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SECRETED PROTEINS AND POLYIVIJCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,XXX
(converted to a provisional application from non-provisional application Ser.
No.
08/825,145), filed March 2 5, 1997, which is incorporated by reference herein.
FIELD OF THI? INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
BACKGROUND Oh THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and; interleukins) has matured rapidly
over the
past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., parti~31 DNA/amino acid sequence of
the protein
2 0 in the case of hybridization cloning; activity of the protein in the case
of expression
cloning). More recent "indirect" cloning techiuques such as signal sequence
cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of thE~ art by making available large
numbers of
2 5 DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 54 to nucleotide 737;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 188 to nucleotide 671;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bf171 6 deposited under accession
number ATCC 98371;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bf171 6 deposited under accession number ATCC 98371;
(~ a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone bf171 6 deposited under accession number
ATCC 98371;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone bf171 6 deposited under accession number ATCC 98371;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising the amino acid sequence from amino acid 109 to amino acid 118 of
2 5 SEQ ID N0:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
3 0 (1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:l from nucleotide 54 to nucleotide 737; the nucleotide sequence of SEQ ID
N0:1 from
nucleotide 188 to nucleotide 671; the nucleotide sequence of the full-length
protein coding
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sequence of clone bf171 6 deposited under accession number ATCC 98371; or the
nucleotide sequence of a mature protein coding sequence of clone bf171 6
deposited
under accession number ATCC 98371. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone bf171 6 deposited under accession number ATCC 98371. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ :fD N0:2 from amino acid 46 to amino
acid
206.
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 46 to
amino acid 206;
(c) fragments of the amino acid sequence of SEQ ID N0:2 comprising
the amino acid sequence from amino acid 109 to amino acid 118 of SEQ ID N0:2;
and
2 0 (d) the amino acid sequence encoded by the cDNA insert of clone
bf171 6 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian protein;. Preferably
such
protein comprises the amino acid sequence o~f SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 46 to amino acid 206.
2 5 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:3 from nucleotide 135 to nucleotide 1169;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1 to nucleotides 875;
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(d) a polynucleotide comprising the nucleotide sequence of the full- ,
length protein coding sequence of clone ck181 7 deposited under accession
number ATCC 98371;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ck181 7 deposited under accession number ATCC 98371;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ck181_7 deposited under accession number
ATCC 98371;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ck181_7 deposited under accession number ATCC 98371;
(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 167 to amino acid 176 of
SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
2 0 of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 135 to nucleotide 1169; the nucleotide sequence of SEQ ID
N0:3
2 5 from nucleotide 1 to nucleotide 875; the nucleotide sequence of the full-
length protein
coding sequence of clone ck181 7 deposited under accession number ATCC 98371;
or the
nucleotide sequence of a mature protein coding sequence of clone ck181 7
deposited
under accession number ATCC 98371. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
3 0 of clone ck181 7 deposited under accession number ATCC 98371. In yet other
preferred
embodiments, the present invention provides a polynucleotfde encoding a
protein
comprising the amino acid sequence of SEQ ID N0:4 from amino acid 1 to amino
acid 247.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
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In other embodiments,, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 1 to
amino acid 247;
{c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
the amino acid sequence from amino acid 167 to amino acid 176 of SEQ ID N0:4;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
ck181 7 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid
sequence
of SEQ ID N0:4 from amino acid 1 to amino acid 247.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:5 from nucleotide 882 to nucleotide 1106;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1050 to nucleotide 1106;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1028 to nucleotide 1395;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone co736_3 deposited under accession
number ATCC 98371;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone co736 3 deposited under accession number ATCC 98371;
3 0 (g} a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone co;736_3 deposited under accession number
ATCC 98371;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone co736_ 3 deposited under accession number ATCC 98371;
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(i) a polynucleotide encoding a protein comprising the amino acid ,
sequence 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 32 to amino acid 41 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 polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 882 to nucleotide 1106; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 1050 to nucleotide 1106; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 1028 to nucleotide 1395; the nucleotide sequence of the full-length
protein
coding sequence of clone co736_3 deposited under accession number ATCC 98371;
or the
nucleotide sequence of a mature protein coding sequence of clone co736_3
deposited
under accession number ATCC 98371. In other preferred embodiments, the
2 0 polynucleotide encodes the full-length or a mature protein encoded by the
cDNA insert
of clone co736 3 deposited under accession number ATCC 98371.
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
2 5 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6 comprising
the amino acid sequence from amino acid 32 to amino acid 41 of SEQ ID N0:6;
and
3 0 (c) the amino acid sequence encoded by the cDNA insert of clone
co736_3 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 2283 to nucleotide 2858;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1164 to nucleotide 1433;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone dm26_2 deposited under accession
number ATCC 98371;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone dm26_2 deposited under accession number ATCC 98371;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone drn26 2 deposited under accession number
ATCC 98371;
(g) a polynucleotide encodiing a mature protein encoded by the cDNA
insert of clone dm26_ 2 deposited under accession number ATCC 98371;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:8;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:8 having biological activity, the fragment
comprising the amino acid sequence from amino acid 91 to amino acid 100 of SEQ
ID N0:8;
2 5 (j) a polynucleotide whidl 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
a polynucleotide capable of hybridizing under stringent conditions
3 0 to any one of the polynucleotides spExified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 2283 to nucleotide 2858; the nucleotide sequence of SEQ
ID N0:7
from nucleotide 1164 to nucleotide 1433; the nucleotide sequence of the full-
length protein
coding sequence of clone dm26_2 deposited under accession number ATCC 98371;
or the
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nucleotide sequence of a mature protein coding sequence of clone dm26_2
deposited
under accession number ATCC 98371. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone dm26_2 deposited under accession number ATCC 98371.
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
the amino acid sequence from amino acid 91 to amino acid 100 of SEQ ID N0:8;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
dm26_2 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
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:
2 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 168 to nucleotide 683;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:9 from nucleotide 318 to nucleotide 683;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone eq229_3 deposited under accession
number ATCC 98371;
(e) a polynucleotide encoding the full-length protein encoded by the
3 0 cDNA insert of clone eq229_3 deposited under accession number ATCC 98371;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone eq229_3 deposited under accession number
ATCC 98371;
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(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone eq229 3 deposited under accession number ATCC 98371;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity, the fragment
comprising the amino acid sequence from amino acid 81 to amino acid 90 of SEQ
ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 168 to nucleotide 683; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 318 to nucleotide 683; the nucleotide sequence of the full-length
protein coding
sequence of clone eq229 3 deposited under accession number ATCC 98371; or the
nucleotide sequence of a mature protein coding sequence of clone eq229 3
deposited
2 0 under accession number ATCC 98371. In other preferred embodiments, the
polynucleotide encodes the full-length or ~~ mature protein encoded by the
cDNA insert
of clone eq229 3 deposited under accession number ATCC 98371. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:10 from amino acid 53 to amino
acid
2 5 172.
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
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 N0:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 53 to
amino acid 172;
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(c) fragments of the amino acid sequence of SEQ ID NO:10 comprising
the amino acid sequence from amino acid 81 to amino acid 90 of SEQ ID N0:10;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
eq229_3 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10 or the amino acid
sequence
of SEQ ID NO:10 from amino acid 53 to amino acid 172.
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 67 to nucleotide 879;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 118 to nucleotide 879;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 1224 to nucleotide 2171;
(e) a polynucleotide comprising the nucleotide sequence of the full
2 0 length protein coding sequence of clone fh3_6 deposited under accession
number
ATCC 98371;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fh3_6 deposited under accession number ATCC 98371;
(g) a polynucleotide comprising the nucleotide sequence of a mature
2 5 protein coding sequence of clone fh3_6 deposited under accession number
ATCC
98371;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fh3 6 deposited under accession number ATCC 98371;
(i) a polynucleotide encoding a protein comprising the amino acid
3 0 sequence of SEQ ID N0:13;
(j) 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 130 to amino acid 139 of
SEQ ID N0:13;
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(k) a polynucleotide which i.s an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:12 from nucleotide 67 to nucleotide 879; the nucleotide sequence of SEQ ID
N0:12
from nucleotide 118 to nucleotide 879; the nucleotide sequence of SEQ ID N0:12
from
nucleotide 1224 to nucleotide 2171; the nucleotide sequence of the full-length
protein
coding sequence of clone fh3 6 deposited under accession number ATCC 98371; or
the
nucleotide sequence of a mature protein coding; sequence of clone fh3 6
deposited under
accession number ATCC 98371. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone fh3_6
deposited under accession number ATCC 98371. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:13 from amino acid 1 to amino acid 119.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:12.
2 0 In other embodiments, the present invE~ntion provides a composition
comprising
a protein, wherein said protein comprises an annino acid sequence selected
from the group
consisting of:
(a} the amino acid sequence of SEQ ID N0:13;
(b) the amino acid sequence of SEQ ID N0:13 from amino acid 1 to
2 S amino acid 119;
(c) fragments of the amino acid sequence of SEQ ID N0:13 comprising
the amino acid sequence from amino acid 130 to amino acid 139 of SEQ ID N0:13;
and
(d) the amino acid sequence encoded by the cDNA insert of clone fh3_6
3 0 deposited under accession number A'CCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:13 or the amino acid
sequence
of SEQ ID N0:13 from amino acid 1 to amino acid 119.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 2 to nucleotide 556;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 53 to nucleotide 556;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 1 to nucleotide 367;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fs87_3 deposited under accession
number
ATCC 98371;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fs87_3 deposited under accession number ATCC 98371;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fs87_3 deposited under accession number ATCC
98371;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone fs87_3 deposited under accession number ATCC 98371;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:15;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 having biological activity, the fragment
2 5 comprising the amino acid sequence from amino acid 87 to amino acid 96 of
SEQ
ID N0:15;
(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
3 0 of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a}-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:14 from nucleotide 2 to nucleotide 556; the nucleotide sequence of SEQ ID
N0:14 from
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nucleotide 53 to nucleotide 556; the nuclleotide sequence of SEQ ID N0:14 from
,
nucleotide 1 to nucleotide 367; the nucleotide sequence of the full-length
protein coding
sequence of clone fs87_3 deposited under accession number ATCC 98371; or the
nucleotide sequence of a mature protein coding sequence of clone fs87_3
deposited under
accession number ATCC 98371. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone fs87_3
deposited under accession number ATCC 98371.
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
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 87 to amino acid 96 of SEQ ID N0:15;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
fs87 3 deposited under accession number ATCC 98371;
the protein being substantially free from otlher mammalian proteins.
Preferably such
2 0 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
N0:17;
2 5 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 492 to nucleotide 602;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fy530 2 deposited under accession
number ATCC 98371;
3 0 (d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fy530 2 deposii:ed under accession number ATCC 98371;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fy~530 2 deposited under accession number
ATCC 98371;
1 ~~
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(f) a polynucleotfde encoding a mature protein encoded by the cDNA
insert of clone fy530 2 deposited under accession number ATCC 98371;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:18;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:18 having biological activity, the fragment
comprising the amino acid sequence from amino acid 13 to amino acid 22 of SEQ
ID N0:18;
(i} a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:17 from nucleotide 492 to nucleotide 602; the nucleotide sequence of the
full-length
protein coding sequence of clone fy530 2 deposited under accession number ATCC
98371;
or the nucleotide sequence of a mature protein coding sequence of clone fy530
2
deposited under accession number ATCC 98371. In other preferred embodiments,
the
2 0 polynucleotide encodes the full-length or a mature protein encoded by the
cDNA insert
of clone fy530 2 deposited under accession number ATCC 98371.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17, SEQ ID N0:16 or SEQ ID N0:19 .
In other embodiments, the present invention provides a composition comprising
2 5 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:18;
(b) fragments of the amino acid sequence of SEQ ID N0:18 comprising
the amino acid sequence from amino acid 13 to amino acid 22 of SEQ ID NO:18;
3 0 and
(c) the amino acid sequence encoded by the cDNA insert of clone
fy530_2 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:18.
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In one embodiment, the present invention provides a composition comprising an
.
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 154 to nucleoside 972;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 1 to nucleotide 341;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ge51_1 deposited under accession
number
ATCC 98371;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ge51_1 deposited under accession number ATCC 98371;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ge51_l deposited under accession number ATCC
98371;
(g) a polynucleotide encodling a mature protein encoded by the cDNA
insert of clone ge51_I deposited undE>r accession number ATCC 98371;
(h} a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:21;
(i) a polynucleotlde encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:21 having biological activity, the fragment
comprising the amino acid sequence from amino acid 131 to amino acid 140 of
SEQ ID N0:21;
2 5 (j) a polynucleotide whidh is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleoHde which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
3 0 to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:20 from nucleotide 154 to nucleotide 972; the nucleotide sequence of SEQ ID
N0:20
from nucleotide 1 to nucleatide 341; the nucleotide sequence of the full-
length protein
coding sequence of clone ge51_1 deposited under accession number ATCC 98371;
or the
l5
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nucleotide sequence of a mature protein coding sequence of clone ge51_1
deposited under ,
accession number ATCC 98371. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone ge51_1
deposited under accession number ATCC 98371. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:21 from amino acid 1 to amino acid 62.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:20.
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 N0:21;
(b) the amino acid sequence of SEQ ID N0:21 from amino acid 1 to
amino acid 62;
(c) fragments of the amino acid sequence of SEQ ID N0:21 comprising
the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID N0:21;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
ge51_1 deposited under accession number ATCC 98371;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:21 or the amino acid
sequence
of SEQ ID N0:21 from amino acid 1 to amino acid 62.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22 from nucleotide 104 to nucleotide 892;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:22 from nucleotide 299 to nucleotide 892;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22 from nucleotide 798 to nucleotide 1261;
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(e) a polynucleotide comprising the nucleotide sequence of the full- ,
length protein coding sequence of clone gx183_1 deposited under accession
number ATCC 9837:1;
(f} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone gx183_1 deposii:ed under accession number ATCC 98371;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone gx183_1 deposited under accession number
ATCC 98371;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gx183_1 deposited under accession number ATCC 98371;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:23;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:a3 having biological activity, the fragment
comprising the amino acid sequence from amino acid 126 to amino acid 135 of
SEQ ID N0:23;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
{l) a polynucleotide which encodes a species homologue of the protein
2 0 of (i} or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:22 from nucleotide 104 to nucleotide 892; the nucleotide sequence of SEQ ID
N0:22
2 5 from nucleotide 299 to nucleotide 892; the nucleotide sequence of SEQ ID
N0:22 from
nucleotide 798 to nucleotide 1261; the nucleotide sequence of the full-length
protein
coding sequence of clone gx183_1 deposited under accession number ATCC 98371;
or the
nucleotide sequence of a mature protein coding sequence of clone gx183_1
deposited
under accession number ATCC 98371. In other preferred embodiments, the
3 0 polynucleotide encodes the full-length or a mature protein encoded by the
cDNA insert
of clone gx183_1 deposited under accession number ATCC 98371. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:23 from amino acid 53 to amino
acid
89.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ,
ID N0:22.
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 N0:23;
(b) the amino acid sequence of SEQ ID N0:23 from amino acid 53 to
amino acid 89;
(c) fragments of the amino acid sequence of SEQ ID N0:23 comprising
the amino acid sequence from amino acid 126 to amino acid 135 of SEQ ID N0:23;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
gx183_1 deposited under accession number ATCC 98371;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:23 or the amino acid
sequence
of SEQ ID N0:23 from amino acid 53 to amino acid 89.
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.
2 0 Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
2 5 polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
3 0 Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
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effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed :in the present application. The
nucleotide
sequence of each clone can readily be detertxiined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
acid sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 0 As used herein a "secreted" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "bf171 6"
A polynucleotide of the present invention has been identified as clone "bf171
6".
bf171 6 was isolated from a human fetal brain 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. bf171 6 is a full-
length clone,
including the entire coding sequence of a :secreted protein (also referred to
herein as
"bf171 6 protein").
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The nucleotide sequence of bf171 6 as presently determined is reported in SEQ
ID
N0:1. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the bf171_6 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:2.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bf171 6 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for bf171 6 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bf171 6 demonstrated at least some similarity with
sequences
identified as AA147377 (zo39b08.r1 Stratagene endothelial cell 937223 Homo
Sapiens
cDNA clone 589239 5'), AA190936 (zp83e01.r1 Stratagene HeLa cell s3 937216
Homo
Sapiens cDNA clone 626808 5'), AA287427 (zs52b05.r1 NCI CGAP_GCB1 Homo Sapiens
cDNA clone), H77893 (ys09f08.r1 Homo sapiens cDNA), N72642 (yv74a12.r1 Homo
Sapiens cDNA clone), T25271 (Human gene signature HUMGS07433), T35346
(EST83197
Homo Sapiens cDNA 5' end similar to None), and W27589 (34h1 Human retina cDNA
randomly primed sublibrary Homo). Based upon sequence similarity, bf271 6
proteins
and each similar protein or peptide may share at least some activity.
Clone "ck181 7"
2 0 A polynucleotide of the present invention has been identified as clone
"ck181 7".
ck181 7 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ck181 7 is a full-
length clone,
2 5 including the entire coding sequence of a secreted protein (also referred
to herein as
"ck181 7 protein")
The nucleotide sequence of ck181 7 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 ck181 7 protein corresponding to the
foregoing
3 0 nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ck181 7 should be approximately 1475 bp.
The nucleotide sequence disclosed herein for ck181 7 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
CA 02285554 1999-09-23
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FASTA search protocols. ck181 7 demonstrated at least some similarity with
sequences
identified as AA150370 (z107e08.r1 Soares pregnant uterus NbHPU Homo Sapiens
cDNA
clone 491654 5'), H00151 (y169h05.r1 Homo sapiens cDNA clone 43510 5'), N21123
(yx52f04.s1 Homo sapiens cDNA clone 265375 3'), N31138 (yx52f04.r1 Homo
sapiens
cDNA clone 265375 5'), 813827 (yf61h04.r1 Homo sapiens cDNA clone 26896 5'
similar to
SP:S42069 542069 TEGT PROTEIN), and T19278 (Human gene signature HUMGS00295).
The predicted amino acid sequence disclosed herein for ck181 7 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted ck181 7 protein demonstrated at least some similarity to
sequences
identified as U88168 (weak similarity to rat TEGT protein (GI 456207)
[Caenorhabditis
elegans]). Based upon sequence similarity, ck181 7 proteins and each similar
protein or
peptide may share at least some activity. The TopPredII computer program
predicts
seven potential transmembrane domains within the ck181 7 protein sequence,
centered
around amino acids 93,136, 168, 206, 229, 258, and 283 of SEQ ID N0:4,
respectively.
Clone "co736 3"
A polynucleotide of the present invention has been identified as clone "co736
3".
co736_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
2 0 identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. co736_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"co736_3 protein").
The nucleotide sequence of co736_3 as presently determined is reported in SEQ
2 5 ID N0:5. What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the co736 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID I'J0:6. Amino acids 44 to 56 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 57, or are a transmembrane domain.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
co736_3 should be approximately 1980 bp.
The nucleotide sequence disclosed herein for co736_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. co736_3 demonstrated at least some similarity with
sequences
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identified as H02676 (yj36g08.r1 Homo sapiens cDNA), H47499 (yp74c10.r1 Homo
Sapiens
cDNA clone 293170 5'), Q53478 (MLL gene 8.3 kb BamHI genomic region), T91862
(yd54b07.s1 Homo Sapiens cDNA clone 112021 3' similar to SP:LIN1 NYCCO P08548
LINE-1 REVERSE TRANSCRIPTASE ;contains Alu repetitive element;contains Ll
repetitive element), U54776 (Human NTT gene, L1, Alu, and MER 38 repeat
regions),
273964 (Human DNA sequence from cosmid V698D2, between markers), and 283843
(Human DNA sequence from PAC 368A4 on chromosome X. Contains ESTs, CELLULAR
NUCLEIC ACID BINDING PROTEIN (CNBP) like gene and STSs). Based upon sequence
similarity, co736_3 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts two potential transmembrane
domains within the co736_3 protein sequence, one centered around amino acid 16
and
another around amino acid 51 of SEQ ID N0:6. The nucleotide sequence of
co736_3
indicates that it may contain one or more copies of the Alu repetitive
element.
Clone "dm26 2"
A polynucleotide of the present invention has been identified as clone "dm26
2".
dm26_2 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
2 0 analysis of the amino acid sequence of the encoded protein. dm26_2 is a
full-length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"dm26_2 protein").
The nucleotide sequence of dm26_2 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
2 5 predicted amino acid sequence of the dm26_2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:8. Amino acids 9 to 21 of SEQ ID
N0:8 are
a possible leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 22, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
3 0 dm26_2 should be approximately 3500 bp.
The nucleotide sequence disclosed herein for dm26_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. dm26_2 demonstrated at least some similarity with
sequences
identified as AC000356 (Human cosmid g1346a312, complete sequence), F03454 (H.
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sapiens partial cDNA sequence; clone c-1xh10), N42290 (yy06a07.r1 Homo sapiens
cDNA
clone 270420 5' similar to contains Ll.t3 Ll repetitive element), N92463
(zb12e05.s1 Homo
Sapiens cDNA clone 301856 3'), N94118 (za25e06.r1 Homo sapiens cDNA clone
293602 5'),
Q60160 (Human brain Expressed Sequencf~ Tag EST02148), 283745 (Human DNA
sequence from PAC 453A3 contains EST and STS), and 299129 (Human DNA sequence
*** SEQUENCING IN PROGRESS *** from clone 425C14; HTGS phase 1.1). The
predicted
amino acid sequence disclosed herein for dm26 2 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted dm26_2 protein demonstrated at least some similarity to sequences
identified
as M22333 (unknown protein [Homo sapiens]), X61294 (L1 retroposon, a portion
of its
ORF2 sequence [Rattus norvegicusj), and 281053 (E02A10.1 [Caenorhabditis
elegans]).
Based upon sequence similarity, dm26 2 proteins and each similar protein or
peptide may
share at least some activity. The nucleotide sequence of dm26_2 indicates that
it may
contain one or more of the following repetitive elements: Alu, L1.
Clone"eq2293"
A polynucleotide of the present inven~don has been identified as clone "eq229
3".
eq229_3 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proi:eins (see U.S. Pat. No. 5,536,637),
or was
2 0 identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. eq229_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"eq229 3 protein').
The nucleotide sequence of the S' portion of eq229 3 as presently determined
is
2 5 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 eq229_3 protein corresponding to the foregoing nucleotide sequence is
reported in
SEQ ID NO:10. Amino acids 38 to 50 are a predicted leader/signal sequence,
with the
predicted mature amino acid sequence beginning at amino acid 51, or are a
3 0 transmembrane domain. Additional nucleotide sequence from the 3' portion
of eq229_3,
including the polyA tail, is reported in SEQ :1D N0:11.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
eq229 3 should be approximately 1900 bp.
2:3
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The nucleotide sequence disclosed herein for eq229_3 was searched against the
,
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. eq229_3 demonstrated at least some similarity with
sequences
identified as N52034 (yz08g04.s1 Homo sapiens cDNA clone 282486 3') and W01791
(za72d06.r1 Soares fetal lung NbHLI9W Homo Sapiens cDNA clone 298091 5').
Based
upon sequence similarity, eq229_3 proteins and each similar protein or peptide
may share
at least some activity.
Clone "fh3 6"
A polynucleotide of the present invention has been identified as clone "fh3
6".
fh3_6 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. fh3_6 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fh3_6 protein").
The nucleotide sequence of fh3 6 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 fh3_6 protein corresponding to the
foregoing
2 0 nucleotide sequence is reported in SEQ ID N0:13. Amino acids 5 to 17 of
SEQ ID N0:13
are a predicted leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 18. Another potential fh3_6 reading frame and
predicted amino
acid sequence is encoded by basepairs 765 to 1556 of SEQ ID N0:12 and is
reported in
SEQ ID N0:34. The overlapping open reading frames that encode SEQ ID N0:13 and
2 5 SEQ ID N0:34 could be joined into a single oepn reading frame if a
frameshift was
introduced into the nucleotide sequence of SEQ ID N0:12 between base pairs 765
and 882.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fh3_6 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for fh3_6 was searched against the
3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fh3_6 demonstrated at least some similarity with
sequences
identified as AA103102 (mo17f02.r1 Life Tech mouse embryo 13 5dpc 10666014 Mus
musculus cDNA clone 553851 5'}, W72947 (zd62g11.s1 Soares fetal heart NbHHI9W
Homo Sapiens cDNA clone 345284 3'}, W74413 (zd62g11.r1 Soares fetal heart
NbHHI9W
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Homo Sapiens cDNA clone 345284 5'), and VV88819 (zh71d11.r1 Soares fetal liver
spleen
1NFLS S2 Homo Sapiens cDNA clone 41752 5 5'). The predicted amino acid
sequence
disclosed herein for fh3_6 was searched against the GenPept and GeneSeq amino
acid
sequence databases using the BLASTX search protocol. The predicted fh3_6
protein
demonstrated at least some similarity to sequences identified as 281052)
D2023.6
[Caenorhabditis elegans]). Based upon sec~,uence similarity, fh3_6 proteins
and each
similar protein or peptide may share at least some activity. The Motifs
computer progras
predicts a prenyl group binding site (CAAX box) at amino acid 268 of SEQ ID
N0:13.
Clone "fs87 3"
A polynucleotide of the present invention has been identified as clone
"fs87_3".
fs87_3 was isolated from a human adult tesi:es 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. fs87_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fs87_3 protein").
The nucleotide sequence of fs87_3 as presently determined is reported in SEQ
ID
N0:14. What applicants presently believe to be the proper reading frame and
the
2 0 predicted amino acid sequence of the fs87_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID 1'J0:15. Amino acids 5 to 17 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 18, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 fs87_3 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for fs87_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fs87_3 demonstrated at least some similarity with
sequences
identified as AA223699 (zr10c04.s1 Stratagf~ne NT2 neuronal precursor 937230
Homo
3 0 Sapiens cDNA clone 651078 3') and AA287263 (zs49h08.r1 NCI CGAP_GCB1 Homo
Sapiens cDNA clone IMAGE:700863 5' similar to SW:CC91 YEAST P41733 CELL
DIVISION CONTROL PROTEIN 91 ). The prE~dicted amino acid sequence disclosed
herein
for fs87_3 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted fs87_3 protein demonstrated at
least
2:5
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some similarity to sequences identified as L31649 (cdc91 [Saccharomyces
cerevisiae]), .
S72417 (E2 ipatient 3) [hepatitis C virus]), U06711 (tracheobronchial mucin
[Homo
Sapiens]), 275550 {T22C1.3 [Caenorhabditis elegans]), and 298598 (hypothetical
protein
[Schizosaccharomyces pombe]). Based upon sequence similarity, fs87_3 proteins
and each
similar protein or peptide may share at least some activity. The TopPredII
computer
program predicts two additional potential transmembrane domains within the
fs87 3
protein sequence, one centered around amino acid 90 and another around amino
acid 170
of SEQ ID N0:15.
Clone "fy530 2"
A polynucleotide of the present invention has been identified as clone "fy530
2".
fy530_2 was isolated from a human adult placenta cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. fy530 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fy530 2 protein").
The nucleotide sequence of the 5' portion of fy530 2 as presently determined
is
reported in SEQ ID N0:16. An additional internal nucleotide sequence from
fy530 2 as
2 0 presently determined is reported in SEQ ID N0:17. What applicants believe
is the proper
reading frame and the predicted amino acid sequence encoded by such internal
sequence
is reported in SEQ ID NO:18. Additional nucleotide sequence from the 3'
portion of
fy530_2, including the polyA tail, is reported in SEQ ID N0:19.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 fy530 2 should be approximately 3550 bp.
The nucleotide sequence disclosed herein for fy530 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fy530 2 demonstrated at least some similarity with
sequences
identified as AA029852 (zk11b04.s1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
3 0 clone 470191 3'), AA118938 (mp64g01.r1 Soares 2NbMT Mus musculus cDNA
clone
574032 5'), L39210 (Human inosine monophosphate dehydrogenase type II gene,
complete
cds), N51229 (yz13b07.s1 Homo sapiens cDNA clone 282901 3'), and X95808
(H.sapiens
mIZNA for protein encoded by a candidate gene, DXS6673E, for mental
retardation). The
predicted amino acid sequence disclosed herein for fy530_2 was searched
against the
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GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol. ,
The predicted fy530_2 protein demonstrated at least some similarity to
sequences
identified as X95808 (X-linked mental retardaition candidate gene [Homo
Sapiens)). Based
upon sequence similarity, fy530 2 proteins and each similar protein or peptide
may share
at least some activity.
Clone " eg-511"
A polynucleotide of the present invention has been identified as clone
"ge51_1".
ge51_1 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ge51 1 is a full-
length clone,
including the entire coding sequence of a :secreted protein (also referred to
herein as
"ge51_1 protein").
The nucleotide sequence of ge51 1 as presently determined is reported in SEQ
ID
N0:20. What applicants presently believes to be the proper reading frame and
the
predicted amino acid sequence of the ge51__1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:21.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 ge51_1 should be approximately 1850 bp.
The nucleotide sequence disclosed herein for ge51_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLA~~TN/BLASTX and
FASTA search protocols. ge51_1 demonstrated at least some similarity with
sequences
identified as AA219716 (zq98d02.r1 Stratag~ene NT2 neuronal precursor 937230
Homo
2 5 Sapiens cDNA clone 650019 5'), AA434286 (zv~30f01.r1 Soares ovary tumor
NbHOT Homo
Sapiens cDNA clone 770809 5' similar to SW:NALS_BOVIN P08037
N-ACETYLLACTOSAMINE SYNTHASE), D61576 (Human fetal brain cDNA 5'-end
GEN-419H03), H30715 (yo78h01.r1 Homo Sapiens cDNA clone 184081 5'), T8U315
(yd07b08.r1 Homo Sapiens cDNA clone 24966 5'), U19889 (callus gallus
3 0 beta-1,4-galactosyltransferase (CKII) mRNA, complete cds), and W90417
(zh72hOl.s1
Soares fetal liver spleen 1NFLS S1 Homo saI>iens cDNA clone 417649 3'). The
predicted
amino acid sequence disclosed herein for ge!il_1 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted ge51 1 protein demonstrated at least some similarity to sequences
identified as
27
CA 02285554 1999-09-23
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M70433 (beta-1,4-galactosyltransferase [Homo sapiens]), R05932 (Human beta-1,4-
galactosyltransferase), and beta-1,4-galactosyltransferases from several other
species.
Based upon sequence similarity, ge51_1 proteins and each similar protein or
peptide may
share at least some activity. The TopPredII computer program predicts two
potential
transmembrane domains within the ge51 1 protein sequence, one centered around
amino
acid X20 and another around amino acid 90 of SEQ ID N0:21.
Clone ";x183 1"
A polynucleotide of the present invention has been identified as clone
"gx183_1".
gx183_1 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. gx183_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"gx183_1 protein").
The nucleotide sequence of gx183_1 as presently determined is reported in SEQ
ID N0:22. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the gx183_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:23. Amino acids 53 to 65 are a
predicted
2 0 leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 66, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
gx183_l should be approximately 2000 bp.
The nucleotide sequence disclosed herein for gx183_1 was searched against the
2 5 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. gx183_1 demonstrated at least some similarity with
sequences
identified as AA010474 (zi09a06.r1 Soares fetal liver spleen 1NFLS S1 Homo
sapiens
cDNA clone 430258 5'), H01847 (yj28f09.r1 Homo sapiens cDNA clone 150089 5'),
L38971
(Mus musculus (E25) mRNA, complete cds), Q60909 (Human brain Expressed
Sequence
3 0 Tag EST00998), W37875 zc13c01.s1 Soares parathyroid tumor NbHPA Homo
sapiens
cDNA clone 322176 3'), and W72197 (zd69e11.s1 Soares fetal heart NbHHI9W Homo
sapiens cDNA clone 345932 3'). The predicted amino acid sequence disclosed
herein for
gx183_1 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted gx183_1 protein demonstrated
at least
28
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
some similarity to sequences identified as AL021786 (dJ696H22.1 (mouse E25
like protein)
[Homo sapiens]) and L38971 (putative [Mus rnusculus]). Based upon sequence
similarity,
gx183_1 proteins and each similar protein or peptide may share at least some
activity.
Deposit of Clones
Clones bf171 6, ck181 7, co736_3, dm26_2, eq229_3, fh3 6, fs87_3, fy530 2,
ge51 1, and gx183_1 were deposited on March 25,1997 with the American Type
Culture
Collection as an original deposit under the Budapest Treaty and were given the
accession
number ATCC 98371, from which each clone comprising a particular
polynucleotide is
obtainable. All restrictions on the availability to the public of the
deposited material will
be irrevocably removed upon the granting of the patent, except for the
requirements
specified in 37 C.F.R. ~ 1.808(b), and the term of the deposit will comply
with 37 C.F.R.
~ 1.806.
Each clone has been transfected into separate bacterial cells (E. coli) in
this
I5 composite deposit. Each clone can be removed from the vector in which it
was deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6"} was derived from
pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman
et al.,
2 0 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from
pMT2
(Kaufman et al., 1989, Mol. Cell. Biol. 9: 94E~-958) by deletion of the DHFR
sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
orientation) in the deposited isolate. In such instances, the cDNA insert can
still be
2 5 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
3 0 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
29
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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
bf171 6 S EQ ID N0:24
ck181_7 SEQ ID N0:25
co736 3 S EQ ID N0:26
dm26 2 S EQ ID N0:27
eq229_3 SEQ ID N0:28
2 0 fh3_6 S EQ ID N0:29
fs87 3 S EQ ID N0:30
fy530_2 SEQ ID N0:31
ge51_1 SEQ ID N0:32
gx183_1 SEQ ID N0:33
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
2 0 diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
2 5 (b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-32P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Uruncorporated
3 0 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.
CA 02285554 1999-09-23
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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
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
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.:'_ g Na citrate/liter, adjusted
to pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnught. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
2 0 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. Oi:her known hybridization methods
can also
be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated
2 5 using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
Fragments of the proteins of the presEmt 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,
3 0 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, 924.5-9253 (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
31
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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
forms) of such protein may be obtained by expression of the disclosed full-
length
polynucleotide (preferably those deposited with ATCC) in a suitable mammalian
cell or
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
2 0 herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
2 5 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 mItNA transcribed
from the
gene (Albert and Morris,1994, Trends Pharmacol. Sci.15(7): 250-254; Lavarosky
et al., 1997,
3 0 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.
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CA 02285554 1999-09-23
WO 98/42741 PCTNS98/05972
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 B1, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al., '1994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,7Ei4; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
2 0 Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
2 5 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%
3 0 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
_S3
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/OS972
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
Species homologues of the disclosed polynucleotides and proteins are also
provided by the present invention. As used herein, a "species homologue" is a
protein or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
the given polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues may be isolated and identified by making suitable probes or primers
from
the sequences provided herein and screening a suitable nucleic acid source
from the
desired species. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hylobates
concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
2 0 Aotus trivirgatus, Sanguinus Oedipus, Microcebus marinas, MbIS muscaclus,
Rattus norvegicus,
Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus
cuniculus, Bos taurus,
Ovis aries, Sus scrofa, and Eguus caballus, for which genetic maps have been
created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
2 5 (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al.,
1993, Nature
Genetics 3:103-112; Johansson et al., 1995, Genomics 25: 682-690; Lyons et
al., 1997, Nature
Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
1997, Genome Research 7:1123-1137; all of which are incorporated by reference
herein).
The invention also encompasses allelic variants of the disclosed
polynucleotides
3 0 or proteins; that is, naturally-occurring alternative forms of the
isolated polynucleotides
which also encode proteins which are identical or have significantly similar
sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
60% sequence identity (more preferably, at least 75% identity; most preferably
at least 90%
34
CA 02285554 1999-09-23
WO 98/42741 PCT/US98105972
identity} with the given polynucleotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
overlap and identity while minimizing sequence gaps. Allelic variants may be
isolated and
identified by making suitable probes or primers from the sequences provided
herein and
screening a suitable nucleic acid source from individuals of the appropriate
species.
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes p~olynucleotides capable of hybridizing
under
reduced stringency conditions, more preferably stringent conditions, and most
preferably
highly stringent conditions, to polynucleotidE>s described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
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CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
~P)t Buffer' and Buffer'
A DNA:DNA z 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; 7xSSC, 50% formamide
D DNA:RNA <50 T~*; lxSSC T"*; IxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; IxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC Tr*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 TH*; 4xSSC TH*; 4xSSC
I DNA:RNA z 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T *; 4xSSC
K RNA:RNA z 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50/~
formamide
L RNA:RNA <50 T~*; 2xSSC T, *; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 T,,*; 6xSSC T~*; 6xSSC
Q RNA:RNA z 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
0
t: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
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
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 *TB - TR: The hybridization temperatwe for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log~o[Na'J) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'J is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
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CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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 Lalboratory 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.~~-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most :preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-9E490 (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, 53',7-566 (1990). As defined herein
"operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria.. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces p~ombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacilhts subtilis, Salmonella typhimurium,
or any bacterial
3'7
CA 02285554 1999-09-23
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PCT/US98/05972
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987 incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX}. Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
38
CA 02285554 1999-09-23
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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 mamrnalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletiion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
:39
CA 02285554 1999-09-23
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USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
described in Gyuris et al., 1993, Cell 75: 791-803 and in Rossi et al., 1997,
Proc. Natl. Acad.
Sci. LISA 94: 8405-8410, all of which are incorporated by reference herein) to
identify
polynucleotides encoding the other protein with which binding occurs or to
identify
3 0 inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
CA 02285554 1999-09-23
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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 a.s research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
j., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel
eds.,1987.
Nutritional Uses
Polynucleotides and proteins of i:he present invention can also be used as
2 0 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 organi~;m or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
2 5 capsules. In the case of microorganisms, the protein or polynucleotide of
the invention
can be added to the medium in or on whic'.h the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention rnay exhibit cytokine, cell proliferation
(either
3 0 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
41
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.22.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon 'y, Schreiber, R.D. In Current
Protocols in
2 0 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
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,
2 5 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
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
3 0 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.
42
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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. h. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulatin" og r Su~pressintr Activi
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
2 5 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
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral {e.g., HIV} as well as
bacterial or fungal
2 0 infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which ma:y be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthriitis, autoimmune pulmonary inflammation,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention ma;y 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
43
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
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
IO inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
I5 Down regulating or preventing one or more antigen functions (including
without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
44
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
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-T92 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis :in MRL/Ipr/Ipr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
CA 02285554 1999-09-23
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PCT/US98/05972
viral infection. In addition, systemic viral diseases such as influenza, the
common cold, ,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivo with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
X32
microglobulin protein or an MHC class II a chain protein and an MHC class II
~3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
46
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
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 o:r splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, VV Strober, Pub. Greene Publishing
Associates
and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa
et al.,
J. Immunol. 135:1564-1572, 1985; Takai et al., j. Immunol. 137:3494-3500,1986;
Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341,1991; Brown et aL, J. Immunol. 153:3079-3092,
1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Th1/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
3 0 Mixed lymphocyte reaction (MLR;1 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, F'ub. Greene Publishing Associates
and Wiley-
Interscience (Chapter 3, In Vitro assays for D~Iouse Lymphocyte Function 3.1-
3.19; Chapter
47
CA 02285554 1999-09-23
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PCT/US98/05972
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,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-b48, 1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re ulating Activity
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
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myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and ,
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post: irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et a:l. Cellular Biology 15:141-
151,1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony fornning assays, Freshney, M.G. In
Culture of
2 5 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-
Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al, eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,
Sutherland,
4~9
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H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regeneration activity that may be attributable
to the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
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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 braiin tissue, i.e, for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson s disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endoi:helium), muscle (smooth,
skeletal or cardiac)
3 0 and vascular (including vascular endotheliwn) 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.
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A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091 /07491 {skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
2 5 Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest.
Dermatol 71:382-84 {1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
(3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
advancementpf 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:
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Assays for activin/inhibin activity W elude, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic ActivitX
A protein of the present invention rr~ay have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by emplloying such protein or peptide
in any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described. in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al.. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
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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
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
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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-InflammatorX 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 chernotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated llyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
2 0 from over production of cytokines such as TrJF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersens;itfvity to an antigenic substance
or material.
Cadherin/Tumor Invasion Su~pressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes v~~ell over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), trut 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
'_i5
CA 02285554 1999-09-23
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first cadherin domain provide the basis for homophilic adhesion; modification
of this ,
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleoHdes
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 polynucleoHdes of the present invention encoding such proteins, can used
to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
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WO 98/42741 PCT/US98/05972
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 descrilbed above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to learn ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
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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,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
2 0 IL-12, IL-13, IL-14, IL-15, IFN, TNFO, 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
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
2 5 or to minimize side effects. Conversely, protein of the present invention
may be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
3 0 A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
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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 stimul~~tory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
2 0 and the like. Preparation of such liposomal formulations is within the
level of skill in the
art, as disclosed, for example, in U.S. Patent :No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,;737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
2 5 amount of each active component of the pharmaceutical composition or
method that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditiions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
3 0 a combination, the term refers to combined amounts of the active
ingredients that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
'.i9
CA 02285554 1999-09-23
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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 factors}, 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 hematopoieHc factor(s), thrombolytic or anti-
thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
2 5 Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
2 0 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
2 5 physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
3 0 When a therapeutically effective amount of protein of the present
invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
CA 02285554 1999-09-23
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pharmaceutical composition for intravenou ,, 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 t:he nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 Itg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
2 0 present invention will vary, depending on the severity of the disease
being treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
2 5 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
3 0 carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et
aL, FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetE~ction of the protein. Neutralizing
monoclonal
~61
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antibodies binding to the protein may also be useful therapeutics for both
conditions .
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
2 0 developing bone and cartilage and optimally capable of being resorbed into
the body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
2 5 application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
3 0 or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
ceramics. Matrices rnay be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
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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 ito 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 hydroxyalkylicelluloses), including
methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
2 0 In further compositions, proteins of the invention may be combined with
other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor (PDGh), transforming growth factors (TGF-
a and
TGF-(3), and insulin-like growth factor (IGF).
2 5 The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
3 0 various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstihition and with inclusion of other
proteins in
63
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the pharmaceutical composition. For example, the addition of other known
growth ,
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
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SEQUhNCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding, Vikk:i
Agostino, Micha<al J.
(ii} TITLE OF INVENTION: SECRET1~D PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 34
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D} STATE: MA
(E} COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzan:ne 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: 1521 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
E~$
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WO 98/42741
(ii) MOLECULE TYPE: cDNA
PCT/US98/05972
(xi) SEQUENCE SEQ ID N0:1:
DESCRIPTION:
GTAACCTTCTTCTGCGCGGCTGCAGCTCGG 60
GACTTCGGCC
TGACCCAGCC
CCCATGGCTT
CAGAAGAGCTACAGAAAGATCTAGAAGAGGTAAAGGTGTT GCTGGAAAAG GCTACTAGGA120
AAAGAGTACGTGATGCCCTTACAGCTGAAAAATCCAAGAT TGAGACAGAA ATCAAGAACA180
AGATGCAACAGAAATCACAGAAGAAAGCAGAACTTCTTGA TAATGAAAAA CCAGCTGCTG240
TGGTTGCTCCCATAACAACGGGCTATACGGTGAAAATCAG TAATTATGGA TGGGATCAGT300
CAGATAAGTTTGTGAAAATCTACATTACCTTAACTGGAGT TCATCAAGTT CCCACTGAGA360
ATGTGCAGGTGCATTTCACAGAGAGGTCATTTGATCTTTT GGTAAAGAAT CTAAATGGGA420
AGAGTTACTCCATGATTGTGAACAATCTCTTGAAACCCAT CTCTGTGGAA GGCAGTTCAA480
AAAAAGTCAAGACTGATACAGTTCTTATATTGTGTAGAAA GAAAGTGGAA AACACAAGGT540
GGGATTACCTGACCCAGGTTGAAAAGGAGTGCAAAGAAAA AGAGAAGCCC TCCTATGACA600
CTGAAACAGATCCTAGTGAGGGATTGATGAATGTTCTAAA GAAAATTTAT GAAGATGGAG660
ACGATGATATGAAGCGAACCATTAATAAAGCCTGGGTGGA ATCAAGAGAG AAGCAAGCCA720
AAGGAGACACGGAATTTTGAGACTTTAAAGTCGTTTTGGG AACTGTGATG TGATGTGGAA780
ATACTGATGTTTCCAGTAAGGGAATATTGGTGAGCTGCAT ATATAAATTT GACAGATAGC840
TATTTACATAGCCTTCTAAGTAAAGGCAATGAATTCTCCA TTTCCTACTG GAGGATTTAT900
TTAAATAAAATATGCTTATTAAACACTCCTGCAAAGATGG TTTTATTAGT ACCCTGGTCA960
TTTTGTTCAAGGAAGGGTTATATTGCATTCTCACGTGAAA TATAAAAAGC AAGTCTTGCC1020
CAATAAAAACGCTACATTGTGTGTATTTTTTGTTCAGCTA AGAATTGGAA AAGTATTTGC1080
TTGCCTTTTAAGTTACTGACATCAGCTTCCACCAGTGTAA AAATTGAGTA AAACCTGAAG1140
TTTTGCATAAAATGCAAATCGGTGCCTGTGCTTGAAGGTT GCTGTAGAGC ATCTGACCCC1200
TTATTACCACCTTAAGCAATGTATATGCCATGCATTACCA TGCACTAATT CAATCACAGG1260
TGTTTCTATCTAGATTTAAATATATTTGTCAATGAATGTG GAATAGAAAA TCTAAACATG1320
ACAATAATAGACATATCTTTGTATGGTACCAGTTAGTTTT GCCGTGGATC AGATGGTTTA1380
TAAAAGTAATAACCATAAAGCAAAAAATAATTTGAAAGCC CGTCTATTCC TATGCTCAAT1440
66
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AAAGTTAAGT TTTTTTTCAA P,~~AAAAAAAA AAAAAAAAAA P,~~AAAAAAAA A,F~AAAAAAAA 15 0 0
A,F~~~1AP.AP A F,F~AAAAAAAA A 15 21
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Ser Glu Glu Leu Gln Lye: Asp Leu Glu Glu Val Lys Val Leu
1 5 10 15
Leu Glu Lys Ala Thr Arg Lys Arcr Val Arg Asp Ala Leu Thr Ala Glu
20 25 30
Lys Ser Lys Ile Glu Thr Glu Ile: Lys Asn Lys Met Gln Gln Lys Ser
35 40 45
Gln Lys Lys Ala Glu Leu Leu Asp Asn Glu Lys Pro Ala Ala Val Val
50 55 60
Ala Pro Ile Thr Thr Gly Tyr Thr Val Lys Ile Ser Asn Tyr Gly Trp
65 70 75 80
Asp Gln Ser Asp Lys Phe Val Ly;: Ile Tyr Ile Thr Leu Thr Gly Val
85 90 95
His Gln Val Pro Thr Glu Asn Val Gln Val His Phe Thr Glu Arg Ser
100 105 110
Phe Asp Leu Leu Val Lys Asn Leu Asn Gly Lys Ser Tyr Ser Met Ile
115 120 125
Val Asn Asn Leu Leu Lys Pro Ile Ser Val Glu Gly Ser Ser Lys Lys
130 135 140
Val Lys Thr Asp Thr Val Leu Ile Leu Cys Arg Lys Lys Val Glu Asn
145 150 155 160
Thr Arg Trp Asp Tyr Leu Thr Gln Val Glu Lys Glu Cys Lys Glu Lys
165 170 175
Glu Lys Pro Ser Tyr Asp Thr Glu Thr Asp Pro Ser Glu Gly Leu Met
180 185 190
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Asn Val Leu Lys Lys Ile Tyr Glu Asp Gly Asp Asp Asp Met Lys Arg
195 200 205
Thr Ile Asn Lys Ala Trp Val Glu Ser Arg Glu Lys Gln Ala Lys Gly
220 215 220
Asp Thr Glu Phe
225
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1394 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
TGCGTCATGCAGTGCGCCGGAGGAACTGTGCTCTTTGAGGCCGACGCTAGGGGCCCGGAA 60
GGGAAACTGCGAGGCGAAGGTGACCGGGGACCGAGCATTTCAGATCTGCTCGGTAGACCT 120
GGTGCACCACCACCATGTTGGCTGCAAGGCTGGTGTGTCTCCGGACACTACCTTCTAGGG 180
TTTTCCACCCAGCTTTCACCAAGGCCTCCCCTGTTGTGAAGAATTCCATCACGAAGAATC 240
AATGGCTGTTAACACCTAGCAGGGAATATGCCACCAAAACAAGAATTGGGATCCGGCGTG 300
GGAGAACTGGCCAAGAACTCAAAGAGGCAGCATTGGAACCATCGATGGAAAAAATATTTA 360
AAATTGATCAGATGGGAAGATGGTTTGTTGCTGGAGGGGCTGCTGTTGGTCTTGGAGCAT 420
TGTGCTACTATGGCTTGGGACTGTCTAATGAGATTGGAGCTATTGAAAAGGCTGTAATTT 480
GGCCTCAGTATGTCAAGGATAGAATTCATTCCACCTATATGTACTTAGCAGGGAGTATTG 540
GTTTAACAGCTTTGTCTGCCATAGCAATCAGCAGAACGCCTGTTCTCATGAACTTCATGA 600
TGAGAGGCTCTTGGGTGACAATTGGTGTGACCTTTGCAGCCATGGTTGGAGCTGGAATGC 660
TGGTACGATCAATACCATATGACCAGAGCCCAGGCCCAAAGCATCTTGCTTGGTTGCTAC 720
ATTCTGGTGTGATGGGTGCAGTGGTGGCTCCTCTGACAATATTAGGGGGTCCTCTTCTCA 780
TCAGAGCTGCATGGTACACAGCTGGCATTGTGGGAGGCCTCTCCACTGTGGCCATGTGTG 840
CGCCCAGTGAAAAGTTTCTGAACATGGGTGCACCCCTGGGAGTGGGCCTGGGTCTCGTCT 900
TTGTGTCCTCATTGGGATCTATGTTTCTTCCACCTACCACCGTGGCTGGTGCCACTCTTT 960
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ACTCAGTGGC AATGTACGGT GGATTAGTTCTTTTCAGCATGTTCCTTCTG TATGATACCC1020
AGAAAGTAAT CAAGCGTGCA GAAGTATCACCAATGTATGGAGTTCAAAAA TATGATCCCA1080
TTAACTCGAT GCTGAGTATC TACATGGATACATTAAATATATTTATGCGA GTTGCAACTA1140
TGCTGGCAAC TGGAGGCAAC AGAAAGAAATGAAGTGACTCAGCTTCTGGC TTCTCTGCTA1200
CATCAAATAT CTTGTTTAAT GGGGCAGATATGCATTAAATAGTTTGTACA AGCAGCTTTC1260
GTTGAAGTTT AGAAGATAAG AAACATGTCATCA'TATTTAAATGTTCCGGT AATGTGATGC1320
CTCAGGTCTG CCTTTTTTTC TGGAGAATAAATGCAGTAATCCTCTCCCAA ATAAGCACAA1380
1394
(2) INFORMATION FOR SEQ
ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ .CD N0:4:
Met Leu Ala Ala Arg Leu Val Cys Leu Arg Thr Leu Pro Ser Arg Val
1 5 10 15
Phe His Pro Ala Phe Thr Lys A1<~ Ser Pro Val Val Lys Asn Ser Ile
20 25 30
Thr Lys Asn Gln Trp Leu Leu Thr Pro Ser Arg Glu Tyr Ala Thr Lys
35 40 45
Thr Arg Ile Gly Tle Arg Arg Gly Arg Thr Gly Gln Glu Leu Lys Glu
50 55 60
Ala Ala Leu Glu Pro Ser Met Glu Lys Ile Phe Lys Ile Asp Gln Met
65 70 75 80
Gly Arg Trp Phe Val Ala Gly Gh,r Ala Ala Val Gly Leu Gly Ala Leu
85 90 95
Cys Tyr Tyr Gly Leu Gly Leu Se_r Asn Glu Ile Gly Ala Ile Glu Lys
1Q0 105 110
Ala Val Ile Trp Pro Gln Tyr Va:1 Lys Asp Arg Ile His Ser Thr Tyr
115 120 125
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Met Tyr Leu Ala Gly Ser Ile Gly Leu Thr Ala Leu Ser Ala Ile Ala ,
130 135 140
Ile Ser Arg Thr Pro Val Leu Met Asn Phe Met Met Arg Gly Ser Trp
145 150 155 160
Val Thr Ile Gly Val Thr Phe Ala Ala Met Val Gly Ala Gly Met Leu
165 170 175
Val Arg Ser Ile Pro Tyr Asp Gln Ser Pro Gly Pro Lys His Leu Ala
180 185 190
Trp Leu Leu His Ser Gly Val Met Gly Ala Val Val Ala Pro Leu Thr
195 200 205
Ile Leu Gly Gly Pro Leu Leu Ile Arg Ala Ala Trp Tyr Thr Ala Gly
210 215 220
Ile Val Gly Gly Leu Ser Thr Val Ala Met Cys Ala Pro Ser Glu Lys
225 230 235 240
Phe Leu Asn Met Gly Ala Pro Leu Gly Val Gly Leu Gly Leu Val Phe
245 250 255
Val Ser Ser Leu Gly Ser Met Phe Leu Pro Pro Thr Thr Val Ala Gly
260 265 270
Ala Thr Leu Tyr Ser Val Ala Met Tyr Gly Gly Leu Val Leu Phe Ser
275 280 285
Met Phe Leu Leu Tyr Asp Thr Gln Lys Val Ile Lys Arg Ala Glu Val
290 295 300
Ser Pro Met Tyr Gly Val Gln Lys Tyr Asp Pro Ile Asn Ser Met Leu
305 310 315 320
Ser Ile Tyr Met Asp Thr Leu Asn Ile Phe Met Arg Val Ala Thr Met
325 330 335
Leu Ala Thr Gly Gly Asn Arg Lys Lys
340 345
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERTSTICS:
(A) LENGTH: 1908 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:5:
GCTTTTTTTTTTTTTTTTGGTTGAGATGGGGTCTCGCCATGTTTCCCACACTGATCTTGA 60
ACTCCTGGGCTCCAGGAATTCTCCTACTTTGGCCTCCCAAAGTGTTGGGAATATTGGCAT 120
GAACCACAGCACCTGACTTGCATATTTGTGAATTCCCCAAATTGCTTTTTTTAAATTGAT 180
TTCTAATTTCATTTCATTGTTATGGGGAACATACTTTGTATGGTTTCAATGTTTTAAAAT 240
TAATTGAGACTTGTTTTATGACTTAGCATATGGTCTGTGTTGAAGAAGGCTCCATGTACA 300
CTTGAGAATAATATGTATACTGTGGTTGTTGGGT'GGATTTTCTATGTATGTTTARGTGAT 360
ATGGTTTTATAGTGTTGTTTAARTCTTCTATTTT'CTTCTTTTTCTGCCCAGTTTTATTTT 420
TGAAAGCATACTGARGTCTCCAACTCARTGCCTT'AGCCTCCTGAGCAGTTGGGACTACAG 480
GCATACGCCACTACACCCAGCAATTTTTTTGTAT'TTTTCTGTAGAGACAGAGTTTCACCA 540
TGTTGCCTAGGCTGGTCTCAGATTCCTGGACTCP.AGTGATCTCGATTCCCGGCCTCTGCC 600
TCCCGGGGTGCTGGGATTGCAGGCATGAGCTACTATGCCTGGCAAATTTTATTTTTCCTT 660
TTATTTTGTCACATAATTAAAGCTACTCCAGAATTCCCTTGATTTCTGCTTGCCTGGTAT 720
ATCTTTTTTCCATTTTTTAACTGTCAGCCTTTTTTGTGCCTGTTAATCTAAAGTATGTGT 780
TTCGTAGATAATATGTAGCTGGATCATATTTTAP,AAATATTTATTCTGCCAAGCTCTGTC 840
TTTTGATTGGAGTATTCTTTCATTTATGTTTGTP,ATTACTGATGAGGGGGGCACTAATGT 900
CTGCTGTTTTGCTATTTGTTTCCCCATGTCTTATGTCTTCATTACTGACTTTTTTATTAA 960
ACAACTATTTTCTTGGGTACCATTTTAAGTCCCTCTCCCACTCATTTTTTAATGTTTTTT 1020
TGTGTTTACTTTTGTTTTTATTGTTTGCCCTGATATTAAAATTAACATTTTACCTTGAAA 1080
TAGTTGGCTTCAGATTAATATCAACTTAGTTTCAATAGCATAGGAAATTTGCTTCACTAT 1140
ATTTCCATTTTCTCCCCGTCCTTTGTGCTATTATTACTATACCAATTAGATCTCTACACA 1200
ATATAGGCATATCAACACATTTTGTAATTATTTC:CTTATCCAGTTGTCTTTTAATATAGA 1260
TCTGTGAAGAAAAGTATTACACAAATAGATCTA9'TCTGTTTTTTATAATTATTTAATTAC 1320
CTTTGGTGGTGCTGTTTATTTTTCATGCATTTGAGTTACTGTCTAGTATTCATTCATTTC 1380
TCTCTGAATCACTCCCTTTAGTATTGCTTGTAGGGCAGGTCTGCTAGCATTGAATTCTTT 1440
TAATTTTTGTGACTCTGCAAATGCCATAATTTC'l:'CTTTTGTTTGTGAAGGATAGTTTTGC 1500
TAGATACAGAATTTGCAGTTGGCATTCTTTTTACTTTAGCAGTTTGAAAATATTTCCCAT 1560.
TGTTGGCCGGGCACAGTGGCTCACGCCTGTGGTCCTAGCACTTTGGGAGGCCGAGGCGGG 1620
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CGGATCGTCT GGGGTCGGGA GTTCGGGACC GGCCTGGCCA ATATGGTGAG GCCCTGTTTC 1680
TGCTAAAATA TAAAAATTGG CTGGGCATGA TGGCGGGTGC CTCTAGTCCC AGCTGCTCGG 1740
GAGGCTGAGG TGGGGGAGTC GCTTGAGCCC GGGAGATGAT GGCTGTGGTG AGCCGGGATG 1800
GCGCCGCTGC ACTCCGGCCT GGGCGGCTGA GTGAGACTCC ATCCCCGAAA AAAAAAAAAA 1860
P,~~~AAAAAAA F,~~~AAA.AAAA AA,AAAP,AAAA P~Ii~.AAAAAAA AAAAAAAA 19 0 8
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Arg Gly Ala Leu Met Ser Ala Val Leu Leu Phe Val Ser Pro Cys
1 5 10 15
Leu Met Ser Ser Leu Leu Thr Phe Leu Leu Asn Asn Tyr Phe Leu Gly
20 25 30
Tyr His Phe Lys Ser Leu Ser His Ser Phe Phe Asn Val Phe Leu Cys
35 40 45
Leu Leu Leu Phe Leu Leu Phe Ala Leu Ile Leu Lys Leu Thr Phe Tyr
50 55 60
Leu Glu Ile Val Gly Phe Arg Leu Ile Ser Thr
65 70 75
(2) INFORMATION FOR SEQ ID N0:7:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3076 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
72
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
CTTTTTTTTTTTTTTTCAATTTCATTTAGTTCTGCCCTGATCTTGGTTATTTCCTTTTTT60
TCTGCTGGGTTTGGGTTTGGTTTGTTCTTATTTCTCTAGTTCCTTGAGGTGTGACCTTAR120
AATGTCAATTTGTGCTCTTTCAATCTTTTTGATGTAGGCGTTGAGGGCTGTGGACTTTTC180
TCTTGGCACTCCCTTTGGTGTATCCCARAGGTTTTGATAGGTTGTGTCATTATTGCAATT240
CAGTTTGAAGAATTTCTTAATCTCCACCTTGATTTTGTTTTTGACCCAATGCTCATTCAG300
GAGCAGGTTATTTACTTTCCATGTACTTGCATGGCTTTGAAGCTTCCTTTTGGAGTTGAT360
TTCCAGTTTTATTCCACTGTGATTTGAGAGAGTGCTTTACATAATTTCAATTTTCTTAAT420
TTTATTAAGGCTCGTTTTATGGCCTATAATATGGTCTATCTTGGAGAAAGTTCCATGCAC480
TGTAGAATAGAATGTGTATTCTGTGGTTGTTGGATGAAATGTTCTGCATATATTCCTAGA540
TTGCCTCCCCACAAAAGGTTGCATCAATGTCTGTGTTTCTCTACACCATCTCACCCTTGC600
CAACTTCGGGTTTCATCAGACCTTACTGATTGTCAGTATGATCTGTGAAACAAATCTCTC660
AGTTTTGATTTGCATTTTTTAAATTATGAGAGCTTGAACACCATTTTACATGTTTATTGG720
CTGTTGTTATTTCCTTTTTGAGATCTGTTCGTTATATGCTTTGCCCGTTTTTCTGTTGGG780
TGGTTATTATTTTTCTTATTGAATGGTATAAGCTCTTTGTAAGTTAAGGACATTAGCCCT840
TAGTCAGATATTTTGACTTAGGTTTTA,ATTTTT'I'TCCACACAGAAGTTTTAAGCTCTGTG900
GCAAATTTATCAGTCTTATATCACTACAGGGTTA.TAAATATTAGYTATCACTTCGGGTTT960
GTGTCTTGCTTAGA.AAGCMTCATTTGAAGATTGTAAATGTTAGTAAGTTTCCCCATATTT1020
TCCTCTAGGACTTCCATGGTTTAATTTGTTTTGTTTAAAYTAGGAATTGGCATTCACATC1080
CTYTTTTGTCCCAGGTCTCAGAGGTCCCTTGTATCTTATAGAGCAGTATTGTTTTATGTT1140
ATTTTCCCATGTATAATTTAAA.AACAAAATACGTTGTTCAAAACAAAATACAGTGGCAGC1200
AGATAATGGCAGTATCTCTGTAACTGCTGGTAAF,CTGTATTTCATAGTGAAGTGTTCATA1260
AACTAAAGAGTCATTGATTTGGTTTCCTGGCTAA,TTAAAATCTGAATTCCATTTGAAGTT1320
CCATTGAAATCATGGTTTTACTCTATAGCAGTGGATGTTTTTTCCCAACCTTTCTGATAT1380
TTTTTTCCTTCCTGAGACAGGGTCTTGCTCTGTC'.ACCTGGGATGGAGTGTAGTTGCACCA1440
TCAAGGCTTACTGCAGTCTCAACTCTCTGAGCTC.'AAGTGATCCTGCCACCTCAGCCTCTT1500
GAGTAGCAAGGATTACAGGCACCTACCACTATGCCTGGCTAATTTTTATATTTTTTGTAG1560
AGATGGATTCTCACTATGTTGCCCGGGCTCATCTTGAACTCGAGCTCAAGCAATCTGTCC1620
ATCTTGGCCTCCCAAAGTGCTGGGATTATAGGCGTGAGCCACTGCACCTGGCCCCTTTCT1680
7:3
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
GATTATTTTA ATCTATCTTT CTAATTCATT TAAAGCATAT1740
AAATGTTCAA AGTGATTTGC
TTAGTTTTTT TTAAATTGAGTGTATTTTATCTAGATATTTTTAAAAGGCA GCATCTAACC1800
TTGGATTTTA TAAATACATCTAAATTTGTTATTTCCAGAATGCTTCAAAA CAGATCTCTG1860
TAGCCTCGTG CTTTGTTATTGTTAGGTTTTTTTTTTTTGTTTTGAGACAG GGTCTTGCTC1920
TATCTGGAGT GCAGTGGCACAGTCATAGCTCACTGTACCCTCAAACTCCT AAACTCAAGT1980
AATCCTCCCA TCTCAGCCTCCTGAGTAGTTGGGACCACAGTCATGCACCA GCATGCCTGG2040
CTAATTTTTT AAATTTTGTTCTTAATAGAGACAGAGTCTTGCTGTGTTGT TCAGGCTGGT2100
CTCAAACTCC TGGGCTCAAGCGATCCTCCCACCTCAGCCTCCTAAAGTGC TGAGATTACG2160
GATGTGAATC ATTACACCCAGCCTATTAATGGTTTTGTATAGCAAGTCTT TTGTGGGTGG2220
TGGAAAGATG AAGTGCTGTGAAATATTGTAGGAGCAGAAACTTGAAATGT GGCAAAAACC2280
ACATGGGCAA AATTTCTGTCTCTTTTCTTATTTTTGCTTTTTTGTTTAAA GGTTTTTCTA2340
TTGGGAAAGC TACTGATCGGATGGATGCTTTCAGGAAAGCAAAGAACAGA GCAGTTCACC2400
ATTTGCATTA TATAGAACGATATGAAGACCATACAATATTCCATGATATT TCATTAAGAT2460
TTAAAAGGAC GCATATCAAGATGAAGAAACAACCCAAAGGTTACGGCCTC CGCTGCCACA2520
GGGCCATCAT CACCATCTGCCGGCTCATTGGCATCAAAGACATGTATGCC AAGGTC'CCTG2580
GGTCCATTAA TATGCTCAGCCTCACCCAGGGCCTCTTCCGTGGGCTCTCC AGACAGGAAA2640
CCCATCAACA GCTGGCTGATAAGAAGGGCCTCCATGTTGTGGAAATCCGG GAGGAATGTG2700
GCCCTCTGCC CATTGTGGTTGCGTCCCCCCGGGGGCCCTTGAGGAAGGAT CCAGAGCCAG2760
AAGATGAGGT TCCAGACGTCAAACTGGACTGGGAAGATGTGAAGACTGCA CAGGGAATGA2820
AGCGCTCTGT GTGGTCTAATTTGAAGAGAGCCGCCACGTAACCTCTCTGG CCTTGTGCAG2880
CCAGTTCCTG TGCTGCCCTGCACCTAGGAGAGACTCAGCCCCTCACAGCT TGGGATGTTA2940
CCTTGCCTTT TGTTTGTTTTGAGGGAAGTTTAATCTTTAAACTCTTTGGA AATAAATAAT3000
TATAGCTTTC P.,F~~AAAAAAAA,F~~AAAAAAAp~~AAAAAAAp,~~~AAAAAA A,~~~ 3
0
6
0
P,~~e3AAAAAAP.
AAAAAA 3076
(2) INFORMATION EQ ID
FOR S N0:8:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 192 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOG Y: linear
74
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Gly Lys Ile Ser Val Ser Phe Leu Ile Phe Ala Phe Leu Phe Lys
1 5 10 15
Gly Phe Ser Ile Gly Lys Ala Thr Asp Arg Met Asp Ala Phe Arg Lys
20 25 30
Ala Lys Asn Arg Ala Val His His Leu His Tyr Ile Glu Arg Tyr Glu
35 40 45
Asp His Thr Ile Phe His Asp Ile Ser Leu Arg Phe Lys Arg Thr His
50 55 60
Ile Lys Met Lys Lys Gln Pro Lys Gly Tyr Gly Leu Arg Cys His Arg
65 70 75 80
Ala Ile Ile Thr Ile Cys Arg Leu Ile Gly Ile Lys Asp Met Tyr Ala
85 90 95
Lys Val Ser Gly Ser Ile Asn Met Leu Ser Leu Thr Gln Gly Leu Phe
100 105 110
Arg Gly Leu Ser Arg Gln Glu Thr His Gln Gln Leu Ala Asp Lys Lys
115 120 125
Gly Leu His Val Val Glu Ile Arg Glu Glu Cys Gly Pro Leu Pro Ile
130 135 140
Val Val Ala Ser Pro Arg Gly Pro Leu Arg Lys Asp Pro Glu Pro Glu
145 150 155 160
Asp Glu Val Pro Asp Val Lys Leu Asp Trp Glu Asp Val Lys Thr Ala
165 170 175
Gln Gly Met Lys Arg Ser Val Trp Ser Asn Leu Lys Arg Ala Ala Thr
180 185 190
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 683 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
7_'i
CA 02285554 1999-09-23
WO 98/42741
PC'T/US98/05972
(xi) SEQUENCE SEQ TD
DESCRIPTION: N0:9:
CGCCAAGTGCGCATGGGGAC GCTATAGCAA GTCCTTCCTC TCCTTCGAAG60
TTCGTTTGCT
ATGACAAGGCCTACCATCGT TTCTTCCTGCCTTTGGGCCGTCAGGCAGTT GGTTGGGACC120
CGCTCCAACCCTCGGTTCTT CCTGCAATACAGTGGATACAATTTGTCATG GCTACTCTGA180
GTGTTATAGGTTCAAGTTCA CTTATTGCCTATGCTGTATTCCATAATATA CAGAAATCTC240
CAGAGATAAGACCACTTTTT TATCTGAGCTTCTGTGACCTGCTCCTGGGA CTTTGCTGGC300
TCACGGAGACACTTCTCTAT GGAGCTTCAGTAGCAAATAAGGACATCATC TGCTATAACC360
TACAAGCAGTTGGACAGATA TTCTACATTTCCTCATTTCTCTACACCGTC AATTACATCT420
GGTATTTGTACACAGAGCTG AGGATGAAACACACCCAGAGTGGACAGAGC ACATCTCCAC480
TGGTGATAGATTATACTTGT CGAGTTGGTCAAATGGCCTTTGTTTTCTCA AGCCTGATAC540
CTCTGCTATTGATGACACCT GTATTCTGTCTGGGAAATACTAGTGAATGT TTCCAAAACT600
TCAGTCAGAGCCACAAGTGT ATCTTGATGCACTCACCACCATCAGCCATG GCTGAACTTC660
CACCTTCTGCCAACACATCT GTC 683
(2) INFORMATION
FOR SEQ
ID NO:10:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 172 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE
TYPE:
protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:10:
Met AlaThrLeu SerValIle GlySer SerSerLeu IleAlaTyr Ala
1 5 10 15
Val PheHisAsn IleGlnLys SerPro GluIleArg ProLeuPhe Tyr
20 25 30
Leu SerPheCys AspLeuLeu LeuGly LeuCysTrp LeuThrGlu Thr
35 40 45
Leu LeuTyrGly AlaSerVal AlaAsn LysAspIle IleCysTyr Asn
50 55 60
Leu GlnAlaVal GlyGlnIle PheTyr IleSerSer PheLeuTyr Thr
76
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
65 70 75 80
Val Asn Tyr Ile Trp Tyr Leu Tyr Thr Glu Leu Arg Met Lys His Thr
85 90 95
Gln Ser Gly Gln Ser Thr Ser Pro Leu Val Ile Asp Tyr Thr Cys Arg
100 105 110
Val Gly Gln Met Ala Phe Val Phe Ser Ser Leu Ile Pro Leu Leu Leu
115 120 125
Met Thr Pro Val Phe Cys Leu Gly Asn Thr Ser Glu Cys Phe GIn Asn
130 135 140
Phe Ser Gln Ser His Lys Cys Ile Leu Met His Ser Pro Pro Ser Ala
145 150 155 160
Met Ala Glu Leu Pro Pro Ser Ala Asn Thr Ser Val
165 170
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 524 base pair
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ I:D N0:11:
ATATGGCTGG ACGCAGCACAAATTCCACCA ACTAAAGCAGGAGGCTCGGCGTGATGCAGA 60
TACCCAGACA CCATTATTATGCTCACAGAA GAGATTCTATAGCAGGGGCTTAAATTCACT 120
GGAATCCACC CTGACTTTTCCTGCCAGTAC TTCTACCATTTTTTGAAACTACAATACTGG 180
AACATCCAGG AACTGGAGTTATTCTACGCT AATC~GATTGGAAAGAATGTTGGGAAAGGAC 240
ATCTTAAATC TTTTCTAACTATGCCCTAAA CTGC'AGAACTCAAAGGAAATATAGTGCCAT 300
TGTTAGTAGT CATTCTAGATGAATTGGGAG TATCTCTCCAGTTATTCCCAGATTCACTAG 360
TGATCCTTAA AGTCTCTATTCAGGGAGAGG AAGACACTTTCCATCTCAGAGATAGACTCG 420
TGTTACCTTG ATGGATATTGGATTTGTCTA AGTCTCTTCTAGAAAAAATAAATTCTAGAT 480
TATTAAAP.AA F~~;AAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAA 5
2
4
(2) INFORMATION
FOR SEQ ID N0:12:
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2171 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQ ID
SEQUENCE N0:12:
DESCRIPTION:
CCCCGCTACCGGGTTGCGGCCGGAAGCCGGGCGCCGCGGCTCTGCTTCCCTCGGGGATCT60
GGCGACATGGCCAGAAAGGCTCTCAAGCTTGCTTCGTGGACCAGCATGGCTCTTGCTGCC120
TCTGGCATCTACTTCTACAGTAACAAGTACTTGGACCCTAATGACTTTGGCGCTGTCAGG180
GTGGGCAGAGCAGTTGCTACGACGGCTGTCATCAGTTACGACTACCTCACTTCCCTGAAG240
AGTGTCCCTTATGGCTCAGAGGAGTACTTGCAGCTGAGATCTAAGGTGCACCTTCGCTCT300
GCCAGGCGTCTCTGTGAGCTCTGCTGTGCCAACCGGGGCACCTTCATCAAGGTGGGCCAG360
CACCTGGGGGCTCTGGACTACCTGTTGCCAGAGGAGTACACCAGCACGCTGAAGGTACTG420
CACAGCCAGGCTCCACAGAGCAGCATGCAAGAGATCCGCCAGGTCATCCGAGAAGATCTG480
GGCAAGGAGGTGCTCGTTCTGGCTGTGAAGCAGCTGTTCCCAGAGTTTGAGTTTATGTGG540
CTTGTGGATGAAGCCAAGAAGAACCTGCCTTTGGAGCTGGATTTCCTCAATGAAGGGAGG600
AATGCTGAGAAGGTGTCCCAGATGCTCAGGCATTTTGACTTCTTGAAGGTCCCCCGAATC660
CACTGGGACCTGTCCACGGAGCGGGTCCTCCTGATGGAGTTTGTGGATGGCGGGCAGGTC720
AATGACAGAGACTACATGGAGAGGAACAAGATCGACGTCAATGAGGTGAGGTCAAGAGCT780
CAGGGCTGCTGTGCCGGGGAACGTGGGCTTGGTCAAGGCTGCCCAGGAAGTGCCTGTGTG840
TCCAGATCTCACGCCACCTGGGCAAGATGTATAGTGAGATGATCTTCGTCAATGGCTTCG900
TGCACTGCGATCCCCACCCCGGCAATGTACTGGTGCGGAAGCACCCCGGCACGGGAAAGG960
CGGAGATTGTCCTGTTGGACCATGGGCTTTACCAGATGCTCACGGAAGAATTCCGCCTGA1020
ATTACTGCCACCTCTGGCAGTCTCTGATCTGGACTGACAGGAAGAGAGTGAAGGAGTACA1080
GCCAGCGACTGGGAGCCGGGGATCTCTACCCCTTGTTTGCCTGCATGCTGACGGCGCGAT1140
CGTGGGACTCGGTCAACAGAGGCATCAGCCAAGCTCCCGTCACTGCCACTGAGGACTTAG1200
AGATTCGCAACAACGCGGCCAACTACCTCCCCCAGATCAGCCATCTCCTCAACCACGTGC1260
7g
CA 02285554 1999-09-23
WO 98/42741 PCT/US98/05972
CGCGCCAGATGCTGCTCATC TTGAAGACCAACGACCTGCTGCGTGGCATT GAGGCCGCCC1320
TGGGCACCCGCGCCAGCGCC AGCTCCTTTCTCAi~CATGTCACGTTGCTGC ATCAGAGCGC1380
TAGCTGAGCACAAGAAGAAG AATACCTGTTCAT'PCTTCAGAAGGACCCAG ATCTCTTTCA1440
GCGAGGCCTTCAACTTATGG CAGATCAACCTCCATGAGCTCATCCTGCGT GTGAAGGGGT1500
TGAAGCTGGCTGACCGGGTC TTGGCCCTAATATGCTGGCTGTTCCCTGCT CCACTCTGAG1560
TGGAATTGCTCTCCCTGCCC CATTCTGGTGTCT'rTCCACTCCTCAGCCCC TCATCTTGCC1620
TCCACCCAGCTGCTCCATTT TTGCCACATCGTGGCCCGCAGCCCCAGAGT CACTGTCCAT1680
GTCACCATCCTCCTCCTCCT TTGGAATCCTCTCCGCACACTGTGGCCCTT GTCTCAGGGC1740
CCACAAGCTGAACTGTGGCA TAGCTCTCTCTTC'rTCTCCAAGAAGACTCA GCAGCCTACA1800
TTCCCATTCCTGGTATGTGC CATTGGGTTGGATGTCCCCACTACTTCCGT TAACCCTTCC1860
CATTGTCAAGATGTGCCACG GGTGCCACTGGGGGCACACTGAACTTGTAG GGAGTGTGAT1920
TTTGTTGGAGGTGCACATGG TCTCTGAATTTGA~~AGAGAACACCTTCCCT TTCCTTGCCA1980
TGTCACCCTCCAGAGGAAGT CACACCTCAGCGAGGTGGTTTGGCATCTGG GGCCAACTCC2040
ATTACAGCTATGAGCTCACT GCTGTCAGTGACGTTTGGTGTTTTCTGTAC TGTGTTTCAA2100
TAAAAACTCCTTCAAGGTTG CAAAAAAAAAAAAAAAAAAAAAAAAAAAAA P~AAAAAAAAA2160
A,~~AAAAAAAAA 2171
(2) INFORMATION
FOR
SEQ
ID N0:13:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 271 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Met Ala Arg Lys Ala Leu Lys Le~u Ala Ser Trp Thr Ser Met Ala Leu
1 5 10 15
Ala Ala Ser Gly Ile Tyr Phe Tyr Ser Asn Lys Tyr Leu Asp Pro Asn
20 25 30
Asp Phe Gly Ala Val Arg Val Gl.y Arg Ala Val Ala Thr Thr Ala Val
35 4(1 45
'79
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
Ile Ser Tyr Asp Tyr Leu Thr Ser Leu Lys Ser Val Pro Tyr Gly Ser
50 55 60
Glu Glu Tyr Leu Gln Leu Arg Ser Lys Val His Leu Arg Ser Ala Arg
65 70 75 80
Arg Leu Cys Glu Leu Cys Cys Ala Asn Arg Gly Thr Phe Ile Lys Val
85 90 95
Gly Gln His Leu Gly Ala Leu Asp Tyr Leu Leu Pro Glu Glu Tyr Thr
100 105 110
Ser Thr Leu Lys Val Leu His Ser Gln Ala Pro Gln Ser Ser Met Gln
115 120 125
Glu Ile Arg Gln Val Ile Arg Glu Asp Leu Gly Lys Glu Val Leu Val
130 135 140
Leu Ala Val Lys Gln Leu Phe Pro Glu Phe Glu Phe Met Trp Leu Val
145 150 155 160
Asp Glu Ala Lys Lys Asn Leu Pro Leu Glu Leu Asp Phe Leu Asn Glu
165 170 175
Gly Arg Asn Ala Glu Lys Val Ser Gln Met Leu Arg His Phe Asp Phe
180 185 190
Leu Lys Val Pro Arg Ile His Trp Asp Leu Ser Thr Glu Arg Val Leu
195 200 205
Leu Met Glu Phe Val Asp Gly Gly Gln Val Asn Asp Arg Asp Tyr Met
210 215 220
Glu Arg Asn Lys Ile Asp Val Asn Glu Val Arg Ser Arg Ala Gln Gly
225 230 235 240
Cys Cys Ala Gly Glu Arg Gly Leu Gly Gln Gly Cys Pro Gly Ser Ala
245 250 255
Cys Val Ser Arg Ser His Ala Thr Trp Ala Arg Cys Ile Val Arg
260 265 270
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1613 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02285554 1999-09-23
WO 98/42741 PCT/LTS98/05972
(xi) SEQUENCE
DESCRIPTION:
SEQ 7.D
N0:24:
CATGGCGGCTCCCTTGGTCCTGGTGCTGGTGGT(3GCTGTGACAGTGCGGGCGGCCTTGTT60
CCGCTCCAGTCTGGCCGAGTTCATTTCCGAGCGGGTGGAGGTGGTGTCCCCACTGAGCTC120
TTGGAAGAGAGTGGTTGAAGGCCTTTCACTGTTGGACTTGGGAGTATCTCCGTATTCTGG180
AGCAGTATTTCATGAAACTCCATTAATAATATAC:CTCTTTCATTTCCTAATTGACTATGC240
TGAATTGGTGTTTATGATAACTGATGCACTCACTGCTATTGCCCTGTATTTTGCAATCCA300
GGACTTCAATAAAGTTGTGTTTAAAAAGCAGAAACTCCTCCTAGAACTGGAACAGTATGC360
CCCAGATGTGGCCGAACTCATCCGGACCCCTATGGAAATGCGTTACATCCCTTTGAAAGT420
GGCCCTGTTCTATCTCTTAAATCCTTACACGAT"."TTGTCTTGTGTTGCCAAGTCTACCTG480
TGCCATCAACAACACCCTCA'PTGCTTTCTTCATS"TTGACTACGATAAAAGTTTCATTATC540
TGTAAAATGGGGACAGTAATTGTACCCACTTCATGGAATTATTGAGAAGACTAAATGGCT600
TAAGGCAGTGCTTTCCTCAGTGCTATTTTTCTTGCCTTAGCGACATACCAGTCTCTGAAC660
CCACTCACCTTGTTTGTCCCAGGACTCCTCTATCTCCTCCAGCGGCAGTACATACCTGTG720
AAAATGAAGAGCAAAGCCTTCTGGATCTTTTCT'CGGGAGTATGCCATGATGTATGTGGGA780
AGCCTAGTGGTAATCATTTGCCTCTCCTTCTTC(:TTCTCAGCTCTTGGGATTTCATCCCC840
GCAGTCTATGGCTTTATACTTTCTGTTCCAGATCTCACTCCAAACATTGGTCTTTTCTGG900
TACTTCTTGGCAGAGATGTTTGAGCACTTCAGCCTCTTCTTTGTATGTGTGTTTCAGATC960
AACGTCTTCTTCTACACCATCCCCTTAGCCATAi~AGCTAAATCCTGAGAAACATCTTTGT1020
CCTCACCTGCATCATCATCGTCTGTTCCCTGCTCTTCCCTGTCCTGTGGCACCTCTGGAT1080
TTATGCAGGAAGTGCCAACTCTAATTTCTTTTA'rGCCATCACACTGACCTTCAACGTTGG1140
GCAGATCCTGCTCATCTCTGATTACTTCTATGCCTTCCTGCGGCGGGAGTACTACCTCAC1200
ACATGGCCTCTACTTGACCGCCAAGGATGGCAC.~GAGGCCATGCTCGTGCTCAAGTAGGC1260
CTGGCTGGCACAGGGCTGCATGGACCTCAGGGGGCTGTGGGGCCAGAAGYTGGGCCAAGC1320
CCTCCAGCCAGAGTTGCCAGCAGGCGAGTGCTTGGGCAGAAGAGGTTCGAGTCCAGGGTC1380
ACAAGTCTCTGGTACCAAAAGGGACCCATGGCTGACTGACAGCAAGGCCTATGGGGAAGA1440
ACTGGGAGYTCCCCAACTTGGACCCCCACCTTGTGGCTCTGCACACCAAGGAGCCCCYTC1500
CCAGACAGGAAGGAGAAGAGGCAGGTGAGCAGGGCTTGTTAGATTGTGGCTACTTAATAA1560
ATGTTTTTTGTTATGAAGTCTA,AAAAAAAAAAAAAAAAAAP~~.AAAAAAAAAAA 1613
~i 1
CA 02285554 1999-09-23
WO 98/42741
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
PCT/US98/05972
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Ala Ala Pro Leu Val Leu Val Leu Val Val Ala Val Thr Val Arg
1 5 10 15
Ala Ala Leu Phe Arg Ser Ser Leu Ala Glu Phe Ile Ser Glu Arg Val
20 25 30
Glu Val Val Ser Pro Leu Ser Ser Trp Lys Arg Val Val Glu Gly Leu
35 40 45
Ser Leu Leu Asp Leu Gly Val Ser Pro Tyr Ser Gly Ala Val Phe His
50 55 60
Glu Thr Pro Leu Ile Ile Tyr Leu Phe His Phe Leu Ile Asp Tyr Ala
65 70 75 80
Glu Leu Val Phe Met Ile Thr Asp Ala Leu Thr Ala Ile Ala Leu Tyr
85 90 95
Phe Ala Ile Gln Asp Phe Asn Lys Val Val Phe Lys Lys Gln Lys Leu
100 105 110
Leu Leu Glu Leu Glu Gln Tyr Ala Pro Asp Val Ala Glu Leu Ile Arg
115 120 125
Thr Pro Met Glu Met Arg Tyr Ile Pro Leu Lys Val Ala Leu Phe Tyr
130 135 140
Leu Leu Asn Pro Tyr Thr Ile Leu Ser Cys Val Ala Lys Ser Thr Cys
145 150 155 160
Ala Ile Asn Asn Thr Leu Ile Ala Phe Phe Ile Leu Thr Thr Ile Lys
165 170 175
Val Ser Leu Ser Val Lys Trp Gly Gln
180 185
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 base pairs
82
CA 02285554 1999-09-23
WO 98/42741 PCTlUS98/05972
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ :LD
N0:16:
AAACCCTGTC GGTCTTGGAG CGACGACGGC TCCCTGGCGG TGCGGCGGGG60
AGAACCAGGG
CCGGCGGGTG CAGCGGAAGC GGCGGCGGCG CGTCGCCGGG AACCCTAAGG120
GCG(3CAGTGA
ACTCTGCAAT ATGAATAATT CCCTAGAGAA TTTGAAGAGT ACATCCGAGT180
CACCATCTCC
AAAGGCACGG TCTGTCCCGC AACACAGGAT CTGGACTCAC TGGCCTCTAA240
GAAGGAATTT
GGGGCCAGAA GCCCTTCAGG AGTTCCAGCA ACTACCATGG TGTACCAACA300
GACAGCCACC
GGGTGGGAAC TGCATATACA CAGACAGCAC GGGTCTTTGC TTGAACTTGC360
TGAAGTGGCT
CTGTCCAATC AC 372
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 602 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:17:
CGGGAAGCTCGAAATGGAGAAGGTGAACCTTATGACCCAG ATGTGCTCTACTATATTTTC 60
CTGTGTATTCAAAAGTATCTTTTTGAAAATGGAAGGGTAG ATGACATTTTCTCCGATCTT 120
TATTATGTTCGGTTCACGGAGTGGCTACATGAAGTTCTGA AGGATGTTCAGCCCCGGGTC 280
ACTCCACTTGGCTATGTCTTGCCCAGCCACGTGACTGAGG AGATGCTATGGGAGTGCAAG 240
CAGCTTGGGGCTCACTCCCCCTCCACCTTGCTGACCACCC TCATGTTCTTTAATACCAAG 300
TAAGTGTTCTAGAGGCTCCACTGCTGGCATCTGTCCAGTG AAGAGTGTGGAAACTATCCA 360
AGAGGCCTTCTGAATTCCTCTGACATATATTTGAGAAACT GGGCTACTGAAAGCCCTAAC 420
CCCACTTGGCTGCATTTTATTTGGTAACCAGTGAGGCAAA CACCCTTGCCAGACCCCTAC 480
F~3
CA 02285554 1999-09-23
WO 98/42741
PCT/US98/05972
CATCCATCTT GATGTGGTTC CTGCACTGGA CACTGCTTGG GTACGGGCCT GCCCAGATCT 540 ,
TGGGAATGTG GGCAGTGGCT CCTCTGAAGC ACCAGTGGGC AGAGGATGAG TCATGGTATC 600
CT
602
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Trp Phe Leu His Trp Thr Leu Leu Gly Tyr Gly Pro Ala Gln Ile
1 5 10 15
Leu Gly Met Trp Ala Val Ala Pro Leu Lys His Gln Trp Ala Glu Asp
20 25 30
Glu Ser Trp Tyr Pro
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:19:
TGGGAAAGGGCTTGGACTGT GAAAAGAAATGTGGCCCCTT TCCATCTTCAAGAGAGATGG 60
AATTAATGATGGATGGACCC TGGAGGGAATCTCCCCAGCC GACTTCCACTGGGCTGACAG 120
ACTTTGCTGACCACAGGGGA ACGATGTTCTTTTCTTTCTT CATGATCAGACATAAACTTA 180
GCATTTTAATGGAAGAAAAA TGAGGGGAACTTCAATTATG ATTTATTAAAGACAATTTCT 240
ATTACACCCTCCTTTATGAC AAGTGACATTTTAGATGTAA AAGTAAAAACTTTACCATGC 300
84
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CTTTTTTTTT TTTGTTGGCC TAACATTGAG GCC2'TAAAAC CTGAGGCTCC TGTGCCTGAT 360
GGAATTCTTG TAACATACAC TTGTGTATCA TATP,AAGATA CCACTCTGTT TCTCTTATGT 420
ATTCTTACTC TAGTTGTTTA TTAAGAATGA CAAGCACGTC TTTTCAACAA P~~,AAAAAAAA 480
AAA 483
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1853 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ I:D
N0:20:
CAGATTCGCTGCTGGAGTGCTGGATGGAGCCTT'CCTCTGCCCTCTGTGACATTTCCAATT 60
TTAGATAATGCCTCACATCTCTGTCCCCCCGGGP.CCCCCTGGAGCCCCCATGATCCCTAA 120
GAAGACAGCTTGAACCTAGATCTCACCCCCAGGF~TGTTGCGGAGGCTGCTGGAGCGGCCT 180
TGCACGCTGGCCCTGCTTGTGGGCTCCCAGCTGG~CTGTCATGATGTACCTGTCACTGGGG 240
GGCTTCCGAAGTCTCAGTGCCCTATTTGGCCGAC:ATCAGGGACCGACATTTGACTATTCT 300
CACCCTCGTGATGTCTACAGTAACCTCAGTCACC.'TGCCTGGGGCCCCAGGGGGTCCTCCA 360
GCTCCTCAAGGTCTGCCCTACTGTCCAGAACGA'I'CTCCTCTCTTAGTGGGTCCTGTGTCG 420
GTGTCCTTTAGCCCAGTGCCATCACTGGCAGAGATTGTGGAGCGGAATCCCCGGGTAGAA 480
CCAGGGGGCCGGTACCGCCCTGCAGGTTGTGAGC,'CCCGCTCCCGAACAGCCATCATTGTG 540
CCTCATCGTGCCCGGGAGCACCACCTGCGCCTGC'.TGCTCTACCACCTGCACCCCTTCTTG 600
CAGCGCCAGCAGCTTGCTTATGGCATCTATGTCATCCACCAGGCTGGAAATGGAACATTT 660
AACAGGGCAAAACTGTTGAACGTTGGGGTGCGAGAGGCCCTGCGTGATGAAGAGTGGGAC 720
TGCCTGTTCTTGCACGATGTGGACCTCTTGCCAGAAAATGACCACAATCTGTATGTGTGT 780
GACCCCCGGGGACCCCGCCATGTTGCCGTTGCTATGAACAAGTTTGGATACAGCCTCCCG 840
TACCCCCAGTACTTCGGAAGAGTCTCAGCACTTACTCCTGACCAGTACCTGAAGATGAAT 900
GGCTTCCCCAATGAATACTGGGGCTGGGGTGGTCJAGGATGACGACTTGCTACCAGGGTGC 960
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GCCTGGCTGGGATGAAGATC TCTCGGCCCCCCACATCTGTAGGACACTAT AAGATGGTGA1020
AGCACCGAGGAGATAAGGGC AATGAGGAAAATCCCCACAGATTTGACCTC CTGGTCCGTA1080
CCCAGAATTCCTGGACGCAA GATGGGATGAACTCACTGACATACCAGTTG CTGGCTCGAG1140
AGCTGGGGCCTCTTTATACC AACATCACAGCAGACATTGGGACTGACCCT CGGGGTCCTC1200
GGGCTCCTTCTGGGCCACGT TACCCACCTGGTTCCTCCCAAGCCTTCCGT CAAGAGATGC1260
TGCAACGCCGGCCCCCAGCC AGGCCTGGGCCTCTATCTACTGCCAACCAC ACAGCCCTCC1320
GAGGTTCACACTGACTCCTC CTTCCTGTCTACCTTAATCATGAAACCGAA TTCATGGGGT1380
TGTATTCTCCCCACCCTCAG CTCCTCACTGTTCTCAGAAGGATGTGAGGG AACTGAACTC1440
TGGTGCCGTGCTAGGGGGTA GGGGCCTCTCCCTCACTGCTGGACTGGAGC TGGGCTCCTG1500
TAGACCTGAGGGTCCNTCTY TCTAGGTCTCCTGTAGGGCTTAKGACTGTG AATCCTTGAT1560
GTCATGATTTTATGTGACGA TTCCTAGGAGTCCCTGCCCCTAGAGTAGGA GCAGGGYTGG1620
ACCCCAAGCCCNTCCYTYTT CCATGGAGAGAAGAGTGATCTGGYTTCTCC TCGGACCTCT1680
GTGAATATTTATTCTATTTA TGGTTCCCGGGAAGTTGTTTGGTGAAGGAA GCCCCTCCCC1740
TGGGCATTTTCTGCCTATGC TGGAATAGCTCCCTCTTCTGGTCCTGGCTC AGGGGGCTGG1800
GATTTTGATATATTTTCTAA TAAAGGACTTTGTCTCGCAAP~~,AAAAAAAA AAA 1853
(2) INFORMATION :
FOR
SEQ
ID N0:21
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 273 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:21:
MetLeu ArgArgLeu LeuGlu Arg Cys ThrLeuAla LeuLeuVal
Pro
2 5 10 15
GlySer GlnLeuAla ValMet Met Leu SerLeuGly GlyPheArg
Tyr
20 25 30
SerLeu SerAlaLeu PheGly Arg Gln GlyProThr PheAspTyr
Asp
35 40 45
SerHis ProArgAsp ValTyr Ser Leu SerHisLeu ProGlyAla
Asn
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50 55 60
Pro Gly Gly Pro Pro Ala Pro Gln Gly Leu Pro Tyr Cys Pro Glu Arg
65 70 75 80
Ser Pro Leu Leu Val Gly Pro Val Ser Val Ser Phe Ser Pro Val Pro
85 90 95
Ser Leu Ala Glu Ile Val Glu Arg Asn Pro Arg Val Glu Pro Gly Gly
100 105 110
Arg Tyr Arg Pro Ala Gly Cys Glu Pro Arg Ser Arg Thr Ala Ile Ile
115 120 125
Val Pro His Arg Ala Arg Glu His His Leu Arg Leu Leu Leu Tyr His
130 135 140
Leu His Pro Phe Leu Gln Arg Glr.. Gln Leu Ala Tyr Gly Ile Tyr Val
145 150 155 160
Ile His Gln Ala Gly Asn Gly Thr Phe Asn Arg Ala Lys Leu Leu Asn
165 170 175
Val Gly Val Arg Glu Ala Leu Arcr Asp Glu Glu Trp Asp Cys Leu Phe
180 185 190
Leu His Asp Val Asp Leu Leu Pro Glu Asn Asp His Asr. Leu Tyr Val
195 200 205
Cys Asp Pro Arg Gly Pro Arg His; Val Ala Val A1a Met Asn Lys Phe
210 215 220
Gly Tyr Ser Leu Pro Tyr Pro Gln Tyr Phe Gly Arg Val Ser Ala Leu
225 230 235 240
Thr Pro Asp Gln Tyr Leu Lys Met. Asn Gly Phe Pro Asn Glu Tyr Trp
245 250 255
Gly Trp Gly Gly Glu Asp Asp Asp Leu Leu Pro Gly Cys Ala Trp Leu
260 265 270
Gly
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1686 base paixs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
g7
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(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:22:
AGATAAAGTAAGTGCTGTTTGGGCTAACAGGATCTCCTCTTGCAGTCTGCAGCCCAGGAC 60
GCTGATTCCAGCAGCGCCTTACCGCGCAGCCCGAAGATTCACTATGGTGAAAATCGCCTT 120
CAATACCCCTACCGCCGTGCAAAAGGAGGAGGCGCGGCAAGACGTGGAGGCCCTCCTGAG 180
CCGCACGGTCAGAACTCAGATACTGACCGGCAAGGAGCTCCGAGTTGCCACCCAGGAAAA 240
AGAGGGCTCCTCTGGGAGATGTATGCTTACTCTCTTAGGCCTTTCATTCATCTTGGCAGG 300
ACTTATTGTTGGTGGAGCCTGCATTTACAAGTACTTCATGCCCAAGAGCACCATTTACCG 360
TGGAGAGATGTKCTTTTTTGATTCTGAGGATCCTGCAAATTCCCTTCGTGGAGGAGAGCC 420
TAACTTCCTGCCTGTGACTGAGGAGGCTGACATTCGTGAGGATGACAACATTGCAATCAT 480
TGATGTGCCTGTCCCCAGTTTCTCTGATAGTGACCCTGCAGCAATTATTCATGACTTTGA 540
AAAGGGAATGACTGCTTACCTGGACTTGTTGCTGGGGAACTGCTATCTGATGCCCCTCAA 600
TACTTCTATTGTTATGCCTCCAAAAAATCTGGTAGAGYTCTTTGGCAAACTGGCGAGTGG 660
CAGATATCTGCYTCAAACTTATGTGGTTCGAGAAGACCTAGTTGCTGTGGAGGAAATTCG 720
TGATGTTAGTAACCTTGGCATCTTTATTTACCAACTTTGCAATAACAGAAAGTCCTTCCG 780
CCTTCGTCGCAGAGACCTCTTGCTGGGTTTCAACAAACGTGCCATTGATAAATGCTGGAA 840
GATTAGACACTTCCCCAACGAATTTATTGTTGAGACCAAGATCTGTCAAGAGTAAGAGGC 900
AACAGATAGAGTGTCCTTGGTAATAAGAAGTCAGAGATTTACAATATGACTTTAACATTA 960
AGGTTTATGGGATACTCAAGATATTTACTCATGCATTTACTCTATTGCTTATGCTTTAAA 1020
AAAAGGAAAAGAAAAAAACTACTAACCACTGCAAGCTCTTGTCAAATTTTAGTTTAATTG 1080
GCATTGCTTGTTTTTTGAAACTGAAATTACATGAGTTTCATTTTTTCTTTGAATTTATAG 1140
GGTTTAGATTTCTGAAAGCAGCATGAATATATCACCTAACATCCTGACAATAAATTCCAT 1200
CCGTTGTTTTTTTTGTTTGTTTGTTTTTTCTTTTCCTTTAAGTAAGCTCTTTATTCATCT 1260
TATGGTGCAGCAATTTTAAAATTTGAAATATTTTAAATTGTTTTTGAACTTTTTGTGTAA 1320
AATATATCAGATCTCAACATTGTTGGTTTCTTTTGTTTTTCATTTTGTACAACTTTCTTG 1380
AATTTAGAAATTACATCTTTGCAGTTCTGTTAGGTGCTCTGTAATTAACCTGACTTATAT 1440
GTGAACAATTTTCATGAGACAGTCATTTTTAACTAATGCAGTGATTCTTTCTCACTACTA 1500
TCTGTATTGTGGAATGCACAAAATTGTGTAGGTGCTGAATGCTGTAAGGAGTTTAGGTTG 1560
gg
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TATGAATTCT ACAACCCTAT AATAAATTTT ACS~CTATAAA AA,AAAAAAAA AAAAAAAAAA 1620
P,1~;~1AAAAAAA P,~~AAAAAAAA P,~~~AAAP.AAA AA~~,AAAAAAA A,~~~AAAAAAA AAAAAAAAAA
16 8 0
AAAAAA 1686
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 263 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Met Val Lys Ile Ala Phe Asn Tlzr Pro Thr Ala Val Gln Lys Glu Glu
1 5 10 15
Ala Arg Gln Asp Val Glu Ala L~au Leu Ser Arg Thr Val Arg Thr Gln
20 25 30
Ile Leu Thr Gly Lys Glu Leu A:rg Val Ala Thr G1r_ Glu Lys Glu Gly
35 40 45
Ser Ser Gly Arg Cys Met Leu T:nr Leu Leu Gly Leu Ser Phe Ile Leu
50 55 60
Ala Gly Leu Ile Val Gly Gly Ala Cys Ile Tyr Lys Tyr Phe Met Pro
65 70 75 80
Lys Ser Thr Ile Tyr Arg Gly Glu Met Xaa Phe Phe Asp Ser Glu Asp
85 90 95
Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro Asn Phe Leu Pro Val Thr
100 105 110
Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn Ile Ala Ile Ile Asp Val
115 120 125
Pro Val Pro Ser Phe Ser Asp Ser Asp Pro Ala Ala Ile Ile His Asp
130 135 140
Phe Glu Lys Gly Met Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys
145 150 155 160
Tyr Leu Met Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn Leu
165 170 175
Val Glu Xaa Phe Gly Lys Leu Ala Ser Gly Arg Tyr Leu Xaa Gln Thr
89
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180 285 190
Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp Val
195 200 205
Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn Asn Arg Lys Ser
210 215 220
Phe Arg Leu Arg Arg Arg Asp Leu Leu Leu Gly Phe Asn Lys Arg Ala
225 230 235 240
Ile Asp Lys Cys Trp Lys Ile Arg His Phe Pro Asn Glu Phe Ile Val
245 250 255
Glu Thr Lys Ile Cys Gln Glu
260
(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:
TNCACATTCTC AGTGGGAACT TGATGAAC 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
ANATATAGGTG GAATGAATTC TATCCTTG 2g
(2) INFORMATION FOR SEQ ID N0:26:
CA 02285554 1999-09-23
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pair.
(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:
GNTATAGTAAT AATAGCACAA AGGACGGG 2g
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pair..
(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:
TNGCCAGGAAA CCAAATCAAT GACTCTTT 29
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pair:a
(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:
TNTAATTGACG GTGTAGAGAA ATGAGGAA 29
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ANAAATGGAGC AGCTGGGTGG AGGCAAGA 29
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
TNCGGAGATAC TCCCAAGTCC AACAGTGA 29
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GNTTAGGGCTT TCAGTAGCCC AGTTTCTC 29
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
92
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
ANTGACAGGTA CATCATGACA GCCAGCTG 29
(2) INFORMATION FOR SEQ ID N0:33:
(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 N0:33:
CNGGATGTTAG GTGATATATT CATGCTGC 29
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 264 amino acids
(B) TYPE: amino acid
(C} STRANDEDNESS:
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Gly Glu Val Lys Ser Ser Gly Leu Leu Cys Arg Gly Thr Trp Ala Trp
1 5 10 15
Ser Arg Leu Pro Arg Lys Cys L~~u Cys Val Gln Ile Ser Arg His Leu
20 25 30
Gly Lys Met Tyr Ser Glu Met Ile Phe Val Asn Gly Phe Val His Cys
35 40 45
93
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Asp Pro His Pro Gly Asn Val Leu Val Arg Lys His Pro Gly Thr Gly .
SO 55 60
Lys Ala Glu Ile Val Leu Leu Asp His Gly Leu Tyr Gln Met Leu Thr
65 70 75 80
Glu Glu Phe Arg Leu Asn Tyr Cys His Leu Trp Gln Ser Leu Ile Trp
85 90 95
Thr Asp Arg Lys Arg Val Lys Glu Tyr Ser Gln Arg Leu Gly Ala Gly
100 105 110
Asp Leu Tyr Pro Leu Phe Ala Cys Met Leu Thr Ala Arg Ser Trp Asp
115 120 125
Ser Val Asn Arg Gly Ile Ser Gln Ala Pro Val Thr Ala Thr Glu Asp
130 135 140
Leu Glu Ile Arg Asn Asn Ala Ala Asn Tyr Leu Pro Gln Ile Ser His
145 150 155 160
Leu Leu Asn His Val Pro Arg Gln Met Leu Leu Ile Leu Lys Thr Asn
165 170 175
Asp Leu Leu Arg Gly Ile Glu Ala Ala Leu Gly Thr Arg Ala Ser Ala
180 185 190
Ser Ser Phe Leu Asn Met Ser Arg Cys Cys Ile Arg Ala Leu Ala Glu
195 200 205
His Lys Lys Lys Asn Thr Cys Ser Phe Phe Arg Arg Thr Gln Ile Ser
210 215 220
Phe Ser Glu Ala Phe Asn Leu Trp Gln Ile Asn Leu His Glu Leu Ile
225 230 235 240
Leu Arg Val Lys Gly Leu Lys Leu Ala Asp Arg Val Leu Ala Leu Ile
245 250 255
Cys Trp Leu Phe Pro Ala Pro Leu
260
94
