Note: Descriptions are shown in the official language in which they were submitted.
CA 02316864 2000-06-28
WO 99/35252 PCTlUS98/27903
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of provisional application Ser. No.
60/070,346, filed January 2,1998, 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
2 0 related to the discovered protein (i.e., partial DNA/amino acid sequence
of the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
2 5 techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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:I;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 257 to nucleotide 622;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ff168_12 deposited under accession
number ATCC 98623;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ff168_12 deposited under accession number ATCC 98623;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ff168_12 deposited under accession number
ATCC 98623;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ff168_12 deposited under accession number ATCC 98623;
(g) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:2;
(h) a polynudeotide 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;
(i) a polynucleotide 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
N0:1 from nucleotide 257 to nucleotide 622; the nucleotide sequence of the
full-length
protein coding sequence of clone ff168_12 deposited under accession number
ATCC
98623; or the nucleotide sequence of a mature protein coding sequence of clone
ff168_12
deposited under accession number ATCC 98623. In other preferred embodiments,
the
2
CA 02316864 2000-06-28
WO 99135252 PCT/US98I27903
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ff168_12 deposited under accession number ATCC 98623. 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: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 56
to amino acid 65 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID N0:1; and
2 0 (ab) the nucleotide sequence of the cDNA insert of done
ff168_12 deposited under accession number ATCC 98623;
(ii} hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
2 5 probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
3 0 the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID NO:1; and
(bb) the nucleotide sequence of the cDNA insert of clone
ff168_12 deposited under accession number ATCC 98623;
3
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
{ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
NO:1 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:1 , but
excluding the
poly(A) tail at the 3' end of SEQ ID NO:1. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID NO:1 from nucleotide 257 to nucleotide 622, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID NO:1 from nucleotide 257 to nucleotide 622, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide
257 to
nucleotide 622.
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) fragments of the amino acid sequence of SEQ ID N0:2, each
fragment comprising eight consecutive amino acids of SEQ ID N0:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone
ff168_12 deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 protein comprises the amino acid sequence of SEQ ID N0:2. In further
preferred
embodiments, the present invention provides 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 protein comprising a fragment of the amino acid
sequence of
3 0 SEQ ID N0:2 having biological activity, the fragment comprising the amino
acid sequence
from amino acid 56 to amino acid 65 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
4
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(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 1323 to nucleotide 1829;
(c} a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 1539 to nucleotide 1829;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ls9_1 deposited under accession number
ATCC 98623;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ls9_1 deposited under accession number ATCC 98623;
(f) a polynucleoHde comprising the nucleotide sequence of a mature
protein coding sequence of clone ls9_1 deposited under accession number ATCC
98623;
1 S (g} a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ls9_1 deposited under accession number ATCC 98623;
{h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID N0:4 having biological activity, the
fragment
comprising eight consecutive amino acids of SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
2 S 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
N0:3 from nucleotide 1323 to nucleotide 1829; the nucleotide sequence of SEQ
ID N0:3
3 0 from nucleotide 1539 to nucleotide 1829; the nucleotide sequence of the
full-length
protein coding sequence of clone ls9_1 deposited under accession number ATCC
98623;
or the nucleotide sequence of a mature protein coding sequence of clone ls9_1
deposited
under accession number ATCC 98623. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
5
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
of clone ls9_1 deposited under accession number ATCC 98623. 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 79
to amino acid 88 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the
3' end of SEQ ID N0:3; and
(ab) the nucleotide sequence of the cDNA insert of clone
2 0 ls9_1 deposited under accession number ATCC 98623;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
2 5 and
{b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
3 0 (ba) SEQ ID N0:3, but excluding the poly(A) tail at the
3' end of SEQ ID N0:3; and
(bb) the nucleotide sequence of the cDNA insert of clone
ls9_1 deposited under accession number ATCC 98623;
6
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:3 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:3 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:3. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:3 from nucleotide 1323 to nucleotide 1829, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:3 from nucleotide 1323 to nucleotide 1829, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide
1323 to
nucleotide 1829. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:3 from nucleotide 1539 to nucleotide 1829, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from
nucleotide 1539 to nucleotide 1829, to a nucleotide sequence corresponding to
the 3' end
2 0 of said sequence of SEQ ID N0:3 from nucleotide 1539 to nucleotide 1829.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
2 5 (b) fragments of the amino acid sequence of SEQ ID N0:4, each
fragment comprising eight consecutive amino acids of SEQ ID N0:4; and
(c) the amino acid sequence encoded by the cDNA insert of clone ls9_1
deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
3 0 protein comprises the amino acid sequence of SEQ ID N0:4. 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
7
CA 02316864 2000-06-28
WO 99/35252 PCTlUS98/27903
SEQ ID N0:4 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 79 to amino acid 88 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 507 to nucleotide 722;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 615 to nucleotide 722;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone na1010_1 deposited under accession
number ATCC 98623;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone na1010_1 deposited under accession number ATCC 98623;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone na1010_1 deposited under accession number
ATCC 98623;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone na1010_1 deposited under accession number ATCC 98623;
(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 polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 507 to nucleotide 722; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 615 to nucleotide 722; the nucleotide sequence of the full-length
protein coding
8
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
sequence of clone na1010_1 deposited under accession number ATCC 98623; or the
nucleotide sequence of a mature protein coding sequence of clone na1010_1
deposited
under accession number ATCC 98623. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone na1010_1 deposited under accession number ATCC 98623. 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 31
to amino acid 40 of SEQ ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:S.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
2 0 consisting of:
(aa) SEQ ID N0:5, but excluding the poly(A) tail at the
3' end of SEQ ID N0:5; and
(ab) the nucleotide sequence of the cDNA insert of clone
na1010_1 deposited under accession number ATCC 98623;
2 5 (ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
3 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
9
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(ba) SEQ ID N0:5, but excluding the poly(A) tail at the
3' end of SEQ ID N0:5; and
(bb) the nucleotide sequence of the cDNA insert of clone
na1010 1 deposited under accession number ATCC 98623;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:5 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:5 from nucleotide 507 to nucleotide 722, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:5 from nucleotide 507 to nucleotide 722, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide
507 to
nucleotide 722. Also preferably the polynucleotide isolated according to the
above
2 0 process comprises a nucleotide sequence corresponding to the cDNA sequence
of SEQ ID
N0:5 from nucleotide 615 to nucleotide 722, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from
nucleotide 615 to nucleotide 722, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:5 from nucleotide b15 to nucleotide 722.
2 5 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6, each
3 0 fragment comprising eight consecutive amino acids of SEQ ID N0:6; and
(c) the amino acid sequence encoded by the cDNA insert of clone
na1010_1 deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6. In further preferred
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
embodiments, 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 having biological activity, the fragment comprising the amino and
sequence
from amino acid 31 to amino acid 40 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 673 to nucleotide 987;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 868 to nucleotide 987;
1S (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone nf87_1 deposited under accession
number
ATCC 98623;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone nf87_1 deposited under accession number ATCC 98623;
2 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone nf87 1 deposited under accession number ATCC
98623;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone nf87 1 deposited under accession number ATCC 98623;
2 5 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:8;
3 0 (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
11
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(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
N0:7 from nucleotide 673 to nucleotide 987; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 868 to nucleotide 987; the nucleotide sequence of the full-length
protein coding
sequence of clone nf87_1 deposited under accession number ATCC 98623; or the
nucleotide sequence of a mature protein coding sequence of clone nf87_1
deposited under
accession number ATCC 98623. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone nf87 1
1 G deposited under accession number ATCC 98623. 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 N0:8 having biological activity,
the
fragment comprising the amino acid sequence from amino acid 47 to amino acid
56 of SEQ
ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
2 0 Further embodiments of the invention provide isolated polynucleotides
produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
{i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
2 5 consisting of:
(aa) SEQ ID N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
(ab) the nucleotide sequence of the cDNA insert of clone
nf87_1 deposited under accession number ATCC 98623;
3 0 (ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at b5 degrees C; and
{iii) isolating the DNA polynucleotides detected with the
probe(s);
and
12
CA 02316864 2000-06-28
WO 99135252 PCT/US98I27903
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
(bb) the nucleotide sequence of the cDNA insert of clone
nf87_1 deposited under accession number ATCC 98623;
(ii) hybridizing said primers) to human genomic DNA in
1 C~ conditions at least as stringent as 4X SSC at 65 degrees C;
(iii} amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and
extending
15 contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:7 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:7 from nucleotide 673 to nucleotide 987, and
extending
2 0 contiguously from a nucleotide sequence corresponding to the 5' end of
said sequence of
SEQ ID N0:7 from nucleotide 673 to nucleotide 987, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide
673 to
nucleotide 987. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
2 5 N0:7 from nucleotide 868 to nucleotide 987, and extending contiguously
from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:7 from
nucleotide 868 to nucleotide 987, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:7 from nucleotide 868 to nucleotide 987.
In other embodiments, the present invention provides a composition comprising
3 0 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8, each
fragment comprising eight consecutive amino acids of SEQ ID N0:8; and
13
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(c) the amino acid sequence encoded by the cDNA insert of clone
nf87_1 deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:8. 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 N0:8, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:8 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 47 to amino acid 56 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 57 to nucleotide 824;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 114 to nucleotide 824;
(d) a polynucleotide comprising the nucleotide sequence of the full-
2 0 length protein coding sequence of clone nh796_1 deposited under accession
number ATCC 98623;
(e} a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone nh796_1 deposited under accession number ATCC 98623;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 5 protein coding sequence of clone nh796_1 deposited under accession number
ATCC 98623;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone nh796_1 deposited under accession number ATCC 98623;
(h) a polynucleotide encoding a protein comprising the amino acid
3 0 sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:10;
14
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
(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
N0:9 from nucleotide 57 to nucleotide 824; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 114 to nucleotide 824; the nucleotide sequence of the full-length
protein coding
sequence of clone nh796_1 deposited under accession number ATCC 98623; or the
nucleotide sequence of a mature protein coding sequence of clone nh796_1
deposited
under accession number ATCC 98623. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone nh796_1 deposited under accession number ATCC 98623. 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:10, or a
polynucleotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10
having
2 0 biological activity, the fragment comprising the amino acid sequence from
amino acid 123
to amino acid 132 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced
2 5 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
3 0 (aa) SEQ ID N0:9, but excluding the poly(A) tail at the
3' end of SEQ ID N0:9; and
(ab) the nucleotide sequence of the cDNA insert of clone
nh796_1 deposited under accession number ATCC 98623;
IS
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:9, but excluding the poly(A) tail at the
3' end of SEQ ID N0:9; and
(bb) the nucleotide sequence of the cDNA insert of clone
nh796_1 deposited under accession number ATCC 98623;
{ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and
extending
2 0 contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID NO:9 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:9 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:9. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:9 from nucleotide 57 to nucleotide 824, and
extending
2 5 contiguously from a nucleotide sequence corresponding to the 5' end of
said sequence of
SEQ ID N0:9 from nucleotide 57 to nucleotide 824, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 57
to
nucleotide 824. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
3 0 N0:9 from nucleotide 114 to nucleotide 824, and extending contiguously
from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from
nucleotide 114 to nucleotide 824, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:9 from nucleotide 114 to nucleotide 824.
16
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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;
(h) fragments of the amino acid sequence of SEQ ID NO:10, each
fragment comprising eight consecutive amino acids of SEQ ID N0:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone
nh796_1 deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10. In further
preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:10 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 having biological activity, the fragment comprising the amino
acid
sequence from amino acid 123 to amino acid 132 of SEQ ID NO:10.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a poiynucleotide comprising the nucleotide sequence of SEQ ID
2 0 NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 297 to nucleotide 542;
{c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 510 to nucleotide 542;
2 S (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone nn229_1 deposited under accession
number ATCC 98623;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone nn229_1 deposited under accession number ATCC 98623;
3 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone nn229_1 deposited under accession number
ATCC 98623;
{g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone nn229_1 deposited under accession number ATCC 98623;
17
CA 02316864 2000-06-28
WO 99/35252 PCT/US98127903
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:12;
(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
N0:11 from nucleotide 297 to nucleotide 542; the nucleotide sequence of SEQ ID
NO:11
from nucleotide 510 to nucleotide 542; the nucleotide sequence of the full-
length protein
I 5 coding sequence of clone nn229_1 deposited under accession number ATCC
98623; or the
nucleotide sequence of a mature protein coding sequence of clone nn229_1
deposited
under accession number ATCC 98623. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone nn229_1 deposited under accession number ATCC 98623. In further
preferred
2 0 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
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
2 5 biological activity, the fragment comprising the amino acid sequence from
amino acid 36
to amino acid 45 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:11.
Further embodiments of the invention provide isolated polynucleotides produced
3 0 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
18
CA 02316864 2000-06-28
WO 99/35252 PCT/US98127903
(aa) SEQ ID N0:11, but excluding the poly(A) tail at the
3' end of SEQ ID NO:11; and
(ab) the nucleotide sequence of the cDNA insert of clone
nn229_1 deposited under accession number ATCC 98623;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID NO:11, but excluding the poly(A) tail at the
3' end of SEQ ID NO:11; and
(bb) the nucleotide sequence of the cDNA insert of clone
nn229_1 deposited under accession number ATCC 98623;
{ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C;
2 0 {iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:11, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
2 5 ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID
NO:11 , but
excluding the poly(A) tail at the 3' end of SEQ ID NO:11. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 297 to
nucleotide
542, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
3 0 of said sequence of SEQ ID NO:11 from nucleotide 297 to nucleotide 542, to
a nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID N0:11 from
nucleotide
297 to nucleotide 542. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
NO:lI from nucleotide 510 to nucleotide 542, and extending contiguously from a
19
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:11 from
nucleotide 510 to nucleotide 542, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID NO:11 from nucleotide 510 to nucleotide 542.
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, each
fragment comprising eight consecutive amino acids of SEQ ID N0:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
nn229_1 deposited under accession number ATCC 98623;
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
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 36 to amino acid 45 of SEQ ID N0:12.
2 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 S N0:13 from nucleotide 547 to nucleotide 750;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 601 to nucleotide 750;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone np156_1 deposited under accession
3 0 number ATCC 98623;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone np156_1 deposited under accession number ATCC 98623;
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone np156_1 deposited under accession number
ATCC 98623;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone np156_1 deposited under accession number ATCC 98623;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity, the fragment
comprising eight consecutive amino acids of SEQ ID N0:14;
(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
N0:13 from nucleotide 547 to nucleotide 750; the nucleotide sequence of SEQ ID
N0:13
from nucleotide 601 to nucleotide 750; the nucleotide sequence of the full-
length protein
2 0 coding sequence of clone np156_1 deposited under accession number ATCC
98623; or the
nucleotide sequence of a mature protein coding sequence of clone np156_1
deposited
under accession number ATCC 98623. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone np156_1 deposited under accession number ATCC 98623. In further
preferred
2 5 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:14 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) consecutive amino acids of SEQ ID N0:14, or a
polynucleotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14
having
3 0 biological activity, the fragment comprising the amino acid sequence from
amino acid 29
to amino acid 38 of SEQ ID N0:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
21
CA 02316864 2000-06-28
WO 99/35252 PCT/US98I27903
Further embodiments of the invention provide isolated polynudeotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:13, but excluding the poly{A) tail at the
3' end of SEQ ID N0:13; and
(ab) the nucleotide sequence of the cDNA insert of clone
np156_1 deposited under accession number ATCC 98623;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 65 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
2 0 {ba) SEQ ID N0:13, but excluding the poly(A) tail at the
3' end of SEQ ID N0:13; and
(bb) the nucleotide sequence of the cDNA insert of clone
np156_1 deposited under accession number ATCC 98623;
(ii) hybridizing said primers) to human genomic DNA in
2 5 conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13, and
3 0 extending contiguously from a nucleotide sequence corresponding to the 5'
end of SEQ
ID N0:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:13
, but
excluding the poly(A) tail at the 3' end of SEQ ID N0:13. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:13 from nucleotide 547 to
nucleotide
22
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
750, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
of said sequence of SEQ ID N0:13 from nucleotide 547 to nucleotide 750, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from
nucleotide
547 to nucleotide 750. Also preferably the polynucleotide isolated according
to the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:13 from nucleotide 601 to nucleotide 750, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:13 from
nucleotide 601 to nucleotide 750, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:13 from nucleotide 601 to nucleotide 750.
1 G In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) fragments of the amino acid sequence of SEQ ID N0:14, each
fragment comprising eight consecutive amino acids of SEQ ID N0:14; and
(c) the amino acid sequence encoded by the cDNA insert of clone
np156_1 deposited under accession number ATCC 98623;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:14. In further
preferred
2 C embodiments, the present invention provides a protein comprising a
fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) consecutive
amino
acids of SEQ ID N0:14, or a protein comprising a fragment of the amino acid
sequence of
SEQ ID N0:14 having biological activity, the fragment comprising the amino
acid
2 5 sequence from amino acid 29 to amino acid 38 of SEQ ID N0:14.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
3 C modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
23
CA 02316864 2000-06-28
WO 99/35252 PCT/US98127903
(b) purifying the protein from the culture.
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 DRAWIN
Figures IA and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
2 C sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature 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
2 5 sequence. For each disclosed protein applicants have identified what they
have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable
host
cell, is transported across or through a membrane, including transport as a
result of signal
3 0 sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
24
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/Z7903
Clone "ff168 12"
A polynucleotide of the present invention has been identified as clone "ff168
12".
ff168_22 was isolated from a human adult testes (teratocarcinoma NCCTT) 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.
ff168_12
is a full-length clone, including the entire coding sequence of a secreted
protein {also
referred to herein as "ff168_12 protein").
The nucleotide sequence of ff168_12 as presently determined is reported in SEQ
ID NO:1, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the ff168_12 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
ff168_12 should be approximately 1600 bp.
The nucleotide sequence disclosed herein for ff168_12 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ff168_12 demonstrated at least some similarity with
sequences
identified as AA025945 (ze91e02.r1 Soares fetal heart NbHHI9W Homo Sapiens
cDNA
clone 366362 5'), AA156237 (z150c09.s1 Soares pregnant uterus NbHPU Homo
sapiens
2 0 cDNA clone 505360 3'), AA420993 (zu08e09.s1 Soares testis NHT Homo Sapiens
cDNA
clone 731272 3'), N78486 (yz78e03.r1 Homo sapiens cDNA clone 289180 5'),
W01843
(za80a01.r1 Soares fetal lung NbHLI9W Homo Sapiens cDNA clone 298824 5'), and
W95777 (ze07e02.r1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone 358298
5').
Based upon sequence similarity, ff168 12 proteins and each similar protein or
peptide
2 5 may share at least some activity.
Clone "ls9 1"
A polynucleotide of the present invention has been identified as clone "ls9
1".
Is9_1 was isolated from a human adult brain (substantia nigra) cDNA library
using
3 0 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. ls9_1
is a full-
length clone, including the entire coding sequence of a secreted protein (also
referred to
herein as "ls9_1 protein").
CA 02316864 2000-06-28
WO 99/35252 PCT/US98I27903
The nucleotide sequence of ls9_1 as presently determined is reported in SEQ ID
N0:3, and includes a poly(A) tail. What applicants presently believe to be the
proper
reading frame and the predicted amino acid sequence of the ls9_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:4. Amino acids
60 to 72
of SEQ ID N0:4 are a predicted leader/signal sequence, with the predicted
mature amino
and sequence beginning at amino acid 73. Due to the hydrophobic nature of the
predicted
leader/signal sequence, it is likely to act as a transmembrane domain should
the predicted
leader/signal sequence not be separated from the remainder of the ls9_1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ls9_1 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for ls9_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ls9_1 demonstrated at least some similarity with
sequences
identified as AA527586 (ng42d05.s1 NCI CGAP_Co3 Homo sapiens cDNA clone
IMAGE:937449), AC000119 (Human BAC clone RG104I04 from 7q21-7q22, complete
sequence), T18551 (Human polycystic kidney disease normal PKDl gene), Y10196
(H.sapiens PEX gene), and 294721 (Human DNA sequence *** SEQUENCING IN
PROGRESS *** from clone 167A14; HTGS phase 1). The predicted amino acid
sequence
disclosed herein for ls9_1 was searched against the GenPept and GeneSeq amino
acid
2 0 sequence databases using the BLASTX search protocol. The predicted ls9_1
protein
demonstrated at least some similarity to sequences identified as AB002375
(KIAA0377
[Homo Sapiens]) and 895913 (Neural thread protein). Based upon sequence
similarity,
ls9_1 proteins and each similar protein or peptide may share at least some
activity. The
TopPredII computer program predicts an additional potential transmembrane
domain
2 5 within the ls9_1 protein sequence centered around amino acid 40 of SEQ ID
N0:4. The
nucleotide sequence of ls9_1 indicates that it may contain an Alu/SVA
repetitive element.
Clone "na1010 1"
A polynucleotide of the present invention has been identified as clone
"na1010_1".
3 0 na1010_1 was isolated from a human adult brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. na1010_1 is a full-
length
26
CA 02316864 2000-06-28
WO 99!35252 PCTIUS98/27903
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "na1010_1 protein").
The nucleotide sequence of na1010_1 as presently determined is reported in SEQ
ID N0:5, and includes a poly(A) tail. What applicants presently believe to be
the proper
S reading frame and the predicted amino acid sequence of the naI010_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino
acids 24 to 36 of SEQ ID N0:6 are a predicted leader/signal sequence, with the
predicted
mature amino acid sequence beginning at amino acid 37. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
na1010_I protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
na1010_1 should be approximately 1050 bp.
The nucleotide sequence disclosed herein for na1010_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. na1010_1 demonstrated at least some similarity with
sequences
identified as AC002091 (Genomic sequence from Human 17, complete sequence),
AC002382 (Human BAC clone RG022J17 from 7q21, complete sequence), and M26434
(Human hypoxanthine phosphoribosyltransferase (HPRT) gene, complete cds).
Based
2 0 upon sequence similarity, na1020_1 proteins and each similar protein or
peptide may
share at least some activity. The nucleotide sequence of na1010_1 indicates
that it may
contain one or more of the following repetitive elements: Ll/A/MIR/SVA/LTRII,
Alu/SVA/A/GAA, or Alu/A/GAAAA.
Clone "nf87 1"
A polynucleotide of the present invention has been identified as clone
"nf87_1".
nf87 1 was isolated from a human adult brain (substantia nigra) 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
3 0 of computer analysis of the amino acid sequence of the encoded protein.
nf87_1 is a full
length clone, including the entire coding sequence of a secreted protein (also
referred to
herein as "nf87_1 protein").
The nucleotide sequence of nf87 1 as presently determined is reported in SEQ
ID
N0:7, and includes a poly(A) tail. What applicants presently believe to be the
proper
27
CA 02316864 2000-06-28
WO 99/35252 PCT/US98I27903
reading frame and the predicted amino acid sequence of the nf87_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:8. Amino acids
53 to 65
of SEQ ID N0:8 are a predicted ieader/signal sequence, with the predicted
mature amino
acid sequence beginning at amino acid 66. Due to the hydrophobic nature of the
predicted
leader/signal sequence, it is likely to act as a transmembrane domain should
the predicted
leader/signal sequence not be separated from the remainder of the nf87 1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
nf87_1 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for nf87 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. nf87_1 demonstrated at least some similarity with
sequences
identified as AA358277 (EST67398 Fetal lung III Homo sapiens cDNA 5' end
similar to
similar to interferon-alpha-inducible gene p27), W52706 (zc55g02.r1 Soares
senescent
fibrobiasts NbHSF Homo Sapiens cDNA clone 326258 5' similar to SW
IIVI7_HLnVIAN
P40305 INTERFERON-ALPHA INDUCED 11.5 KD PROTEIN), and X67325 (H.sapiens
p27 mRNA). The predicted amino acid sequence disclosed herein for nf87_1 was
searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted nf87_1 protein demonstrated at least some
similarity to
sequences identified as X67325 (p27 gene product [Homo sapiens]). The
interferon alpha-
2 0 inducible gene is localized on human chromosome 14q32 and expresses the
highly
hydrophobic p27 gene product in breast carcinoma cells. Based upon sequence
similarity,
nf87 1 proteins and each similar protein or peptide may share at least some
activity.
nf87 1 protein was expressed in a COS cell expression system, and an expressed
protein band of approximately 16 kDa was detected in membrane fractions using
SDS
2 5 polyacrylamide gel electrophoresis.
Clone "nh796 1"
A polynucleotide of the present invention has been identified as clone
"nh796_1".
nh796_1 was isolated from a human adult brain (thalamus) cDNA library using
methods
3 0 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. nh796_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"nh796_1 protein").
28
CA 02316864 2000-06-28
WO 99!35252 PCT/US98127903
The nucleotide sequence of nh796_1 as presently determined is reported in SEQ
ID N0:9, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the nh796_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:10. Amino
acids 7 to 19 of SEQ ID N0:10 are a predicted leader/signal sequence, with the
predicted
mature amino acid sequence beginning at amino acid 20. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
nh796_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
done
nh796_1 should be approximately 1050 bp.
The nucleotide sequence disclosed herein for nh796_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. nh796_1 demonstrated at least some similarity with
sequences
identified as AA315985 (EST18772 Lung Homo sapiens cDNA 5' end), N23239
(yw47b07.s1 Homo sapiens cDNA clone 255349 3'), N27741 (yw51c06.s1 Homo
sapiens
cDNA clone 255754 3'), U69172 (Mus musculus unknown protein mRNA, complete
cds),
and 224371 (H. sapiens (D20S195) DNA segment containing (CA) repeat; clone
AFM321xc1; single read). The predicted amino acid sequence disclosed herein
for
2 0 nh796_l was searched against the GenPept and GeneSeq amino acid sequence
databases
using the BLASTX search protocol. The predicted nh796_1 protein demonstrated
at least
some similarity to sequences identified as U69172 (unknown [Mus musculus]).
The
mouse protein of unknown function {U69172) is expressed in late palate
development.
Based upon sequence similarity, nh796_1 proteins and each similar protein or
peptide
2 5 may share at least some activity.
nh796_1 protein was expressed in a COS cell expression system, and an
expressed
protein band of approximately 25 kDa was detected in conditioned media and
membrane
fractions using SDS polyacrylamide gel electrophoresis.
3 0 Clone "nn229 1"
A polynucleotide of the present invention has been identified as clone
"nn229_1".
nn229_1 was isolated from a human fetal kidney (293 cell line) 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
29
CA 02316864 2000-06-28
WO 99/35252 PCT/US98127903
of computer analysis of the amino acid sequence of the encoded protein.
nn229_1 is a full-
length clone, including the entire coding sequence of a secreted protein (also
referred to
herein as "nn229_1 pxoteiri').
The nucleotide sequence of nn229_1 as presently determined is reported in SEQ
ID N0:11, and includes a poly(A) tail. What applicants presently believe to be
the proper
reading frame and the predicted amino acid sequence of the nn229_l protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:12. Amino
acids 59 to 7I of SEQ ID N0:12 are a predicted leader/signal sequence, with
the predicted
mature amino acid sequence beginning at amino acid 72. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
nn229_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
nn229_1 should be approximately 1050 bp.
The nucleotide sequence disclosed herein for nn229_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. nn229_1 demonstrated at least some similarity with
sequences
identified as H24014 (ym49f02.s1 Homo Sapiens cDNA clone 51480 3'), 808508
(ye95hOl.r1
Homo sapiens cDNA clone 125521 5' similar to gb I M87910 I HUMALNE34 Human
2 0 carcinoma cell-derived Alu RNA transcript, (rRNA); gb J02931 TISSUE FACTOR
PRECURSOR (HUMAN)), and 296508 (H.sapiens telomeric DNA sequence, clone
22QTEL030, read 22QTEL00030.seq). Based upon sequence similarity, nn229_1
proteins
and each similar protein or peptide may share at least some activity. The
TopPredII
computer program predicts a potential transmembrane domain within the nn229_1
2 S protein sequence centered around amino acid 20 of SEQ ID N0:12. The
nucleotide
sequence of nn229_1 indicates that it may contain a MER20 repetitive element.
Clone "n 1
A polynucleotide of the present invention has been identified as clone
"np156_1".
3 0 np156_1 was isolated from a human fetal kidney (293 cell line) 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.
np156_1 is a full-
CA 02316864 2000-06-28
WO 99135252 PCTIUS98I27903
length clone, including the entire coding sequence of a secreted protein (also
referred to
herein as "np156_1 protein').
The nucleotide sequence of np156_1 as presently determined is reported in SEQ
ID N0:13, and includes a poly(A) tail. What applicants presently believe to be
the proper
S reading frame and the predicted amino acid sequence of the np156_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:14. Amino
acids 6 to 18 of SEQ ID N0:14 are a predicted leader/signal sequence, with the
predicted
mature amino acid sequence beginning at amino acid 19. Due to the hydrophobic
nature
of the predicted leader/signal sequence, it is likely to act as a
transmembrane domain
should the predicted leader/signal sequence not be separated from the
remainder of the
np156_1 protein.
The EcoRI /NotI restriction fragment obtainable from the deposit containing
clone
np156_1 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for np156_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. np156_1 demonstrated at least some similarity with
sequences
identified as AA298580 (EST114211 HSC172 cells I Homo sapiens cDNA 5' end),
AA447514 (zw81a05.s1 Soares testis NHT Homo sapiens cDNA clone 782576 3'),
AC002309 (*** SEQUENCING IN PROGRESS *** Human Chromosome 22q11 Cosmid
2 0 Clone 63e9; HTGS phase 1, 3 unordered pieces), AF007269 (Arabidopsis
thaliana BAC
IG002N01), and N53641 (yz04g03.r1 Homo sapiens cDNA clone 282100 5'). Based
upon
sequence similarity, np156_1 proteins and each similar protein or peptide may
share at
least some activity.
Deposit of Clones
Clones ff168_12, ls9_1, na1010_1, nf87_l, nh796_l, nn229_1, and np156_1 were
deposited on December 31, 1997 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 98623, from which
each
3 0 clone comprising a particular polynucleotide is obtainable. All
restrictions on the
availability to the public of the deposited material will be irrevocably
removed upon the
granting of the patent, except for the requirements specified in 37 C.F.R. ~
1.808(b), and
the term of the deposit will comply with 37 C.F.R. ~ 1.806.
31
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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 1A 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. CeII. 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
can still be isolated by digestion with EcoRI and NotI. However, NotI will
then produce
the 5' site and EcoRI will produce the 3' site for placement of the cDNA in
proper
orientation for expression in a suitable vector. The cDNA may also be
expressed from the
vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
2 0 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
identified below, and should be most reliable in isolating the clone of
interest.
Clone P_rQb_ a Seauence
ff168_12 SEQ ID N0:15
ls9_1 SEQ ID N0:16
na1010_1 SEQ ID N0:17
~8? 1 sEQ ro No:lB
nh796_1 SEQ ID N0:19
3 0 nn229_1 SEQ ID N0:20
np156_1 SEQ iD N0:21
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
32
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98I27903
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 y 32P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 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
2 0 grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
2 5 known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/Iiter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
3 C NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to le+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
33
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
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
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 ~ 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
1 S immunoglobulins for many purposes, including increasing the valency of
protein binding
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
2 0 decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature
forms) of such protein may be obtained by expression of the disclosed full-
length
2 5 polynucleotide (preferably those deposited with ATCC) in a suitable
mammalian cell or
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
3 0 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
34
CA 02316864 2000-06-28
WO 99/35252 PCTNS98127903
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
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.
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 "UruGene clusters" of overlapping sequences. Many of the "UniGene
clusters"
2 0 so identified will already have been mapped to particular chromosomal
sites.
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
2 5 gene (Albert and Morris,1994, Trends Pharmacol. Sci. 15(7): 250-254;
Lavarosky et al.,1997,
Biochem. Mol. Med. 62(1):11-22; and I-iampel,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
3 0 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
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
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 (Piasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative genetic selection strategies (Mansour
et al.,1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
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
2 0 transmembrane domains of proteins of the invention can be identified in
accordance with
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
2 5 amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
3 0 identity) with that disclosed protein, where sequence identity is
determined by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
36
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
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
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
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
2 0 routines are integral to the database search itself, and thus yield much
better sensitivity and
selectivity while producing the more easily interpreted output. Gapping can
optionally be
fumed 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
integer value including zero, one through eight, nine, ten, eleven, twelve
through twenty,
2 5 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
3 0 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.
37
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
Species homologues of the disclosed polynucleotides and proteins are also
provided by the present invention. As used herein, a "species homologue" is a
protein or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
the given polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues may be isolated and identified by making suitable probes or primers
from
the sequences provided herein and screening a suitable nucleic acid source
from the
desired species. Preferably, species homologues are those isolated from
mammalian
spe~es. Most preferably, speaes homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hylobates
concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus
norvegicus,
Cricetulus griseus, Felis catus, Mustela vison, Canis famiIiaris, Oryctolagus
cuniculus, Bos taurus,
2 0 Ovis aries, Sus scrofa, and Eguus caballus, for which genetic maps have
been created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
(O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al.,
1993, Nature
Genetics 3:103-1I2; Johansson et al., 1995, Genomics 25: 682-690; Lyons et
al., 1997, Nature
2 5 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 sigruficantiy similar
sequences to
3 0 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
38
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
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 polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
1 G least as stringent as, for example, conditions A-F; stringent conditions
are at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
39
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
StringencyPolynucleotideHybrid Hybridization TemperatureWash
ConditionHybrid Length and Temperature
~P); Buffer' and Buffer'
A DNA:DNA 2 50 65 C;1 xSSC -or- 65 C; 0.3xSSC
42C; lxSSC, 50%
formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50%
formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA 2 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50%
formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA 2 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50%
formamide
1 H DNA:DNA <50 TN*; 4xSSC TH*; 4xSSC
L7
I DNA:RNA 2 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50%
formamide
J DNA:RNA <50- T~*; 4xSSC T~*; 4xSSC
K RNA:RNA s 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50%
formamide
L RNA:RNA <50 TL*; 2xSSC TL*; ~~
M DNA:DNA 2 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50%
formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z SO 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50%
formamide
P DNA:RNA <50 T,,*; 6xSSC TP*; 6xSSC
Q RNA:RNA 2 50 60C; 4xSSC -or- 6pC; ~~
45C; 6xSSC, 50%
formamide
2 R RNA:RNA <50 TR*; 4xSSC TR~ 4~~
~
;: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing poiynucleotides. When
hybridizing a polynucleotide to a target polynucleoHde of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is O.15M NaCI, lOmM NaHZPO;, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases}. For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,°[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).
CA 02316864 2000-06-28
WO 99/35252 PCT/US98I27903
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 ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vi o 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
41
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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,
1 G e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and
such methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987, incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein rnay 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
Sepharose0; 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 ("Flag") is commercially available from the Eastman
Kodak
Company (New Haven, CT).
42
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
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
S 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
43
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/2'7903
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
1 C 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.
44
CA 02316864 2000-06-28
WO 99135252 PCTIUS98/27903
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.
Cytokine and Pll Proliferation/Differentiation Activitv
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
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98/27903
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor-dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761,1994.
Assays for rytokine production and/or proliferation of spleen cells, lymph
node
2 0 cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, 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 I pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto.1994.
2 5 Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
3 0 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin I1- 8ennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
46
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, j.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiiey-
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.
Immure.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Suppressing Activit~t
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
2 0 deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
2 5 diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
3 0 treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
47
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to regulate 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
1 S 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
2 0 limitation B lymphocyte antigen functions (such as , for example, B7)),
e.g., preventing
high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
2 5 through its recognition as foreign by T cells, followed by an immune
reaction that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
3 0 1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
48
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et aL,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
2 0 activation of autoreactive T cells may reduce or eliminate disease
symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
2 5 antigen-specific tolerance of autoreactive T cells which could lead to
long-term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythrnatosis in MRL/Ipr/Ipr mice or NZB hybrid
mice,
3 0 marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a 8 lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
49
CA 02316864 2000-06-28
WO 99/35252 PCT/US98I27903
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costirnulating 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,
2 C carcinoma) transfected with a nucleic acid encoding at least one peptide
of the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
3 0 lymphocyte antigens) on the surface of the tumor cell provides the
necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
(3Z
microglobulin protein or an MHC class II a chain protein and an MHC class II
~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.
2 0 USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974,1982;
Handa et al.,
J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-3500,1986;
Takai et al.,
j. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
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.
2 5 Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et ai., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Th1 /Th2 profiles) include, without limitation,
those described
3 0 in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Taronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
51
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
those described in: Current Protocols in Immunology, Ed by j. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., j. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783,1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989;
Bhardwaj
et al., journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
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,
2 0 Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-
897, 1993;
Gorczyca et al., International journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
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
2 5 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,
1991.
Hematopoiesis Re ulating 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
3 0 marginal biological activity in support of colony forming cells or of
factor-dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
52
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
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.
2 0 Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis} include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-
151,1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
2 5 Assays for stem cell survival and differentiation (which will identify,
among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
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
3 0 hematopoietic colony forming cells with high proliferative potential,
McNiece, LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et a1. 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
53
CA 02316864 2000-06-28
WO 99!35252 PCT/US98/27903
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
I62, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
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.
2 0 A protein of this invention may also be used in the treatment of
periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
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
2 5 of bone and/or cartilage repair or by blocking inflammation or processes
of tissue
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
3 0 invention, which induces tendon/ligament-like tissue or other tissue
formation in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
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
54
CA 02316864 2000-06-28
WO 99!35252 PCT/US98/27903
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivv to effect
tissue repair. The
1 C compositions of the invention may also be useful in the treatment of
tendirutis, 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
2 0 localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson s disease, Huntingtori 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
2 5 from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
3 0 It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs {including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing,, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin 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.
56
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98I27903
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, eosinophiIs, 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.
57
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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-
176$,1994.
Hemostatic and ThrombolvHc Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolyHc activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474,1988.
Receptor/Ligand Acti
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:
58
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
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 Acti
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
59
CA 02316864 2000-06-28
WO 99/35252 PCTIUS98I27903
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.
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-
1038,1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
antibody-dependent cell-mediated cytotoxicity (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
2 0 inhibiting angiogenesis), by causing production of other factors, agents
or cell types which
inhibit tumor growth, or by suppressing, eliminating or inhibiting factors,
agents or cell
types which promote tumor growth.
Other Activities
2 5 A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing} bodily characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
3 0 or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
61
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders}, depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
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
2 0 effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, Ih-2, IL-3, IL,-4, I2,-5, ILr6, IL-7, IL-8,
IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
2 5 cell factor, and erythropoietin. The pharmaceutical composition may
further contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
3 0 in formulations of the particular cytokine, lymphokine, other
hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
62
CA 02316864 2000-06-28
WO 99/35252 PCT/US98127903
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers} or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
2 0 pharmaceutically acceptable carriers, with amphipathic agents such as
lipids which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
2 5 art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent
No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
3 0 sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
63
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytolcines,
lympholcines
or other hematopoietic factors. When co-administered with one or more
cytolcines,
lympholcines 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
cytolcine(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.
2 G Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
2 5 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid Garner 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
3 0 physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
64
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringer s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
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
2 0 increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 lZg
to about 100
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
2 5 present invention will vary, depending on the severity of the disease
being treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
3 0 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
CA 02316864 2000-06-28
WO 99135252 PCT/US98/2~903
SEQUENCE LISTING
<110> Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Merberg, David
Treacy, Maurice
Agostino, Michael J.
Steininger II, Robert J.
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> 6058A
<140>
<141>
<160> 21
<I70> PatentIn Ver. 2.0
<210> 1
<211> 1527
<212> DNA
<213> Homo Sapiens
<400> 1
tgaacaacaa gctaaaatgg aatagcacag aatggctgag gagccactgt gaagaaaggc 60
atgcagccat gaaactgcag tgtcccttgc tgttagtggg gtggctctga ccaatctgga 120
agatacagaa aatgccaaga gagcctacgc agaagcagtc cacctggata agtgtaaccc 180
tttagtaaac ctgaactatg ctgtgctgct gtacaaccag ggcgagaaga agaacgccct 240
ggcccaatat caggagatgg agaagaaagt cagcctactc aaggacaata gctctctgga 300
atttgactct gagatggtgg agatggctca gaagttggga gctgctctcc aggttgggga 360
ggcactggtc tggaccaaac cagttaaaga tcccaaatca aagcaccaga ccacttcaac 420
cagcaaacct gccagtttcc agcagcctct gggctctaat caagctctag gacaggcaat 480
gtcttcagca gctgcataca ggacgctccc ctcaggtgct ggaggaacat cccagttcac 540
aaagccccca tctcttcctc tggagccaga gcctgcggtg gaatcaagtc caactgaaac 600
atcagaacaa ataagagaga aataagaata gaatgaatga ccccaaaata gggttttctt 660
gggcgaggat gtgctggatt aggaaaggtg acatgacaca ggcagagcag agtggcaccc 720
accacagaat acagtgtgtg ttattacgag gagccagcag ttgagcctaa ggtccttcta 780
cctacctggt attggcattt gaggtcggaa accctctact gccccataag ccaggaaaag 840
tgaaaagaga acacagttcc tttaagaact ggcagcaagg cttgaggcct tatgtatgta 900
gctgagtcag caaggtacat gatgctgtct gctttcaaaa ggacttttct ctcctagctg 960
actgactcct tccttagttc aaggaacagc tgagacagac ctctgctgag tagctctgtg 1020
atgacsaagc cttggtttaa ctgaggtgat cctcaggttg tgaggtttat tagtccccaa 1080
ggcaaacaca aatattagat taataatcca actttaatag tatacattta aaagaaaaaa 1140
aacaaaagcc ctggaagttg aggccaagcc tgctgagtat tgcagctgca tttgcccaaa 1200
gggaatccag aacaagtccc tccmtgtatt ttgttcttga gaggggtcag tctagaagct 1260
agatcctatc aggatgagga gcagcagccc agggcttgtc tggatmagca ccaacgattt 1320
taaagaaaaa aggaagagtt tcttagatga gtaattgtta ttgaagatag tcagtgataa 1380
ccactgacca gatgctatca atacastatg tgtccttttt agaataaaga ttacatatca 1440
tcatttcctt tggggaaaat tgttattcag gtataaaaac nagagatcat aataaaaacc 1500
taaaagaacc taaaaaaaaa aaaaaaa 1527
<210> 2
<211> 122
<212> PRT
<213> Homo Sapiens
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
<400> 2
Met Glu Lys Lys Val Ser Leu Leu Lys Asp Asn Ser Ser Leu Glu Phe
1 5 10 15
Asp Ser Glu Met Val Glu Met Ala Gln Lys Leu Gly Ala Ala Leu Gln
20 25 30
Val Gly Glu Ala Leu Val Trp Thr Lys Pro Val Lys Asp Pro Lys Ser
35 40 45
Lys His Gln Thr Thr Ser Thr Ser Lys Pro Ala Ser Phe Gln Gln Pro
50 55 60
Leu Gly Ser Asn Gln Ala Leu Gly Gln Ala Met Ser Ser Ala Ala Ala
65 70 75 80
Tyr Arg Thr Leu Pro Ser Gly Ala Gly Gly Thr Ser Gln Phe Thr Lys
85 90 95
Pro Pro Ser Leu Pro Leu Glu Pro Glu Pro Ala Val Glu Ser Ser Pro
100 105 110
Thr Glu Thr Ser Glu Gln Ile Arg Glu Lys
115 120
<210> 3
<211> 2352
<212> DNA
<213> Homo sapiens
<400> 3
agcagagtga gctgaagctc ctgaggaggg ttcccgaagg ggggcgctca gagatggggg 60
cagggggcgg ggagaggaga gtctgcctta tgtcccttcc ttgtggactt cacatggtca 120
tgcaggaagt gaggatgggt gtccagcggg ggccgaggcc actagtatcc tcctgcttcc 180
ccctgccatt ctccagggct ggactgaccc tatggactgg gagagagtgc ctgaggccac 240
catgccacag tcaaaggggg tcctatctca gaaggtggca gcatccactg agatatcctc 300
acccgaaggg aaggaggctg ctgggtagca aataagcccc ttcttttctt ggtgagttga 360
tgacctccaa tagctcccag tgtcatgggt acccagtacg cattagctgg tgttgggttg 420
attgagacct ggggcagttc ctggggcasg aagccagatg ggagatgaga tagaaagtgt 480
taggagttat cctctttgcc tggcctttga gaataactta ctgtgtgact ttgggcaagt 540
tccttcccca ctctgggcct cagtttctca cttgggaaag caaggagttt gaccagatga 600
tcacaatggg ccttcctagc tctggccacc aagaatttgt gaacattaga gctcctggtc 660
tggtgggtag agccagagct gctgactggt ctctctgcct ccagagggga tttattggac 720
ctcagaggtg gcagggccct atggagcacc aactgccctc aaccccaccc tgtgcccaag 780
actgggaagg gattgatgtc aggctgtggc cataggtagc atgagttgcc caaggaggga 840
cagagcatat ctttgctgag gcttggctga ggggcttatg atagggcttg cagtacctca 900
cagccccctg tgggcacaga caccctgagg tttacccagg caaatatatt gattagcagg 960
acaagggctc tctcctcagt ttctgctccc ttccatctct ctcccatact tgtctgaaaa 1020
gggagacaaa aaatcttata caagtggcat ctcaatcctt tccagtccag cacctcctgg 1080
ggcaggcagt gtgacttatt tcctgtggtg aaatcacctg tttcacaagc ctggcagcgc 1140
gacttctgag tctcatgacc actcaaccca agggacccct ccccagacca gaaccaagtc 1200
agctgggggc tgtcgtacta ccctgtccag tcttgagggc ctagttgcag gtcccccagg 1260
catccagccc ctcctagagc ttgctgggca ggctgcacct catctgggca ggcgcagagc 1320
tgatgaaatg ctggagcaat gcatggcaaa catatgccct ccagtgtctt ctgaaacctt 1380
tggggctgac acaagatcct ttagtgtttg ggatgacctc tttcctgcag acttcttccc 1440
ctatccctaa ctcatgcatg gaaaacgttt gtcaggctgg tttcccgagc ctcctgcacc 1500
tcaacatcac gctcaccctt ttgggtttag cccagtgtta tttagcaaat ttctccagct 1560
gcagggaagg atcagagcac tatctttttt tttttttttt ctcctggagc caggactgca 1620
2
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
caaggcaatg gccaaattta gttgaattca gcctaccatc ctttgctgat gactcagctc 1680
tatgccaagt actggagcca cagagatggg tcagtcccag cccctgtcct caggaagccc 1740
atggtcaggg aaacgttgta gggataagta atagagggca gttgccttca gggctcctgg 1800
tggctgctgg tccctatggt gccttgatgt gaattagaag acggtgccct ttccaggtgg 1860
attcagacct acactagaac gcacagcttt gggagtgaca cacaggttgg attttagcac 1920
cccttgcccc ttggccagag gtgccctgct gcacggccat acgctgcagc ctcgagggac 1980
acacaggcca aagtgtttcc ttcagcctct tcctggagag gasgccgcag gtcatgtttc 2040
caagcttctg gtctcaaact cttggcctca agggatcctc ctacctcggc ctccgaaagt 2100
gctgggatta caggtgtgag ccaccatgcc tggcctcact gtgtagttgt gaatagctta 2160
atagtttgca atgtggtgct tctcacagct cttctctgta atgggaacat gaaaaattac 2220
ctggtacagt tttatgcttt gtggtgtggc ttttaatttt tataaacatg tcttactgct 2280
attgccaggg atttagattt ttaataaact tccagataca acagtaaaaa aaaaaaaaaa 2340
aaaaaaaaaa as 2352
<210> 4
<211> 169
<212> PRT
<213> Homo sapiens
<400> 4
Met Lys Cys Tzp Ser Asn Ala Trp Gln Thr Tyr Ala Leu Gln Cys Leu
1 5 10 15
Leu Lys Pro Leu Gly Leu Thr Gln Asp Pro Leu Val Phe Gly Met Thr
20 25 30
Ser Phe Leu Gln Thr Ser Ser Pro Ile Pro Asn Ser Cys Met Glu Asn
35 40 45
Val Cys Gln Ala Gly Phe Pro Ser Leu Leu His Leu Asn Ile Thr Leu
50 55 60
Thr Leu Leu Gly Leu Ala Gln Cys Tyr Leu Ala Asn Phe Ser Ser Cys
65 70 75 80
Arg Glu Gly Ser Glu His Tyr Leu Phe Phe Phe Phe Phe Ser Trp Ser
85 90 95
Gln Asp Cys Thr Arg Gln Trp Pro Asn Leu Val Glu Phe Ser Leu Pro
100 105 110
Ser Phe Ala Asp Asp Ser Ala Leu Cys Gln Val Leu Glu Pro Gln Arg
115 120 125
Trp Val Ser Pro Ser Pro Cys Pro Gln Glu Ala His Gly Gln Gly Asn
130 135 140
Val Val Gly Ile Ser Asn Arg Gly Gln Leu Pro Ser Gly Leu Leu Val
145 150 155 160
Ala Ala Gly Pro Tyr Gly Ala Leu Met
165
<210> 5
<211> 995
<212> DNA
<213> Homo sapiens
<220>
3
CA 02316864 2000-06-28
WO 99135252 PCTIUS98127903
<221> unsure
<222> (852)
<400> 5
ctaaaccctt cctccagcct ctacctcctg caaaccatcg tccatattgc aagcaatcag 60
atttatttat ttattttttg aggcaggaga atggcgtgaa cccgggaggc aaagcttgca 120
gtgagccaag atcgcaccac tgcactccag cctgggtgac agagcgagac tctgtctcaa 180
aaaaaaaaaa saaaaagaaa agaaaaaaac ctattgccta cctcccaagg gcaaatgcag 240
cctggtgttt ggctccaagt ctgcttcagc tttggctccc atcactccgc tttccttttg 300
cctcaactta agatcttgcc acatgtacac ttcccataac attccagctg agaggctttt 360
gtatacgagg ggtttttttt tgtttgtttt gccwagaatg atcctccctg gtgaatctta 420
gcttaaatca ccaggcagtt aagcaggctt ttctctatga tttcaccccc actttgtata 480
tttctgtgat tagtcctgaa catcccatgt tgtactgttt acctctctca ctggacttag 540
aaattctgaa gaacagaaac aaaaagtttt ctctttctct gtatgttctt tttttgttgt 600
tattattatt gacttggtat atcttctttc agatgtattt tcttttattc tcaacacaaa 660
gtaattttaa catgatcttt ctgggccaaa attttcttat ctgtaaaatg aagatgttgg ?20
actaggattc agggcttctt aactaaagaa ttcaatagat gatgctggga caagtgtata 780
tctacctgta aaggaatgaa gttggacccc ttcctcatac tatacacaaa aattaactca 640
aaatggatca tngacctaaa cataagagct aaaactataa gactttcaga agaaaacaca 900
ggagtaagtc ttcatgacct tggattaagg aatggttgct tagatatgac acccaaaaaa 960
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa gg5
<210> 6
<211> 72
<212> PRT
<213> Homo sapiens
<400> 6
Met Leu Tyr Cys Leu Pro Leu Ser Leu Asp Leu Glu Ile Leu Lys Asn
1 5 10 15
Arg Asn Lys Lys Phe Ser Leu Ser Leu Tyr Val Leu Phe Leu Leu Leu
20 25 30
Leu Leu Leu Thr Trp Tyr Ile Phe Phe Gln Met Tyr Phe Leu Leu Phe
35 40 45
Ser Thr Gln Ser Asn Phe Asn Met Ile Phe Leu Gly Gln Asn Phe Leu
50 55 60
Ile Cys Lys Met Lys Met Leu Asp
65 70
<210> 7
<211> 1038
<212> DNA
<213> Homo sapiens
<400> 7
gacggcctca ccatgatgaa acgggcagct gctgctgcag tgggaggagg taagttaccc 60
ggatcgcctg tctccaggcc ctcacctagc ctggtccccg ggctgctggg agaacgcaga 120
gatgaggcgc tgggctggct ctcaccctcc acttccgaag ctgcccgagt agcctgagtg 180
agccacagca tcaaaatact ccagggaaaa gctcactccc attcctgacc cagcttctct 240
tctagtcctt atgtcgaata agcataggag gaagatcgtt tgaaagarga tttgcagcta 300
aactccacgt ggcttatttc acatttatgc gtggacacac acacacacac acacacacac 360
acacaaattt gagaccaatg aagggtattg acttcctcag catcacacag caagttagag 420
acaaaccagg gccatggctg gtccttctat gacatctttg cttcacctgg ctccacactc 480
caccttttct tcaccagaag accactaagt tgccatctct gtattgctca agctgacagt 540
ctccggaaac tgtcaaggaa ttcctaagcg gggggcgggg ggaagggtcc cttctcctga 600
4
CA 02316864 2000-06-28
WO 99135252 PCTIUS98127903
gcccacctct gcactcagct tctctctccc acagccctgg cagtgggggc tgtgcccgtg 660
gtgctcagtg ccatgggctt cactggggca ggaatcgccg cgtcctccat agcagccaag 720
atgatgtccg cagcagccat tgccaacggg ggtggtgttt ctgcggggag cctggtggct 780
actctgcagt ccgtgggggc agctggactc tccacatcat ccaacatcct cctggcctct 840
gttgggtcag tgtkgggggc ctgctkgggg aattcacctt cttcttctct cccagctgaa 900
cccgaggcta aagaagatga ggcaagagaa aatgtacccc aaggtgaacc tccaaaaccc 960
ccactcaagt cagagaaaca tgaggaataa aggtcacatg cagatgcata aaaaaaaaaa 1020
aaaaaaaaaa aaaaaaaa 1038
<210> 8
<211> 105
<212> PRT
<213> Homo sapiens
<220>
<221> UNSURE
<222> (61)
<220>
<221> UNSURE
<222> (65)
<400> 8
Met Gly Phe Thr Gly Ala Gly Ile Ala Ala Ser Ser Ile Ala Ala Lys
1 5 10 15
Met Met Ser Ala Ala Ala Ile Ala Asn Gly Gly Gly Val Ser Ala Gly
20 25 30
Ser Leu Val Ala Thr Leu Gln Ser Val Gly Ala Ala Gly Leu Ser Thr
35 40 45
Ser Ser Asn Ile Leu Leu Ala Ser Val Gly Ser Val Xaa Gly Ala Cys
50 55 60
Xaa Gly Asn Ser Pro Ser Ser Ser Leu Pro Ala Glu Pro Glu Ala Lys
65 70 75 80
Glu Asp Glu Ala Arg Glu Asn Val Pro Gln Gly Glu Pro Pro Lys Pro
85 90 95
Pro Leu Lys Ser Glu Lys His Glu Glu
100 105
<210> 9
<211> 1060
<212> DNA
<213> Homo sapiens
<400> 9
gaggagacca ggacagctgc tgagacctct aagaagtcca gatactaaga gcaaagatgt 60
ttcsaactgg gggcctcatt gtcttctacg ggctgttagc ccagaccatg gcccagtttg 120
gaggcctgcc cgtgcccctg gaccagaccc tgcccttgaa tgtgaatcca gccctgccct 180
tgagtcccac aggtcttgca ggaagcttga caaatgccct cagcaatggc ctgctgtctg 240
ggggcctgtt gggcattctg gaaaaccttc cgctcctgga catcctgaag cctggaggag 300
gtacttctgg tggcctcctt gggggactgc ttggaaaagt gacgtcagtg attcctggcc 360
tgaacaacat cattgacata aaggtcactg acccccagct gctggaactt ggccttgtgc 420
agagccctga tggccaccgt ctctatgtca ccatccctct cggcataaag ctccaagtga 480
atacgcccct ggtcggtgca agtctgttga ggctggctgt gaagctggac atcactgcag 540
CA 02316864 2000-06-28
WO 99135252 PCT/US98127903
aaatcttagctgtgagagat aagcaggaga ggtccttggtgactgcaccc600
ggatceacct
attcccctggaagcctgcaa atttctctgc tggccccctccccattcaag660
ttgatggact
gtcttctggacagcctcaca gggatcttga gcctgagttggttcagggca720
ataaagtcct
acgtgtgccctctggtcaat gaggttctca catcaccctggtgcatgaca780
gaggcttgga
ttgttaacatgctgatccac ggactacagt ggtctaagccttccaggaag840
ttgtcatcaa
gggctggcctctgctgagct gaactatttc atccatttcctctggcccag900
ttgctgctca
cttcccagtgctcacagatg gctggcccat atgacacagttgccttctct960
gtgctggaag
ccgaggaacctgccccctct cctttcccac taacatcccatgtgcctcac1020
caggcgtgtg
rtaataaaatggctcttctt ctgcaaaaaa 1060
aaaaaaaaaa
<210>
<211>
256
<212>
PRT
<213> sapiens
Homo
<400>
10
Met Phe Thr Gly Gly Leu Ile Val GIy Leu Ala Gln
Gln Phe Tyr Leu
1 5 10 15
Thr Met Gln Phe Gly Gly Leu Pro Leu Asp Thr Leu
Ala Val Pro Gln
20 25 30
Pro Leu Val Asn Pro Ala Leu Pro Pro Thr Leu Ala
Asn Leu Ser Gly
35 40 45
Gly Ser Thr Asn Ala Leu Ser Asn Leu Ser Gly Leu
Leu Gly Leu Gly
50 55 60
Leu Gly Leu Glu Asn Leu Pro Leu IIe Leu Pro Gly
Ile Leu Asp Lys
65 ?0 75 80
Gly Gly Ser Gly Gly Leu Leu Gly Leu Gly Val Thr
Thr Gly Leu Lys
85 90 95
Ser Val Pro Gly Leu Asn Asn IIe Ile Lys Thr Asp
Ile Ile Asp Val
100 105 110
Pro Gln Leu Glu Leu Gly Leu Val Pro Asp His Arg
Leu Gln Ser Gly
115 120 125
Leu Tyr Thr Ile Pro Leu Gly Ile Gln Val Thr Pro
Val Lys Leu Asn
130 135 140
Leu Val Ala Ser Leu Leu Arg Leu Lys Leu Ile Thr
Gly Ala Val Asp
145 150 155 160
Ala Glu Leu Ala Val Arg Asp Lys Arg Ile Leu Val
Ile Gln Glu His
165 170 175
Leu Gly Cys Thr His Ser Pro Gly Gln Ile Leu Leu
Asp Ser Leu Ser
180 185 190
Asp Gly Gly Pro Leu Pro Ile Gln Leu Asp Leu Thr
Leu Gly Leu Ser
195 200 205
Gly Ile Asn Lys Val Leu Pro Glu Gln Gly Val Cys
Leu Leu Val Asn
210 215 220
Pro Leu Asn Glu Val Leu Arg Gly Ile Thr Val His
Val Leu Asp Leu
225 230 235 240
6
CA 02316864 2000-06-28
WO 99135252 PCT/US98/27903
Asp Ile Val Asn Met Leu Ile His Gly Leu Gln Phe Val Ile Lys Val
245 250 255
<210> 11
<211> 992
<212> DNA
<213> Homo sapiens
<400> 11
gcagaatggg gctctggtct ctgggcattc atttccctca tagaggctga gaataaaaca 60
aggacttatt cacacatgtt ctagaacccc agaatggccc aagttacctg agaccagggt 120
ttctcaacct tgacaccatt gacattttgg actgggtaat tctttgttct gcagagctgt 180
cctttgcact gtaggagatt tactaatatc cctggcctct acccagtagt accactagca 240
cctattcccc acccagcgtg tctccagata ttgtcaaata tcccatcggg tgcaaaatga 300
tccctggtca agatctgttg cccaagatgt tacaggtcac aatgaccaca tttgaaattg 360
ttttcccttt cattttaccc tgtgaaagca tctctcctag agccttgcaa gaggcaggtg 420
acattgtgtc catatttctt cctgtttcag aacttctgtt tcacaacaat ttctctctcg 480
ctacaagtat tctttcactc agcactgggg aagttgggaa cagctggtca ccatcatccc 540
tttaatcaac tcacacctgt ttaaagagtg tttctgattt gaccttcatc ccttagttta 600
ctggggttaa aaaaagtctc agcaattttc attatttctc gtgggtctca ttatcaaacc 660
tttacttatt tcggcatatt tcctctgggc ttcttctagt ttctgcctta caagcaatgc 720
tgttctgtaa atttattgaa aactctggaa catttcacct ttagagatgg aggatggaag 780
gattggtacc agaagagggc taagatacgt tttctgtctt gagctgaaag cacagtctac 840
tctccttcgt tttgtcgatg agaaagttga ggccagaggg gaggtgacat gtttagagtc 900
acccagctgg ttagtgacag aaaaagcgtg agagttgtct aggattcctg ccactttcaa 960
taaagacctg acttggaaaa aaaaaaaaaa as 992
<210> 12
<211> 82
<212> PRT
<213> Homo sapiens
<400> 12
Met Ile Pro Gly Gln Asp Leu Leu Pro Lys Met Leu Gln Val Thr Met
1 5 10 15
Thr Thr Phe Glu Ile Val Phe Pro Phe Ile Leu Pro Cys Glu Ser Ile
20 25 30
Ser Pro Arg Ala Leu Gln Glu Ala Gly Asp Ile Val Ser Ile Phe Leu
35 40 45
Pro Val Ser Glu Leu Leu Phe His Asn Asn Phe Ser Leu Ala Thr Ser
50 55 60
Ile Leu Ser Leu Ser Thr Gly Glu Val Gly Asn Ser Trp Ser Pro Ser
65 70 75 80
Ser Leu
<210> 13
<211> 1095
<212> DNA
<213> Homo sapiens
<400> 13
7
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
gtcttaatga gcaacagcaa cagcagtctc cagttaagaa agagagaatt aaatacagca 60
gagatttcct gttgaagctc tcaagtgttt ccatctgcag aaaaaaacca gactttctgc 120
ctgatcatcc cattgtactg caaaaaccag aaaacaacca aagttttaag tagcatttta 180
agaacagatg aatttaagtt tggacatctg caaatgaggt ggatctagca acaataactg 240
taatggactg tgacaattca atttattctt aattttgatg gttggctatt tgacttctct 300
aaaaatgaga aagagctatt ttaaaatata aagaattttc taatcagttt cagctttgca 360
ggaggtttcc tgcataaatt gggaagtaac actggaaagt aggaatttgg ttagtgaagt 420
gggaagactg tatatttata atttgcatac tacttgcaat tttttgtttt tcatcacttg 480
taataatgga atggaaatgt aagctgtaaa gactctcaaa tataaaatat ttgctacagt 540
gtatatatgg tacataattg cttgttgctt ttaaagttcc ttctgttgtt ctgcttccca 600
ctgatttcat accagctcat gaatggatca ttacagtctc tccagaggct tagaatgatt 660
cagaatgttc aatgcatagt tctcaataaa caggaggcag aatttttaat gggtatttct 720
tttcagatat atgattggtc tctaggtttt tgataataat atggtcttaa attcataatt 780
actagcagag attgataatt tggaaacaat ggtagtgaat gaaactgaag ttgaaaaacg 840
gctgctactt atgtcactaa tcagaccata tgaatagcag aagttgagca atttcaaagt 900
aaaactgata tttttatttc caaaggaatt tagacatttg aaaataattg acatacatta 960
agttttaatt cgataatttc ttatatatgg atgaacaatt tttgggttta agcttttaat 1020
tcctagaaat tttatacatt aaatctcctg caatttgtca ctctggatgt tactgtttaa 1080
aaaaaaaaaa aaaaa 1095
<210> 14
<211> 68
<212> PRT
<213> Homo sapiens
<400> 14
Met Val His Asn Cys Leu Leu Leu Leu Lys Phe Leu Leu Leu Phe Cys
1 5 10 15
Phe Pro Leu Ile Ser Tyr Gln Leu Met Asn Gly Ser Leu Gln Ser Leu
20 25 30
Gln Arg Leu Arg Met Ile Gln Asn Val Gln Cys Ile Val Leu Asn Lys
35 40 45
Gln Glu Ala Glu Phe Leu Met Gly Ile Ser Phe Gln Ile Tyr Asp Trp
50 55 60
Ser Leu Gly Phe
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> ;2)
<223> biotinylated phosphoaramidite residue
<400> 15
cngagagcta ttgtccttga gtaggctga 29
<210> 16
<211> 29
8
CA 02316864 2000-06-28
WO 99/35252 PCT/US98/27903
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 16
gnatcttgtg tcagccccaa aggtttcag 2g
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 17
antacaacat gggatgttca ggactaatc 29
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 18
cngcagcagc agctgcccgt ttcatcatg 29
<210> 19
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylsted phosphoaramidite residue
<400> 19
9
CA 02316864 2000-06-28
WO 99/35252 PCT/1JS98/27903
cngggctaac agcccgtaga agacaatga 2g
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
c221> misc_feature
c222> (2)
c223> biotinylated phosphoaramidite residue
<400> 20
cnctaggaga gatgctttca cagggtaas
29
<210> 21
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 21
cngtgggaag cagaacaaca gaaggaact 2g
lO
