Note: Descriptions are shown in the official language in which they were submitted.
CA 02305689 2000-03-29
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/090,100
(converted to a provisional application from non-provisional application Ser.
No.
08/942,813), filed October 2,1997, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA/amino acid sequence of
the protein
2 0 in the case of hybridization cloning; activity of the protein in the case
of expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 25 to nucleotide 1458;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 21 to nucleotide 730;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bd107_16 deposited under accession
number ATCC 98898;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bd107_16 deposited under accession number ATCC 98898;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone bd107_16 deposited under accession number
ATCC 98898;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone bd107_16 deposited under accession number ATCC 98898;
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 eight consecutive amino acids of SEQ ID N0:2;
2 5 (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 that hybridizes under stringent conditions to any
3 0 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:1 from nucleotide 25 to nucleotide 1458; the nucleotide sequence of SEQ ID
NO:1
from nucleotide 21 to nucleotide 730; the nucleotide sequence of the full-
length protein
coding sequence of clone bd107_16 deposited under accession number ATCC 98898;
or
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the nucleotide sequence of a mature protein coding sequence of clone bd107_16
deposited
under accession number ATCC 98898. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone bd107_I6 deposited under accession number ATCC 98898. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 2 to amino
acid 118.
In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:2
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:2, or 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 234 to amino acid 243 of SEQ ID N0:2.
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;
2 0 (b) the amino acid sequence of SEQ ID N0:2 from amino acid 2 to
amino acid 118;
(c) fragments of the amino acid sequence of SEQ ID N0:2 comprising
eight consecutive amino acids of SEQ ID N0:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 5 bd107_16 deposited under accession number ATCC 98898;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 2 to amino acid 118. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
3 0 sequence of SEQ ID N0:2 having biological activity, the fragment
preferably comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
N0:2, or 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 234 to amino acid 243 of SEQ ID N0:2.
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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
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 6 to nucleotide 977;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 87 to nucleotide 977;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 8 to nucleotide 630;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bm41 7 deposited under accession
number ATCC 98898;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bm41 7 deposited under accession number ATCC 98898;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone bm41 7 deposited under accession number
ATCC 98898;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone bm41 7 deposited under accession number ATCC 98898;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
2 5 comprising eight consecutive amino acids of SEQ ID N0:4;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleoHde which encodes a species homologue of the protein
of (i) or (j) above ; and
3 0 (m) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 6 to nucleotide 977; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 87 to nucleotide 977; the nucleotide sequence of SEQ ID N0:3 from
nucleotide
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8 to nucleotide 630; the nucleotide sequence of the full-length protein coding
sequence of
clone bm41_7 deposited under accession number ATCC 98898; or the nucleotide
sequence
of a mature protein coding sequence of clone bm41 7 deposited under accession
number
ATCC 98898. In other preferred embodiments, the polynucieotide encodes the
full-length
or a mature protein encoded by the cDNA insert of clone bm41 7 deposited under
accession number ATCC 98898. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising the amino
acid
sequence of SEQ ID N0:4 from amino acid 211 to amino acid 315. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:4 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:4, or 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 157
to amino acid 166 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 211 to
amino acid 315;
(c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
2 5 eight consecutive amino acids of SEQ ID N0:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone
bm41 7 deposited under accession number ATCC 98898;
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
3 0 of SEQ ID N0:4 from amino acid 211 to amino acid 315. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:4, or a protein comprising a fragment of the amino acid
sequence of
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SEQ ID N0:4 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 157 to amino acid 166 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 168 to nucleotide 962;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 351 to nucleotide 962;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone br342_ll deposited under accession
number ATCC 98551;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone br342_ll deposited under accession number ATCC 98551;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone br342_ll deposited under accession number
ATCC 98551;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone br342_l l deposited under accession number ATCC 98551;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
2 5 comprising eight consecutive amino acids of SEQ ID N0:6;
(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 that hybridizes under stringent conditions to any
one of the polynucieotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 168 to nucleotide 962; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 351 to nucleotide 962; the nucleotide sequence of the full-length
protein coding
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sequence of clone br342_ll deposited under accession number ATCC 98551; or the
nucleotide sequence of a mature protein coding sequence of clone br342_ll
deposited
under accession number ATCC 98551. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone br342_11 deposited under accession number ATCC 98551. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:6 from amino acid 1 to amino
acid 78.
In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:6
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:6, or 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 127 to amino acid 136 of SEQ ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
2 0 (a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ ID N0:6 from amino acid 1 to
amino acid 78;
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
eight consecutive amino acids of SEQ ID N0:6; and
2 5 (d} the amino acid sequence encoded by the cDNA insert of clone
br342_ll deposited under accession number ATCC 98551;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6 or the amino acid
sequence
of SEQ ID N0:6 from amino acid 1 to amino acid 78. In further preferred
embodiments,
3 0 the present invention provides a protein comprising a fragment of the
amino acid
sequence of SEQ ID N0:6 having biological activity, the fragment preferably
comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
N0:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:6
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having biological activity, the fragment comprising the amino acid sequence
from amino
acid 127 to amino acid 136 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 134 to nucleotide 493;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ej258 11 deposited under accession
number ATCC 98551;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ej258_ll deposited under accession number ATCC 98551;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ej258_ll deposited under accession number
ATCC 98551;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ej258_ll deposited under accession number ATCC 98551;
(g) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:8;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:8;
(i) a polynucleotfde which is an allelic variant of a polynucleotide of
2 5 (a)-(f) above;
{j) a polynucleotide which encodes a species homologue of the protein
of {g) or (h) above ; and
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(h}.
3 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:7 from nucleotide 134 to nucleotide 493; the nucleotide sequence of the
full-length
protein coding sequence of clone ej258_ll deposited under accession number
ATCC
98551; or the nucleotide sequence of a mature protein coding sequence of clone
ej258 11
deposited under accession number ATCC 98551. In other preferred embodiments,
the
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polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ej258_11 deposited under accession number ATCC 98551. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino
acid 64.
In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:8
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:8, or a
polynucleotide encoding a protein comprising a fragment of the amino acid
sequence of
SEQ iD NO:B having biological activity, the fragment comprising the amino acid
sequence
from amino acid 55 to amino acid 64 of SEQ ID N0:8.
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) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to
amino acid 64;
2 0 (c) fragments of the amino acid sequence of SEQ ID N0:8 comprising
eight consecutive amino acids of SEQ ID N0:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone
ej258_ll deposited under accession number ATCC 98551;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 protein comprises the amino acid sequence of SEQ ID N0:8 or the amino acid
sequence
of SEQ ID N0:8 from amino acid 1 to amino acid 64. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
sequence of SEQ ID N0:8 having biological activity, the fragment preferably
comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
3 0 N0:8, or a protein comprising a fragment of the amino acid sequence of SEQ
ID NO:B
having biological activity, the fragment comprising the amino acid sequence
from amino
acid 55 to amino acid 64 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
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(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 14 to nucleotide 406;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 62 to nucleotide 406;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone k232_2x deposited under accession
number ATCC 98551;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone k232 2x deposited under accession number ATCC 98551;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone k232 2x deposited under accession number
ATCC 98551;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone k232_2x deposited under accession number ATCC 98551;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID NO:10 having biological activity, the
fragment
comprising eight consecutive amino acids 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
2 5 of (h) or (i) above ; and
(1) a polynucleotide that hybridizes 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 14 to nucleotide 406; the nucleotide sequence of SEQ ID
N0:9 from
3 0 nucleotide 62 to nucleotide 406; the nucleotide sequence of the full-
length protein coding
sequence of clone k232 2x deposited under accession number ATCC 98551; or the
nucleotide sequence of a mature protein coding sequence of clone k232_2x
deposited
under accession number ATCC 98551. In other preferred .embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
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of clone 1c232 2x deposited under accession number ATCC 98551. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino
acid
81. In further preferred embodiments, the present invention provides a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
NO:10
having biological activity, the fragment preferably comprising eight (more
preferably
twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:IO, or a
poiynucleotide 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 60 to amino acid 69 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID N0:10 from amino acid 1 to
amino acid 81;
(c) fragments of the amino acid sequence of SEQ ID NO:10 comprising
2 0 eight consecutive amino acids of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
kZ32_2x deposited under accession number ATCC 98551;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid
sequence
2 5 of SEQ ID NO:10 from amino acid 1 to amino acid 81. In further preferred
embodiments,
the present invention provides a protein comprising a fragment of the amino
acid
sequence of SEQ ID NO:10 having biological activity, the fragment preferably
comprising
eight (more preferably twenty, most preferably thirty) consecutive amino acids
of SEQ ID
NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID
NO:10
3 0 having biological activity, the fragment comprising the amino acid
sequence from amino
acid 60 to amino acid 69 of SEQ ID N0:10.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
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(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 580 to nucleotide 816;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone 1f307_5 deposited under accession
number
ATCC 98551;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone 1f307_5 deposited under accession number ATCC 98551;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone 1f307 5 deposited under accession number ATCC
98551;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone 1f307_5 deposited under accession number ATCC 98551;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:12;
2 0 (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 that hybridizes under stringent conditions to any
2 5 one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:11 from nucleotide 580 to nucleotide 816; the nucleotide sequence of the
full-length
protein coding sequence of clone 1f307_5 deposited under accession number ATCC
98551;
or the nucleotide sequence of a mature protein coding sequence of clone
1f307_5 deposited
3 0 under accession number ATCC 98551. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone 1f307_5 deposited under accession number ATCC 98551. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:12 having
biological
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activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:12, or a
polynucleotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 34
to amino acid 43 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:11 or SEQ ID N0:13.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:12;
(b) fragments of the amino acid sequence of SEQ ID N0:12 comprising
eight consecutive amino acids of SEQ ID N0:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
1f307_5 deposited under accession number ATCC 98551;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
preferably
2 0 comprising eight (more preferably twenty, most preferably thirty)
consecutive amino
acids of SEQ ID N0:12, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:12 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 34 to amino acid 43 of SEQ ID N0:12.
In one embodiment, the present invention provides a composition comprising an
2 5 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 127 to nucleotide 627;
3 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 250 to nucleotide 627;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone 1r204_1 deposited under accession
number ATCC 98551;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone 1r204_1 deposited under accession number ATCC 98551;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone 1r204_1 deposited under accession number
ATCC 98551;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone 1r204_1 deposited under accession number ATCC 98551;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:15;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:15;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
2 0 N0:14 from nucleotide 127 to nucleotide 627; the nucleotide sequence of
SEQ ID N0:14
from nucleotide 250 to nucleotide 627; the nucleotide sequence of the full-
length protein
coding sequence of clone 1r204_1 deposited under accession number ATCC 98551;
or the
nucleotide sequence of a mature protein coding sequence of clone 1r204_1
deposited under
accession number ATCC 98551. In other preferred embodiments, the
polynucleotide
2 5 encodes the full-length or a mature protein encoded by the cDNA insert of
clone 1r204_1
deposited under accession number ATCC 98551. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:15 from amino acid 23 to amino acid 106. In further
preferred embodiments, the present invention provides a polynucleotide
encoding a
3 0 protein comprising a fragment of the amino acid sequence of SEQ ID N0:15
having
biological activity, the fragment preferably comprising eight (more preferably
twenty,
most preferably thirty) consecutive amino acids of SEQ ID N0:15, or a
polynucleotide
encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:15
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having biological activity, the fragment comprising the amino acid sequence
from amino
acid 78 to amino acid 87 of SEQ ID N0:15.
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) the amino acid sequence of SEQ ID N0:15 from amino acid 23 to
amino acid 106;
(c) fragments of the amino acid sequence of SEQ ID N0:15 comprising
eight consecutive amino acids of SEQ ID N0:15; and
(d) the amino acid sequence encoded by the cDNA insert of clone
1r204_1 deposited under accession number ATCC 98551;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:15 or the amino acid
sequence
of SEQ ID N0:15 from amino acid 23 to amino acid 106. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:15 having biological activity, the fragment
preferably
2 0 comprising eight {more preferably twenty, most preferably thirty)
consecutive amino
acids of SEQ ID NO:15, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:15 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 78 to amino acid 87 of SEQ ID N0:15.
In certain preferred embodiments, the polynucleotide is operably linked to an
2 5 expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
3 0 Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
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The protein produced according to such methods is also provided by the present
invention.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
2 0 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
2 5 determined to be the reading frame best identifiable with sequence
information available
at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable
host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
3 0 proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
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Close "bd107 16"
A polynucleotide of the present invention has been identified as clone
"bd107_16".
bd107_16 was isolated from a human fetal kidney cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. bd107_I6 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "bd107_16 protein").
The nucleotide sequence of bd107_16 as presently determined is reported in SEQ
ID NO:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the bd107_16 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
bd107_16 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for bd107_16 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bd107_16 demonstrated at least some similarity with
sequences
identified as AA261841 (zs17h09.r1 NCI CGAP_GCB1 Homo sapiens cDNA clone
IMAGE:685505 5'), AA424094 (zv80d05.r1 Soares total fetus Nb2HF8 9w Homo
sapiens
2 0 cDNA clone 759945 5'), AA449717 (zx09b06.s1 Soares total fetus Nb2HF8 9w
Homo
Sapiens cDNA clone 785939 3' similar to TR:E246888 E246888 CHROMOSOME XVI
READING FRAME ORF YPL146C), AA875866 (ob34d08.s1 NCI CGAP_Kids Homo
Sapiens cDNA clone IMAGE:1325583 3' similar to TR:Q12080 Q12080 P2610), H80410
(yu97b09.r1 Homo sapiens cDNA clone 241145 5'), N39747 (yx92h07.r1 Homo
Sapiens
2 5 cDNA clone 269245 5'), 897655 (yq59d12.r1 Homo sapiens cDNA clone 200087
5'), T19822
(Human gene signature HUMGS00904; standard; cDNA to mRNA), and W68551
(zd36h03.r1 Soares fetal heart NbHHI9W Homo Sapiens cDNA clone 342773 5'). The
predicted amino acid sequence disclosed herein for bd107_16 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
3 0 The predicted bd107_16 protein demonstrated at least some similarity to
the sequence
identified as U43703 (Lpi2p [Saccharomyces cerevisiae]). Based upon sequence
similarity,
bd107_16 proteins and each similar protein or peptide may share at least some
activity.
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CloBe "bm41 7"
A polynucleotide of the present invention has been identified as clone "bm41
7".
bm41 7 was isolated from a human adult muscle 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. bm41 7 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"bm41 7 protein").
The nucleotide sequence of bm41 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 bm41 7 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:4. Amino acids 15 to 27 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 28, or
are within a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bm41 7 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for bm41 7 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bm41 7 demonstrated at least some similarity with
sequences
2 0 identified as AF047439 (Homo sapiens unknown mRNA, complete cds), H44519
(yo74d10.r1 Homo sapiens cDNA clone 183667 5'), N29833 (yw93d10.s1 Homo
sapiens
cDNA clone 259795 3'), T23021 (Human gene signature HUMGS04750; standard; cDNA
to mRNA), W58059 (zd22f10.r1 Soares fetal heart NbHHI9W Homo Sapiens cDNA
clone),
and 278368 (H.sapiens mItNA, expressed sequence tag ICRFp507F18226, mRIVA
2 5 sequence). The predicted amino acid sequence disclosed herein for bm41 7
was searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted bm41 7 protein demonstrated at least some
similarity to
the sequence identified as AF047439 (unknown [Homo sapiens]). Based upon
sequence
similarity, bm41 7 proteins and each similar protein or peptide may share at
least some
3 0 activity. The TopPredII computer program predicts an additional potential
trans-
membrane domain within the bm41 7 protein sequence centered around' amino acid
252
of SEQ ID N0:4.
1$
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Clone"br342 11"
A polynucleotide of the present invention has been identified as clone
"br342_11".
br342_11 was isolated from a human fetal kidney cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. br342_11 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "br342_11 protein').
The nucleotide sequence of br342_11 as presently determined is reported in SEQ
ID N0:5. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the br342_11 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6. Amino acids 49 to 61 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 62, or are within a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
br342_ll should be approximately 1400 bp.
The nucleotide sequence disclosed herein for br342_ll was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. br342_l l demonstrated at least some similarity with
sequences
2 0 identified as 269722 (Human DNA sequence from cosmid U212C1, between
markers
DXS366 and DXS87 on chromosome X), 293019 Human DNA sequence *** SEQUENCING
IN PROGRESS *** from clone 49C23; HTGS phase 1; Human DNA sequence from PAC
49C23 on chromosome X contains malate dehydrogenase pseudogene and STS), and
295126 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 30P20;
2 5 HTGS phase 1; Human DNA sequence from PAC 30P20 on chromosome Xq21.1-
Xq21.3.
Contains set pseudogene, ESTs and STS). The predicted amino acid sequence
disclosed
herein for br342_11 was searched against the GenPept and GeneSeq amino acid
sequence
databases using the BLASTX search protocol. The predicted br342_11 protein
demonstrated at least some similarity to sequences identified as D89049
(lectin-like
3 0 oxidized LDL receptor [Bos taurus]) and 899586 (Low density lipoprotein
receptor).
Based upon sequence similarity, br342_11 proteins and each similar protein or
peptide
may share at least some activity. The nucleotide sequence of br342_11
indicates that it
may contain one or more of the following repetitive elements: MER4A, MER4B.
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Clone "ej258 11"
A polynucleotide of the present invention has been identified as clone
"ej258_ll".
ej258_ll 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. ej258_11 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ej258_ll protein").
The nucleotide sequence of ej258_11 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the ej258_11 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ej258_11 should be approximately 670 bp.
The nucleotide sequence disclosed herein for ej258_ll was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ej258_l l demonstrated at least some similarity with
sequences
identified as AA217161 (mu86g11.r1 Soares mouse lymph node NbMLN Mus musculus
cDNA clone 652484 5' similar to WP:F35H12.2 CE04511), AA330720 (EST34452
Embryo,
2 0 6 week I Homo sapiens cDNA 5' end), AJ000649 (Oryctolagus cuniculus
unknown
differentially expressed mRNA), U17432 (Bos taurus beta-mannosidase mRNA,
complete
cds), U91321 (Human chromosome 16p13 BAC clone CIT987SK-363E6, complete
sequence), 274031 (Caenorhabditis elegans cosmid F32D8), and 299127 (Human DNA
sequence *** SEQUENCING IN PROGRESS *** from clone 102620; HTGS phase 1; Human
DNA sequence from PAC 102620 on chromosome 1q24-q25. Contains ESTS, STSs and a
predicted CpG island). The predicted amino acid sequence disclosed herein for
ej258_ll
was searched against the GenPept and GeneSeq amino acid sequence databases
using the
BLASTX search protocol. The predicted ej258_ll protein demonstrated at least
some
similarity to the sequence identified as U41540 (coded for by C. elegans cDNA
yk42d 12.5;
3 0 coded for by C. elegans cDNA yk27e 10.5; coded for by C. elegans cDNA
cm08h6; coded
for by C. elegans cDNA yk88e 12.5). Based upon sequence similarity, ej258_11
proteins
and each similar protein or peptide may share at least some activity.
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Cane "k232 2x"
A polynucleotide of the present invention has been identified as clone "k232
2x".
A cDNA clone was first isolated from a murine adult bone marrow 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. This
murine
cDNA was then used to isolate k232_2x, a full-length human cDNA clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"k232_2x protein').
The nucleotide sequence of k232_2x as presently determined is reported in SEQ
ID N0:9. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the k232 2x protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID NO:10. Amino acids 4 to 16 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 17, or are within a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
k232_2x should be approximately 555 bp.
The nucleotide sequence disclosed herein for k232_2x was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. k232_2x demonstrated at least some similarity with
sequences
2 0 identified as AA087828 (mn94b04.r1 Stratagene mouse lung 937302 Mus
musculus cDNA
clone 551695 5'), AA095731 (15720.seq.F Fetal heart, Lambda ZAP Express Homo
Sapiens
cDNA 5'), AA398859 (zt80e12.r1 Soares testis NHT Homo Sapiens cDNA clone
728686 5'),
N78829 (zb17a05.s1 Homo Sapiens cDNA clone 302288 3'), T21965 (Human gene
signature
HUMGS03508), and W17346 (zb18c05.r1 Soares fetal lung NbHLI9W Homo sapiens
2 5 cDNA clone 302408 5'). Based upon sequence similarity, k232 2x proteins
and each
similar protein or peptide may share at least some activity. The TopPredII
computer
program predicts two potential transmembrane domains within the k232_2x
protein
sequence, one near the signal sequence and another near the C-terminus of SEQ
ID NO:10.
3 0 Clone "1f307 5"
A polynucleotide of the present invention has been identified as clone "1f307
5".
1f307_5 was isolated from a human adult spinal cord cDNA library using methods
which
are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
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analysis of the amino acid sequence of the encoded protein. 1f307 5 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"1f307 5 protein').
The nucleotide sequence of the 5' portion of 1f307_5 as presently determined
is
reported in SEQ ID NO:11. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID NOa2. The predicted amino acid
sequence
of the 1f307_5 protein corresponding to the foregoing nucleotide sequence is
reported in
SEQ ID N0:12. Additional nucleotide sequence from the 3' portion of 1f307 5,
including
the polyA tail, is reported in SEQ ID N0:13.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
1f307_5 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for If307 5 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. 1f307_5 demonstrated at least some similarity with
sequences
identified as AA039895 (zk46a02.r1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
clone 485834 5') and AA513783 (nh89a05.r1 NCI_CGAP_Brl.1 Homo Sapiens cDNA
clone
965648). Based upon sequence similarity,1f307_5 proteins and each similar
protein or
peptide may share at least some activity. The TopPredII computer program
predicts a
potential transmembrane domain within the 1f307_5 protein sequence centered
around
2 0 amino acid 50 of SEQ ID N0:12.
Clone "1r204 1"
A polynucleotide of the present invention has been identified as clone
"1r204_1".
1r204_1 was isolated from a human adult lymph node cDNA library using methods
which
2 5 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. 1r204_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"1r204_1 protein").
3 0 The nucleotide sequence of 1r204_1 as presently determined is reported in
SEQ ID
N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the 1r204_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15. Amino acids 29 to 41 are a
predicted
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leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 42, or are within a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
1r204_1 should be approximately 900 bp.
The nucleotide sequence disclosed herein for 1r204_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. 1r204_1 demonstrated at least some similarity with
sequences
identified as N47763 (yy55e07.r1 Homo Sapiens cDNA clone 277476 5') and N56875
(yy55e07.s1 Homo sapiens cDNA clone 277476 3' similar to SW:CYTO_BOVIN P01035
CYSTATIN, COLOSTRUM). The predicted amino acid sequence disclosed herein for
1r204_1 was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted 1r204_1 protein demonstrated
at least
some similarity to sequences. identified as M27891 (cystatin C [Homo
Sapiens]), P94392
(Sequence of complete recombinant cystatin C in E.coli), 843323 (Cystatin
polypeptide),
and X62412 (cystatin [unidentified]). Based upon sequence similarity, 1r204_1
proteins
and each similar protein or peptide may share at least some activity. The
predicted
1r204_1 protein contains a sequence highly conserved in all cystatins (QIVAG
in human
cystatin C, QIVKG in the predicted 1r204_1 protein). Cystatins are inhibitors
of
papain-like cysteine proteinases such as cathepsins. Cystatin C belongs to
family 2 of the
2 0 cystatin superfamily. The family 2 cystatins are secreted proteins of
about 120 amino
acids. All cystatins have important roles in processes involving cysteine
proteinase
activity like bone resorption. They are also implicated in a variety of
diseases (e.g. sepsis,
cancer metastasis, rheumatoid arthritis etc.) since they regulate potentially
harmful
proteinase activity. The predicted 1r204_1 protein appears to be a novel
cystatin C related
2 5 inhibitor of cysteine proteinases. The TopPredII computer program predicts
a potential
transmembrane domain within the 1r204_1 protein sequence near amino acid 42 of
SEQ
ID N0:15.
Deposit of Clones
3 0 Clones bd107_16 and bm41 7 were deposited on September 25, 1998 with the
American Type Culture Collection (10801 University Boulevard, Manassas,
Virginia
20110-2209 U.S.A.) as an original deposit under the Budapest Treaty and were
given the
accession number ATCC 98898, from which each clone comprising a particular
polynucleotide is obtainable. Clones bd107_13, bm41_3, br342_11, ej258_11,
k232_2x,
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1f307_5, and 1r204_1 were deposited on October 2,1997 with the American Type
Culture
Collection as an original deposit under the Budapest Treaty and were given the
accession
number ATCC 98551, from which each clone comprising a particular
polynucleotide is
obtainable. Clones bd107_13 and bm41 3 are additional isolates of clones
bd107_16 and
bm41 7, respectively. 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
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 Figures lA and 1B, respectively. The pED6dpc2 vector
("pED6") was derived from pED6dpc1 by insertion of a new polylinker to
facilitate
cDNA cloning (Kaufman et al., 1991, Nucleic Acids Res. 19: 4485-4490); the
pNOTs vector
was derived from pMT2 (Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by
deletion of
the DHFR sequences, insertion of a new polylinker, and insertion of the M13
origin of
replication in the CIaI site. In some instances, the deposited clone can
become "flipped"
(i.e., in the reverse orientation) in the deposited isolate. In such
instances, the cDNA insert
2 0 can still be isolated by digestion with EcoRI and NotI. However, NotI will
then produce
the 5' site and EcoRI will produce the 3' site for placement of the cDNA in
proper
orientation for expression in a suitable vector. The cDNA may also be
expressed from the
vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
2 5 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 an
oligonucleotide probe that was used to isolate or to sequence each full-length
clone is
3 0 identified below, and should be most reliable in isolating the clone of
interest.
lon Probe uence
bd107_16 SEQ ID N0:16
bm41 7 SEQ ID N0:17
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br342_11 SEQ ID N0:18
ej258_l l SEQ ID N0:19
k232_2x SEQ ID N0:20
1f307_5 SEQ ID N0:21
1r204_l SEQ ID N0:22
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's'"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-'2P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
2 0 labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
2 5 The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 ul 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
3 0 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;
CA 02305689 2000-03-29
WO 99!18127 PCT/US98/20793
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
S NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to le+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
10 by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
2 0 protein may be in linear form or they may be cyclized using known methods,
for example,
as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 {1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc.114 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
2 5 sites. For example, fragments of the protein may be fused through "linkei'
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
3 0 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
26
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
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
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.
The chromosomal location corresponding to the polynucleotide sequences
disclosed herein may also be determined, for example by hybridizing
appropriately
labeled polynucleotides of the present invention to chromosomes in situ. It
may also be
possible to determine the corresponding chromosomal location for a disclosed
2 0 polynucleotide by identifying significantly similar nucleotide sequences
in public
databases, such as expressed sequence tags (ESTs), that have already been
mapped to
particular chromosomal locations. For at least some of the polynucleotide
sequences
disclosed herein, public database sequences having at least some similarity to
the
polynucleotide of the present invention have been listed by database accession
number.
2 5 Searches using the GenBank accession numbers of these public database
sequences can
then be performed at an Internet site provided by the National Center for
Biotechnology
Information having the address http://www.ncbi.nlm.nih.gov/UniGene/, in order
to
identify "UniGene clusters" of overlapping sequences. Many of the "UruGene
clusters"
so identified will already have been mapped to particular chromosomal sites.
3 0 Organisms that have enhanced, reduced, or modified expression of the
genes)
corresponding to the polynucleotide sequences disclosed herein are provided.
The
desired change in gene expression can be achieved through the use of antisense
polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from
the
gene (Albert and Morris,1994, Trends Pharmacvl. Sci.15(7): 250-254; Lavarosky
et al.,1997,
27
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
Biochem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. Mol.
Biol. 58: 1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 Bl, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
2 0 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 5 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
3 0 known techniques for determination of such domains from sequence
information. For
example, the TopPredII computer program can be used to predict the location of
transmembrane domains in an amino acid sequence, domains which are described
by the
location of the center of the transmsmbrane domain, with at least ten
transmembrane
amino acids on each side of the reported central residue(s).
28
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
In particular, sequence identity may be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
version 1.4, which in turn is based on the public domain NCBI-BLAST version
1.4
(Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods
in Enzymology
266: 460-480; Altschul et al., 1990, Basic local alignment search tool,
Journal of
Molecular Biology 215: 403-410; Gish and States, 1993, Identification of
protein coding
regions by database similarity search, Nature Genetics 3: 266-272; Karlin and
Altschul,
1993, Applications and statistics for multiple high-scoring segments in
molecular
2 0 sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are
incorporated by
reference herein). WU-BLAST version 2.0 executable programs for several UNIX
platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The
complete
suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is
provided at that site, in addition to several support programs. WU-BLAST 2.0
is
2 5 copyrighted and may not be sold or redistributed in any form or manner
without the
express written consent of the author; but the posted executables may
otherwise be freely
used for commercial, nonprofit, or academic purposes. In all search programs
in the suite
-- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX -- the gapped alignment
routines are integral to the database search itself, and thus yield much
better sensitivity and
3 0 selectivity while producing the more easily interpreted output. Gapping
can optionally be
turned off in all of these programs, if desired. The default penalty (Q) for a
gap of length
one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to
any
29
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
integer value including zero, one through eight, nine, ten, eleven, twelve
through twenty,
twenty-one through fifty, fifty-one through one hundred, etc. The default per-
residue
penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for
BLASTN,
but may be changed to any integer value including zero, one, two, three, four,
five, six,
seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through
fifty, fifty-one
through one hundred, etc. Any combination of values for Q and R can be used in
order to
align sequences so as to maximize overlap and identity while minimizing
sequence gaps.
The default amino acid comparison matrix is BLOSUM62, but other amino acid
comparison matrices such as PAM can be utilized.
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
2 0 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
2 5 species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hylobates
concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatics, Sanguinus Oedipus, Microcebus murinus, Mus musculus,
Rattus norvegicus,
Cricetulus griseus, Felis catccs, Mccstela vison, Canis familiaris,
Oryctolagus cuniculus, Bos taunts,
Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been
created
3 0 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
(O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al.,
1993, Nakure
Genetics 3:103-112; Johansson et al., 1995, Genomics 25: 682-690; Lyons et
al., 1997, Nature
CA 02305689 2000-03-29
WO 99/18127 PCTNS98/20793
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
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%
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 polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
stringent conditions, to polynucieotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
2 0 stringent as, for example, conditions G-L; and reduced stringency
conditions are at least
as stringent as, for example, conditions M-R.
31
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
StringencyPolynucleotideHybrid Hybridization TemperatureWash
ConditionHybrid Length and Temperature
(bp)= BufferT and Buffer'
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50%
formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
S C DNA:RNA >_ 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50%
formamide
D DNA:RNA <50 Tp"; lxSSC Tp*; lxSSC
E RNA:RNA s 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 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 T"*; 4xSSC T"*; 4xSSC
I DNA:RNA >- 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50%
formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA >_ 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 a 50 55C; 4xSSC -or- 55'C; 2xSSC
42C; 6xSSC, 50%
formamide
P DNA:RNA <50 T~*; 6xSSC T,,*; 6xSSC
Q RNA:RNA s 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50%
formamide
2 R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
0
x: 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 NaHZPOy, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 *TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,o[Na']) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
32
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 9 4485-4490 (1991), in order to produce the
protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 1$5 537-566 (1990). As defined herein "operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the Iigated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
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CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
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 A~icultural
Experiment
Station Bulletin No. 1555 (1987J, incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
1 S 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 BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 Invitrogen Corporation (Carlsbad, CA), 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 ("Fiag") is commercially available from the Eastman
Kodak
Company (New Haven, CT). ,
34
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The_protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
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.
2 0 The proteins provided herein also include proteins characterized by amino
acid
sequences similar to those of purified proteins but into which modification
are naturally
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,
2 5 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
amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
3 0 insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be useful
for screening
or other immunological methodologies may also be easily made by those skilled
in the art
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The 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
2 0 (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"
known sequences in the process of discovering other novel polynucleotides; for
selecting
2 5 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
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
3 0 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
inhibitors of the binding interaction.
36
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
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
2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
Kimmel eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 5 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
3 0 can be added to the medium in or on which the microorganism is cultured.
C3rtokine and Cell Proliferation/Differentiation ActivitX
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
37
CA 02305689 2000-03-29
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induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DAl, 123, T1165, HT2, CTLL2, TF-1, Mo7e and
CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
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; Bertagnoili, 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
2 0 stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
2 5 cells include, without limitation, those described in: Measurement of
Human and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
3 0 Measurement of mouse and human interleukin 6 - Nordan, R. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, john Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto.1991;
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WO 99/18127 PCT/US98/20793
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Su~vressing Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
2 0 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
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
2 5 protein of the present invention, inciuding 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
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
3 0 Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
39
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WO 99/18127 PCT/U59$/20793
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
2 0 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
2 5 the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
3 0 molecule to the natural ligand(s) on the immune cells without transmitting
the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
CA 02305689 2000-03-29
WO 99/18127 PCTNS98/20793
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992}. In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 0 Administration of reagents which block costimulation of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease prcxess. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
2 S the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr mice or NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
3 0 murine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
41
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 0 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
2 5 transfected tumor cells are returned to the patient to result in
expression of the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
3 0 costimulation signal to T cells to induce a T cell mediated immune
response against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.~., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
ji2
42
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having,
the activity- of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa
et al.,
2 0 J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-
3500,1986; Takai et al.,
J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
2 5 Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell
function: In vitro
3 0 antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
43
CA 02305689 2000-03-29
WO 99/18127 PCT/US98I20793
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.I-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 0 Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation, those described in: Antica et al.,
Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991
Hematgpoiesis RegylatinQ ActivitX
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
3 0 lines indicates involvement in regulating hematopoiesis, e.g. in
supporting the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
44
CA 02305689 2000-03-29
WO 99!18127 PCT/US98/20793
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 0 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
2 5 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
3 0 Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et at. 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
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/Z0793
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY.1994.
Tissue Growth ActivitX
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 0 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
2 5 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
3 0 circumstances where such tissue is not normally formed, has application in
the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
46
CA 02305689 2000-03-29
WO 99/18127 PCT/US98l20793
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/liga_ment cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 0 Parkinsons disease, Huntington s disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
2 5 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
3 0 generation or regeneration of other tissues, such as organs (including,
for example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
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of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. WQ95/05$46 {nerve, neuronal);
International Patent
Publication No. W091 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
2 0 Activin/Inhibin ActivitX
A protein of the present invention may also exhibit activin- or inhibin-
related
activities. Inhibins are characterized by their ability to inhibit the release
of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
2 5 invention, alone or in heterodimers with a member of the inhibin a family,
may be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
3 0 ~i group, may be useful as a fertility inducing therapeutic, based upon
the ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
United States Patent 4,798,885. A protein of the invention may also be useful
for
advancement of the onset of fertility in sexually immature mammals, so as to
increase,the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
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The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-
782,1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 0 population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
of cells can be readily determined by employing such protein or peptide in any
known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
2 5 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
3 0 include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
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APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol.152:5860-5867,1994; johnston et al. J. of Immunol. 153: 1762-1768,
1994.
Hemostatic and ThrombolXtic ActivitX
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/L~and 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
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
2 5 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
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
3 0 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.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med.169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activi
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
2 0 inflammatory response syndrome (SIRS)), ischemia-reperfusion injury,
endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
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
3 0 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.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
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extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
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
2 0 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
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
2 5 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
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
3 0 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
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.
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Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activitv
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
2 0 factors, agents or cell types which inhibit tumor growth, or by
suppressing, eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following
additional
2 5 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,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
3 0 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,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
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cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
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
2 0 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,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may further
contain other
2 5 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,
or to minimize side effects. Conversely, protein of the present invention may
be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
3 0 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.
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
54
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunoigobulin
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 Garners, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
2 0 in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
2 5 by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
3 0 prevention or amelioration of such conditions. When applied to an
individual active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
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.
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
2 0 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
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.
2 5 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
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
3 0 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.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
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CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
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
2 0 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 lZg
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
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
2 5 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
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
3 0 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
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocy~nin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
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CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
R.P. Merrifield, J. Amer.Chem.Soc. ~ 2149-2154 (1963); j.L. Krstenansky, et
aL, FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
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
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
2 5 applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
3 0 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
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
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ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
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
2 0 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.
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
2 5 question. These agents include various growth factors such as epidermal
growth factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors {TGF-
a and
TGF-p), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
3 0 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
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
59
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
SEQUENCE LISTING
<110> Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Evans, Cheryl
Merberg, David
Treacy, Maurice
Agostino, Michael J.
Spaulding, Vikki
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI6053A .
<140>
<141>
<160> 22
<170> PatentIn Ver. 2.0
<210> 1
<211> 1528
<212> DNA
<213> Homo sapiens
<400> 1
cgcttttttt ttttttttga caagatggcg gcaggaggca gtggcgttgg tgggaagcgc 60
agctcgaaaa gcgatgccga ttctggtttc ctggggctgc ggcccacttc ggtggaccca 120
gcgctgaggc ggcggcggcg aggcccaaga aataagaagc ggggctggcg gcggcttgct 180
caggagccgc tggggctgga ggttgaccag ttcctggaag acgtgcggct acaggagcgc 240
acgagcggtg gcttgttgtc agaggcccca aatgaaaaac tcttcttcgt ggacactggc 300
tccaaggaaa aagggctgac aaagaagaga accaaagtcc agaagaagtc actgcttctc 360
aagaaacccc ttcgggttga cctcatcctc gagaacacat ccaaagtccc tgcccccaaa 420
gacgtcctcg cccaccaggt ccccaacgcc aagaagctca ggcggaagga gcagctatgg 480
gagaagctgg ccaagcaggg cgagctgccc cgggaggtgc gcagggccca ggcccggctc 540
ctcaaccctt ctgcaacaag ggccaagccc gggccccagg acaccgtaga gcggcccttc 600
tacgacctct gggcctcaga caaccccctg gacaggccgt tggttggcca ggatgagttt 660
ttcctggagc agaccaagaa gaaaggagtg aagcggccag cacgcctgca caccaagccg 720
tcccaggcgc ccgccgtgga ggtggcgcct gccggagctt cctacaatcc atcctttgaa 780
gaccaccaga ccctgctctc agcggcccac gaggtggagt tgcagcggca gaaggaggcg 840
gagaagctgg agcggcagct ggccctgccc gccatggagc aggccgccac ccaggagtcc 900
acattccagg agctgtgcga ggggctgctg gaggagtcgg atggtgaggg ggagccaggc 960
cagggcgagg ggccggaggc tggggatgcc gaggtctgtc ccacgcccgc ccgcctggcc 1020
accacagaga agaagacgga gcagcagcgg cggcgggaga aggctgtgca caggctgcgg 1080
gtacagcagg ccgcgttgcg ggccgcccgg ctccggcacc aggagctgtt ccggctgcgc 1140
gggatcaagg cccaggtggc cctgaggctg gcggagctgg cgcggcggcg gaggcggcgg 1200
caggcgcggc gggaggctga ggctgacaag ccccgaaggc tgggacggct caagtaccag 1260
gcacctgaca tcgacgtgca gctgagctcg gagctgacag actcgctcag gaccctgaag 1320
cccgagggca acatccttcg agaccggttc aagagcttcc agaggaggaa tatgatcgag 1380
cctcgagaga gagccaagtt caaacgcaag tacaaggtga agctggtgga gaagcgggcg 1440
ttccgtgaga tccagttgta gctgccatca gatgccggag actcgccctt caataaaaaa 1500
tctcttctag ctgaaaaaaa aaaaaaaa 1528
<210> 2
<211> 478
<212> PRT
<213> Homo Sapiens
<400> 2 '
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/Z0793
Met Ala Ala Gly Gly Ser Gly Val Gly Gly Lys Arg Ser Ser Lys Ser
1 5 10 15
Asp Ala Asp Ser Gly Phe Leu Gly Leu Arg Pro Thr Ser Val Asp Pro
20 25 30
Ala Leu Arg Arg Arg Arg Arg Gly Pro Arg Asn Lys Lys Arg Gly Trp
35 40 45
Arg Arg Leu Ala Gln Glu Pro Leu Gly Leu Glu Val Asp Gln Phe Leu
50 55 60
Glu Asp Val Arg Leu Gln Glu Arg Thr Ser Gly Gly Leu Leu Ser Glu
65 70 75 80
Ala Pro Asn Glu Lys Leu Phe Phe Val Asp Thr Gly Ser Lys Glu Lys
85 90 95
Gly Leu Thr Lys Lys Arg Thr Lys Val Gln Lys Lys Ser Leu Leu Leu
100 105 110
Lys Lys Pro Leu Arg Val Asp Leu Ile Leu Glu Asn Thr Ser Lys Val
115 120 125
Pro Ala Pro Lys Asp Val Leu Ala His Gln Val Pro Asn Ala Lys Lys
130 135 140
Leu Arg Arg Lys Glu Gln Leu Trp Glu Lys Leu Ala Lys Gln Gly Glu
145 150 155 160
Leu Pro Arg Glu Val Arg Arg Ala Gln Ala Arg Leu Leu Asn Pro Ser
165 170 175
Ala Thr Arg Ala Lys Pro Gly Pro Gln Asp Thr Val Glu Arg Pro Phe
180 185 190
Tyr Asp Leu Trp Ala Ser Asp Asn Pro Leu Asp Arg Pro Leu Val Gly
195 200 205
Gln Asp Glu Phe Phe Leu Glu Gln Thr Lys Lys Lys Gly Val Lys Arg
210 215 220
Pro Ala Arg Leu His Thr Lys Pro Ser Gln Ala Pro Ala Val Glu Val
225 230 235 240
Ala Pro Ala Gly Ala Ser Tyr Asn Pro Ser Phe Glu Asp His Gln Thr
245 250 255
Leu Leu Ser Ala Ala His Glu Val Glu Leu Gln Arg Gln Lys Glu Ala
260 265 270
Glu Lys Leu Glu Arg Gln Leu Ala Leu Pro Ala Met Glu Gln Ala Ala
275 280 285
Thr Gln Glu Ser Thr Phe Gln Glu Leu Cys Glu Gly Leu Leu Glu Glu
290 295 300
Ser Asp Gly Glu Gly Glu Pro Gly Gln Gly Glu Gly Pro Glu Ala Gly
305 310 315 320
Asp Ala Glu Val Cys Pro Thr Pro Ala Arg Leu Ala Thr Thr Glu Lys
325 330 335
2
CA 02305689 2000-03-29
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Lys Thr Glu Gln Gln Arg Arg Arg Glu Lys Ala Val His Arg Leu Arg
340 345 350
Val Gln Gln Ala Ala Leu Arg Ala Ala Arg Leu Arg His Gln Glu Leu
355 360 365
Phe Arg Leu Arg Gly Ile Lys Ala Gln Val Ala Leu Arg Leu Ala Glu
370 375 380
Leu Ala Arg Arg Arg Arg Arg Arg Gln Ala Arg Arg Glu Ala Glu Ala
385 390 395 400
Asp Lys Pro Arg Arg Leu Gly Arg Leu Lys Tyr Gln Ala Pro Asp Ile
405 410 415
Asp Val Gln Leu Ser Ser Glu Leu Thr Asp Ser Leu Arg Thr Leu Lys
420 425 430
Pro Glu Gly Asn Ile Leu Arg Asp Arg Phe Lys Ser Phe Gln Arg Arg
435 440 445
Asn Met Ile Glu Pro Arg Glu Arg Ala Lys Phe Lys Arg Lys Tyr Lys
450 455 460
Val Lys Leu Val Glu Lys Arg Ala Phe Arg Glu Ile Gln Leu
465 470 475
<210> 3
<211> 1472
<212> DNA
<213> Homo sapiens
<400> 3
acaagatggc ggcgccgaag gggagcctct gggtgaggac ccaactgggg ctcccgccgc 60
tgctgctgct gaccatggcc ttggccggag gttcggggac cgcttcggct gaagcatttg 120
actcggtctt gggtgatacg gcgtcttgcc accgggcctg tcagttgacc taccccttgc 180
acacctaccc taaggaagag gagttgtacg catgtcagag aggttgcagg ctgttttcaa 240
tttgtcagtt tgtggatgat ggaattgact taaatcgaac taaattggaa tgtgaatctg 300
catgtacaga agcatattcc caatctgatg agcaatatgc ttgccatctt ggttgccaga 360
atcagctgcc attcgctgaa ctgagacaag aacaacttat gtccctgatg ccaaaaatgc 420
acctactctt tcctctaact ctggtgaggt cattctggag tgacatgatg gactccgcac 480
agagcttcat aacctcttca tggacttttt atcttcaagc cgatgacgga aaaatagtta 540
tattccagtc taagccagaa atccagtacg caccacattt ggagcaggag cctacaaatt 600
tgagagaatc atctctaagc aaaatgtcct cagatctgca aatgagaaat tcacaagcgc 660
acaggaattt tcttgaagat ggagaaagtg atggcttttt aagatgcctc tctcttaact 720
ctgggtggat tttaactaca actcttgtcc tctcggtgat ggtattgctt tggatttgtt 780
gtgcaactgt tgctacagct gtggagcagt atgttccctc tgagaagctg agtatctatg 840
gtgacttgga gtttatgaat gaacaaaagc taaacagata tccagcttct tctcttgtgg 900
ttgttagatc taaaactgaa gatcatgaag aagcagggcc tctacctaca aaagtgaatc 960
ttgctcattc tgaaatttaa gcatttttct tttaaaagac aagtgtaata gacatctaaa 1020
attccactcc tcatagagct tttaaaatgg tttcattgga tataggcctt aagaaatcac 1080
tataaaatgc aaataaagtt actcaaatct gtgaagactg tatttgctat aactttattg 1140
gtattgtttt tgtagtaatt taagaggtgg atgtttggga ttgtattatt attttactaa 1200
tatctgtagc tattttgttt tttgctttgg ttattgtttt tttccctttt cttagctatg 1260
agctgatcat tgctccttct cacctcctgc catgatactg tcagttacct tagttaacaa 1320
gctgaatatt tagtagaaat gatgcttctg ctcaggaatg gcccacaaat ctgtaatttg 1380
aaatttagca ggaaatgacc tttaatgaca ctacattttc aggaactgaa atcattaaaa 1440
ttttatttga ataattaaaa aaaaaaaaaa as 1472
<210> 4
<211> 324 '
<212> PRT
3
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
<213> Homo sapiens
<400> 4
Met Ala Ala Pro Lys Gly Ser Leu Trp Val Arg Thr Gln Leu Gly Leu
1 5 10 15
Pro Pro Leu Leu Leu Leu Thr Met Ala Leu Ala Gly Gly Ser Gly Thr
20 25 30
Ala Ser Ala Glu Ala Phe Asp Ser Val Leu Gly Asp Thr Ala Ser Cys
35 40 45
His Arg Ala Cys Gln Leu Thr Tyr Pro Leu His Thr Tyr Pro Lys Glu
50 55 60
Glu Glu Leu Tyr Ala Cys Gln Arg Gly Cys Arg Leu Phe Ser Ile Cys
65 70 75 80
Gln Phe Val Asp Asp Gly Ile Asp Leu Asn Arg Thr Lys Leu Glu Cys
85 90 95
Glu Ser Ala Cys Thr Glu Ala Tyr Ser Gln Ser Asp Glu Gln Tyr Ala
100 105 110
Cys His Leu Gly Cys Gln Asn Gln Leu Pro Phe Ala Glu Leu Arg Gln
115 120 125
Glu Gln Leu Met Ser Leu Met Pro Lys Met His Leu Leu Phe Pro Leu
130 135 140
Thr Leu Val Arg Ser Phe Trp Ser Asp Met Met Asp Ser Ala Gln Ser
145 150 155 160
Phe Ile Thr Ser Ser Trp Thr Phe Tyr Leu Gln Ala Asp Asp Gly Lys
165 170 175
Ile Val Ile Phe Gln Ser Lys Pro Glu Ile Gln Tyr Ala Pro His Leu
180 185 190
Glu Gln Glu Pro Thr Asn Leu Arg Glu Ser Ser Leu Ser Lys Met Ser
195 200 205
Ser Asp Leu Gln Met Arg Asn Ser Gln Ala His Arg Asn Phe Leu Glu
210 215 220
Asp Gly Glu Ser Asp Gly Phe Leu Arg Cys Leu Ser Leu Asn Ser Gly
225 230 235 240
Trp Ile Leu Thr Thr Thr Leu Val Leu Ser Val Met Val Leu Leu Trp
245 250 255
Ile Cys Cys Ala Thr Val Ala Thr Ala Val Glu Gln Tyr Val Pro Ser
260 265 270
Glu Lys Leu Ser Ile Tyr Gly Asp Leu Glu Phe Met Asn Glu Gln Lys
275 280 2B5
Leu Asn Arg Tyr Pro Ala Ser Ser Leu Val Val Val Arg Ser Lys Thr
290 295 300
Glu Asp His Glu Glu Ala Gly Pro Leu Pro Thr Lys Val Asn Leu Ala
305 310 315 320
4
CA 02305689 2000-03-29
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His Ser Glu Ile
<210> 5
<211> 1573
<212> DNA
<213> Homo sapiens
<400> 5
gtgattggta cagtaggttt ataaacagaa gtttaaactt gtaagcttaa gcttccgttt 60
ataaacagaa gtttaaaatt ataggtcctg tttaacattc agctctgtta actcactcat 120
ctttttgtgt ttttacactt tgtcaagatt tctttacata ttcatcaatg tctgaagaag 180
ttacttatgc agatcttcaa ttccagaact ccagtgagat ggaaaaaatc ccagaaattg 240
gcaaatttgg ggaaaaagca cctccagctc cctctcatgt atggcgtcca gcagccttgt 300
ttctgactct tctgtgcctt ctgttgctca ttggattggg agtcttggca agcatgtttc 360
atgtaacttt gaagatagaa atgaaaaaaa tgaacaaact acaaaacatc agtgaagagc 420
tccagagaaa tatttctcta caactgatga gtaacatgaa tatctccaac aagatcagga 480
acctctccac cacactgcaa acaatagcca ccaaattatg tcgtgagcta tatagcaaag 540
aacaagagca caaatgtaag ccttgtccaa ggagatggat ttggcataag gacagctgtt 600
atttcctaag tgatgatgtc caaacatggc aggagagtaa aatggcctgt gctgctcaga 660
atgccagcct gttgaagata aacaacaaaa atgcattgga atttataaaa tcccagagta 720
gatcatatga ctattggctg ggattatctc ctgaagaaga ttccactcgt ggtatgagag 780
tggataatat aatccactcc tctgcctggg ttataagaaa cgcacctgac ttaaataaca 840
tgtattgtgg atatataaat agactatatg ttcaatatta tcactgcact tataaacaaa 900
gaatgatatg tgagaagatg gccaatccag tgcagcttgg ttctacatat tttagggagg 960
catgaggcat caatcaaata cattgaagga gtgtaggggg tgggggttct aggctatagg 1020
taaatttaaa tattttctgg ttgacaatta gttgagtttg tctgaagacc tgggatttta 1080
tcatgcagat gaaacatcca ggtagcaagc ttcagagaga atagactgtg aatgttaatg 1140
ccagagaggt ataatgaagc atgtccmacy tcccactttc catcatggcy tgaaccykgg 1200
rggaagagga agtccattca gatagttgtg gggggcctts gaattttcat tttcatwwac 1260
gttcttcccc ttctggccaa gatttgccag aggcaacatc aaaaaccagc aaattktaat 1320
tttgtcccac agsgttgcta gggtggcatg gytccccatt tsgggtccat cctawacttc 1380
catgggactc cctatggctg aaggccttat gagtcaaagg acttatagcc aattgattgt 1440
tttaggccag gtaagaatgg atatggacat gcatttatta cytyttaaaa ttattatttt 1500
aagtaaaagc caataaacaa aaangnaaag gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560
aaaaaaaaaa aaa 1573
<210> 6
<211> 265
<212> PRT
<213> Homo Sapiens
<400> 6
Met Ser Glu Glu Val Thr Tyr Ala Asp Leu Gln Phe Gln Asn Ser Ser
1 5 10 15
Glu Met Glu Lys Ile Pro Glu Ile Gly Lys Phe Gly Glu Lys Ala Pro
20 25 30
Pro Ala Pro Ser His Val Trp Arg Pro Ala Ala Leu Phe Leu Thr Leu
35 40 45
Leu Cys Leu Leu Leu Leu Ile Gly Leu Gly Val Leu Ala Ser Met Phe
50 55 60
His Val Thr Leu Lys Ile Glu Met Lys Lys Met Asn Lys Leu Gln Asn
65 70 75 80
Ile Ser Glu Glu Leu Gln Arg Asn Ile Ser Leu Gln Leu Met Ser Asn
85 90 95
Met Asn Ile Ser Asn Lys Ile Arg Asn Leu Ser Thr Thr Leu Gln Thr
CA 02305689 2000-03-29
WO 99/18127 PC'T/US98/20793
100 105 110
Ile Ala Thr Lys Leu Cys Arg Glu Leu Tyr Ser Lys Glu Gln Glu His
115 120 125
Lys Cys Lys Pro Cys Pro Arg Arg Trp Ile Trp His Lys Asp Ser Cys
130 135 140
Tyr Phe Leu Ser Asp Asp Val Gln Thr Trp Gln Glu Ser Lys Met Ala
145 150 155 160
Cys Ala Ala Gln Asn Ala Ser Leu Leu Lys Ile Asn Asn Lys Asn Ala
165 170 175
Leu Glu Phe Ile Lys Ser Gln Sex Arg Ser Tyr Asp Tyr Trp Leu Gly
180 185 190
Leu Ser Pro Glu Glu Asp Ser Thr Arg Gly Met Arg Val Asp Asn Ile
195 200 205
Ile His Ser Ser Ala Trp Val Ile Arg Asn Ala Pro Asp Leu Asn Asn
210 215 220
Met Tyr Cys Gly Tyr Ile Asn Arg Leu Tyr Val Gln Tyr Tyr His Cys
225 230 235 240
Thr Tyr Lys Gln Arg Met Ile Cys Glu Lys Met Ala Asn Pro Val Gln
245 250 255
Leu Gly Ser Thr Tyr Phe Arg Glu Ala
260 265
<210> 7
<211> 618
<212> DNA
<213> Homo Sapiens
<400> 7
gagttatatg acactcaaag gaaaagcaaa agagcattaa gaagtgtctg tttttgttat 60
tgccatttca taaatatttt agtaggtgtt caatttcatt ggatattctt tttttttaat 120
tgtctttgta cctatgattg aaaacagtag ttggtctatg acttttgagg agagggagaa 180
ccgaagatta caggaggcca gcatgaggtt ggaacaagag aatgatgacc ttgcccatga 240
actagtaaca agcaaaattg ctctacggaa tgacttggat caggcagaag acaaggcaga 300
tgtgttgaat aaagagctcc ttttgaccaa acagaggctg gtggagactg aagaggagaa 360
gaggaagcaa gaggaagaga ctgcccagct aaaagaagtc ttcaggaaac agctagagaa 420
ggcagaatat gaaataaaga agactacagc tatcattgct gagtataaac aggtaatgta 480
cttctgtggc acatagagct agttatagtt tgctgctata aaagtaattt tttttttttt 540
ttgcttgagg ccaggagttt gagactagcc tgagcaacat agcaggactc cgtcccaagg 600
aaaaaaaaaa aaaaaaaa 618
<210> 8
<211> 120
<212> PRT
<213> Homo Sapiens
<400> 8
Met Ile Glu Asn Ser Ser Trp Ser Met Thr Phe Glu Glu Arg Glu Asn
1 5 10 15
Arg Arg Leu Gln Glu Ala Ser Met Arg Leu Glu Gln Glu Asn Asp Asp
20 25 30
6
CA 02305689 2000-03-29
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Leu Ala His Glu Leu Val Thr Ser Lys Ile Ala Leu Arg Asn Asp Leu
35 40 45
Asp Gln Ala Glu Asp Lys Ala Asp Val Leu Asn Lys Glu Leu Leu Leu
50 55 60
Thr Lys Gln Arg Leu Val Glu Thr Glu Glu Glu Lys Arg Lys Gln Glu
65 70 75 80
Glu Glu Thr Ala Gln Leu Lys Glu Val Phe Arg Lys Gln Leu Glu Lys
85 90 95
Ala Glu Tyr Glu Ile Lys Lys Thr Thr Ala Ile Ile Ala Glu Tyr Lys
100 105 110
Gln Val Met Tyr Phe Cys Gly Thr
115 120
<210> 9
<211> 510
<212> DNA
<213> Homo Sapiens
<400> 9
tgcagaatcc agaatggatg tcctctttgt agccatcttt gctgtgccac ttatcctggg 60
acaagaatat gaggatgaag aaagactggg agaggatgaa tattatcagg tggtctatta 120
ttatacagtc acccccagtt atgatgactt tagtgcagat ttcaccattg attactccat 180
atttgagtca gaggacaggc tgaacaggtt ggataaggac ataacagaag caatagagac 240
taccattagt cttgaaacag cacgtgcaga ccatccgaag cctgtaactg tgaaaccagt 300
aacaacggaa cctagtccag atctgaacga tgccgtgtcc agtttgcgaa gtcctattcc 360
cctcctcctg tcgtgtgcct ttgttcaggt ggggatgtat ttcatgtaga aggtggaaga 420
aggctgctat gactctttgg atgggagtct ggcaagagga aattggaaga taaaataaat 480
aataagtgaa ataaaaaaaa aaaaaaaaaa 510
<210> 10
<211> 131
<212> PRT
<213> Homo Sapiens
<400> 10
Met Asp Val Leu Phe Val Ala Ile Phe Ala Val Pro Leu Ile Leu Gly
1 5 10 15
Gln Glu Tyr Glu Asp Glu Glu Arg Leu Gly Glu Asp Glu Tyr Tyr Gln
20 25 30
Val Val Tyr Tyr Tyr Thr Val Thr Pro Ser Tyr Asp Asp Phe Ser Ala
35 40 45
Asp Phe Thr Ile Asp Tyr Ser Ile Phe Glu Ser Glu Asp Arg Leu Asn
50 55 60
Arg Leu Asp Lys Asp Ile Thr Glu Ala Ile Glu Thr Thr Ile Ser Leu
65 70 75 80
Glu Thr Ala Arg Ala Asp His Pro Lys Pro Val Thr Val Lys Pro Val
85 90 95
Thr Thr Glu Pro Ser Pro Asp Leu Asn Asp Ala Val Ser Ser Leu Arg
100 105 1I0
Ser Pro Ile Pro Leu Leu Leu Ser Cys Ala Phe Val Gln Val Gly Met
7
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
115 120 125
Tyr Phe Met
130
<210> 11
<211> 883
<212> DNA
<213> Homo sapiens
<400> 11
catctgacca tccatatcca atgttctcat ttaaacatta cccagcatca ttgtttataa 60
tcagaaactc tggtccttct gtctggtggc acttagagtc ttttgtgcca taatgcagca 120
gtatggaggg aggattttat ggagaaatgg ggatagtctt catgaccaca aataaataaa 180
ggaaaactaa gctgcattgt gggttttgaa aaggttatta tacttcttaa caattctttt 240
tttcagggac ttttctagct gtatgactgt tacttgacct tctttgaaaa gcattcccaa 300
aatgctctat tttagataga ttaacattaa ccaacataat tttttttaga tcgagtcagc 360
ataaatttct aagtcagcct ctagtcgtgg ttcatctctt tcacctgcat tttatttggt 420
gtttgtctga agaaaggaaa gaggaaagca aatacgaatt gtactatttg taccaaatct 480
ttgggattca ttggcaaata atttcagtgt ggtgtattat taaatagaaa aaaaaaattt 540
tgtttcctag gttgaaggtc taattgatac gtttgactta tgatgaccat ttatgcactt 600
tcaaatgaat ttgctttcaa aataaatgaa gagcagctgt ccttctttcc tcttttaagt 660
gttcagctgt ggcatgctca gaggttcctg ctggattcca gctggagcgg tgtgataccc 720
ttctttttca gctgttcgtg ccttcctttc ttgtatccac caaagtggag acaaatacat 780
gatctcaaag atacacagta cctacttaat tccagctgat gggagaccaa agaatttgca 840
agtggatggt ttggtatcac tgtaaataaa aagagggcct ggg 8g3
<210> 12
<211> 79
<212> PRT
<213> Homo Sapiens
<400> 12
Met Met Thr Ile Tyr Ala Leu Ser Asn Glu Phe Ala Phe Lys Ile Asn
1 5 10 15
Glu Glu Gln Leu Ser Phe Phe Pro Leu Leu Ser Val Gln Leu Trp His
20 25 30
Ala Gln Arg Phe Leu Leu Asp Ser Ser Trp Ser Gly Val Ile Pro Phe
35 40 45
Phe Phe Ser Cys Ser Cys Leu Pro Phe Leu Tyr Pro Pro Lys Trp Arg
50 55 60
Gln Ile His Asp Leu Lys Asp Thr Gln Tyr Leu Leu Asn Ser Ser
65 70 75
<210> 13
<211> 110
<212> DNA
<213> Homo Sapiens
<400> 13
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 110
<210> 14
<211> 861
<212> DNA
<213> Homo Sapiens
8
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
<400> 14
taggcctctt tggccggtgc tgcctgagaa ggattggcac gggcacagac cactgccccc 60
acctgccctg cgccatctac ccaagaaggc tcggcacggg caccaaccac tgcctccaac 120
tgccccatgc tgcctgagaa ggcactgcac ggccaccccc aactgccccg cactgtccct I80
acccgggcag ccatgcgagc ggctggaact ctgctggcct tctgctgcct ggtcttgagc 240
accactgggg gcccttcccc agatacttgt tcccaggacc ttaactcacg tgtgaagcca 300
ggatttccta aaacaataaa gaccaatgac ccaggagtcc tccaagcagc cagatacagt 360
gttgaaaagt tcaacaactg cacgaacgac atgttcttgt tcaaggagtc ccgcatcaca 420
agggccctag ttcagatagt gaaaggcctg aaatatatgc tcgargtgga aattggcaga 480
actacctgca agaaaaacca gcacctgcgt ctggatgact gtgacttcca aaccaaccac 540
accttgaagc agactctgag ctgctactct gaagtctggg tcgtgccctg gctccagcac 600
ttcgaggtgc ctgttctccg ttgtcactga cccccgcctc ttcagcaaga ccacagccat 660
gacaaacacc aggatgcatg ctccttgtcc cctcccaccc gcywsrtgac ccrrcctsac 720
agaccctctc rggcctcwga cgagtgagcg grtgaagtgc maytgggtsa cmgcagggca 780
gctrgaatgg cagcwtggta gcgcctccta acagrttaaa trgatcacat gtgmttctaa 840
aattraaaaa aaaaaaaaaa a 861
<210> 15
<211> 167
<212> PRT
<213> Homo sapiens
<400> 15
Met Leu Pro Glu Lys Ala Leu His Gly His Pro Gln Leu Pro Arg Thr
1 5 10 15
Val Pro Thr Arg Ala Ala Met Arg Ala Ala Gly Thr Leu Leu Ala Phe
20 25 30
Cys Cys Leu Val Leu Ser Thr Thr Gly Gly Pro Ser Pro Asp Thr Cys
35 40 45
Ser Gln Asp Leu Asn Ser Arg Val Lys Pro Gly Phe Pro Lys Thr Ile
50 55 60
Lys Thr Asn Asp Pro Gly Val Leu Gln Ala Ala Arg Tyr Ser Val Glu
65 70 75 80
Lys Phe Asn Asn Cys Thr Asn Asp Met Phe Leu Phe Lys Glu Ser Arg
85 90 95
Ile Thr Arg Ala Leu Val Gln Ile Val Lys Gly Leu Lys Tyr Met Leu
100 105 110
Glu Val Glu Ile Gly Arg Thr Thr Cys Lys Lys Asn Gln His Leu Arg
115 120 125
Leu Asp Asp Cys Asp Phe Gln Thr Asn His Thr Leu Lys Gln Thr Leu
130 135 140
Ser Cys Tyr Ser Glu Val Trp Val Val Pro Trp Leu Gln His Phe Glu
145 150 155 160
Val Pro Val Leu Arg Cys His
165
<210> 16
<211> 29
<212> DNA
<213> Homo sapiens
<400> 16
9
CA 02305689 2000-03-29
WO 99/18127 PCT/US98/20793
anccagaatc ggcatcgctt ttcgagctg 2g
<210>
17
<211>
29
<212>
DNA
<213> Sapiens
Homo
<400>
17
tntggtgcgtactggatttctggcttaga 29
<210>
18
<211>
29
<212>
DNA
<213> Sapiens
Homo
<400>
18
gntaacagagctgaatgttaaacaggacc 29
<210>
19
<211>
29
<212>
DNA
<213> Sapiens ,
Homo
<400>
19
tntcctcaaaagtcatagaccaactactg 29
<210>
20
<211>
29
<212>
DNA
<213> Sapiens
Homo
<400>
20
gntcagcctgtcctctgactcaaatatgg 29
<210>
21
<211>
29
<212>
DNA
<213> Sapiens
Homo
<400>
21
tnacctgcattttatttggtgtttgtctg 29
<210>
22
<211>
29
<212>
DNA
<213> Sapiens
Homo
<400>
22
tnaacactgtatctggctgcttggaggac 29
