val Ala Leu Pro Gly Ala Glu Lys Ser
210 215 220
Lys Ser Pro Asp Pro Asp Pro Asn Leu Ser His Asp Arg Ile Val His
225 230 235 240
Ile Asn Ser Ile Pro Thr Asn Glu Lys Ala Asp Pro Phe Leu Arg Ser
245 250 255
Ser Lys Ile Ile Gln Ile Ser Ser Gly Arg Glu Leu Arg Val Ile Gln
260 265 270
Glu Ser Glu Ala Gly Asp Ala Gly Leu Pro Arg Val Glu Val Ile Leu
275 280 285
Asp Cys Ser Asp Arg Gln Lys Thr Glu Gly Cys Arg Leu Gln Ala Gly
290 295 300
Lys Glu Cys Val Asp Ser Pro Val Glu Gly Gly Gln Ser Glu Ala Pro
305 310 315 320
Pro Ser Leu Val Ser Phe Ala Val Ser Ser Glu Gly Thr Glu Gln Gly
325 330 335
Glu Asp Pro Arg Ser Glu Lys Asp His Ser Arg Pro His Lys His Arg
340 345 350
Ala Arg His Ala Arg Leu Arg Arg Ser GIu Ser Leu Ser Glu Lys GIn
355 360 365
Val Lys Glu Ala Lys Ser Lys Cys Lys Ser Ile Ala Leu Leu Leu Thr
370 375 380
Asp Ala Pro Asn Pro Asn Ser Lys Gly Val Leu Met Phe Lys Lys Arg
385 390 395 400
Arg Arg Arg Ala Arg Lys
405
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1329 base pairs .
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
gl
CA 02281015 1999-08-13
WO 98/3$209 PCTNS98/03697
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
CCTTCTTCCT CACTCACACA TTTTTTGTAC ATCTGGGCCC TTAGTTTTTA 60
TTCTGTTTAT
TATATGTCTC TGTCTCTCTC TATTGTGTGT GTGTGTGTGT GTGTGTGTGT 120
GTGTGTGTGT
GTGTGTGTGT GGTGCAGGAG TGCCACCCCC AGGGCCCTGT CAACCTCTCT 180
TTTCTCCTCC
ATGGCTGTCT GCCTGCGTAT CTGTCTCTGA GAATCCTCGG GGCGGTCAGG 240
GGATGTCAGG
AGGGGAAGGA GCCGCCCTCC CTATCTTGCT GCTCCTCTTG GCACTCAGGG 300
GCACCTTCCA
TGGAGCCAGA CCGGGTGGAG GGGCTTCTGG GATTTGGTGT CTGCTGCTGC 360
CAGAGCAGGA
ACCCCCAGTC TAGGACTTGG GCATTTTAAC AGGGAGAAAG TAGTGGCTTC 420
CCTTTTCTCT
CTCTCCTCCT TTTTCCCTTT AAGCCCACAG ATTCAGGTCA TGCCAAAAGC 480
TCTCTGGTTG
TAACCTGGAG ACATGTGGAG GGGAATGGCG ATGGGATTAT AGGACTCTCC 540
CCATCTCGGG
CCCTGACCCT GACCCTTGCC ACCAACCCAA AGACAGCTGG TGGGTTTCCC 600
CTTGGAGACA
ATCCTGCGTT TGCCTGGGCC GGCCCTGGCT GCCCTCAGCT TTCGCTGATC 660
TGCCCGGCCT
GGAGCCTCCC ATCACCCCGC TTCTTGTTGG GCCTCAGGCA CTGGTTACCA 720
GAAGGGGGTC
TGGGTCTGCT CAGGATCATG TTTTGTAGCA CCTCCTGTTG GAGGGGTGGA 780
GGGATGTTCC
CCTGAGCCAG GCTGAGACTA GAACCCCATC TTCCCTGAGC CAGGCTGAGA 840
CTAGAACCCC
ATCTTCCCCA CCACGCCACC CCTGTGGCTG CTACAGGAGC ACAGTAGTGA 900
AGGCCTGAGC
TCCAGGTTTG AAAGACCCAA CTGGAGCGTG GGGCGGGCAG GCAGGGGTTA 960
GTGAAAGGAC
ACTTCCAGGG TTAGGACAGA GCATTTAGCC TTCTGGAAGA ACCCCTGCCT 1020
GGGGTGGGAC
TGTGCAGGCC AGAGAAGGTG GCATGGGCCT GAACCCACCT GGACTGACTT 1080
CTGCACTGAA
GCCACAGATG GAGGGTAGGC TGGTGGGTGG GGGTGGTTCG TTCTCTAGCC 1140
GGGGCAGACA
CCCAGCTGGC TGGGTCCTTC YTCAGCCTTG CCTCCTCCTG TCCCCAACCC 1200
TTTCCTTTCC
TCCTGCTTGC GGACTGCTGG TCCCCTCTCC TTCCCTCCTT CCAGCTGTTT 1260
CTAGTTACCA
CCTACCCCTG GCCGTGGACT GATCAGACCA GCATTCAAAA TAAAAGTTTG 1320
TTCCP.AAP~1
1329
A~
(2) INFORMATION FOR SEQ ID N0:19:
g7
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: _
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Ser Gly Gly Glu Gly Ala Ala Leu Pro Ile Leu Leu Leu Leu Leu
1 5 10 15
Ala Leu Arg Gly Thr Phe His Gly Ala Arg Pro Gly Gly Gly Ala Ser
20 25 30
Gly Ile Trp Cys Leu Leu Leu Pro Glu Gln Glu Pro Pro Val
35 40 45
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
ANCCCAAGGAT GTACATGAAT GCTTGGAG 29
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
83
CA 02281015 1999-08-13
W O 98/38209 PCT/US98/03697
GNTCACTGGGC TGGTATATCT GCTCACGT 29
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A.) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
CNGGATAAAAT GCAAAATGAC CAGGAGTG 29
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
ANATGGGTTTC TAGACACTGT TGTTCATG 29
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GNGAAGCACCA AGCAAAATAT CTGCAAGT 29
84
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(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:
ANCCGTAACTA_TCTGGGTCAA GAGGAGGC 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:
CNGGAATGAGG AGTTTGGATG TTCGTAGA 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
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
TNGCAACTTGT AAGGGCTGCT TCTGCTCC 2g
(2) INFORMATION FOR SEQ ID N0:28:
CA 02281015 1999-08-13
WO 98/38209 PCT/US98/03697
(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:
CNCAGAGACAG ATACGCAGGC AGACAGCC 29
86
