Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND P'OLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of provisional application Ser. No.
60/075,118, filed February 11,1998, which is incorporated by reference herein.
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
2 0 past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA/amino acid sequence of
the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
2 5 isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
3 0 cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 29 to nucleotide 253;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone yal5_1 deposited under accession
number
ATCC 98650;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yal5_1 deposited under accession number ATCC 98650;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone yal5_1 deposited under accession number ATCC
98650;
(f) a polynucleofide encoding a mature protein encoded by the cDNA
insert of clone yal5_1 deposited under accession number ATCC 98650;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO~;
2 0 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:2;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(f) above;
2 5 (j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a~(h); and
(1) a polynucleotide that hybridizes under stringent conditions to any
3 0 one of the polynucleotides specified in {a)-(h) and that has a length that
is at least
25% of the length of SEQ ID NO:1.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:1 from nucleotide 29 to nucleotide 253; the nucleotide sequence of the full-
length
protein coding sequence of clone yal5_1 deposited under accession number ATCC
98650;
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or the nucleotide sequence of a mature protein coding sequence of clone yal5_i
deposited
under accession number ATCC 98650. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone yal5_1 deposited under accession number ATCC 98650. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:2 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) contiguous 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 32
to amino acid 41 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO: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:
2 0 (aa) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID N0:1; and
(ab) the nucleotide sequence of the cDNA insert of done
yal5_1 deposited under accession number ATCC 9$650;
(ii} hybridizing said probes) to human genomic DNA in
2 5 conditions at least as stringent as 4X SSC at 50 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
3 0 (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:1, but excluding the poly(A) tail at the
3' end of SEQ ID N0:1; and
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(bb) the nucleotide sequence of the cDNA insert of clone
yal5_1 deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 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
N0:1 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 N0:1. 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 29 to nucleotide 253, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:1 from nucleotide 29 to nucleotide 253, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 29
to
nucleotide 253.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) a fragment of the amino acid sequence of SEQ ID N0:2, the
fragment comprising eight contiguous amino acids of SEQ ID N0:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone
2 5 yal5_1 deposited under accession number ATCC 98650;
the protein being substantially free from other mammalian proteins. Preferably
such
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
3 0 comprising eight (more preferably twenty, most preferably thirty)
contiguous amino acids
of SEQ ID N0:2, or a protein comprising a fragment of the amino acid sequence
of SEQ
ID N0:2 having biological activity, the fragment comprising the amino acid
sequence from
amino acid 32 to amino acid 41 of SEQ ID N0:2.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 151 to nucleotide 288;
{c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 196 to nucleotide 288;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ya24_1 deposited under accession
number
ATCC 98650;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ya24_1 deposited under accession number ATCC 98650;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ya24_1 deposited under accession number ATCC
98650;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ya24_1 deposited under accession number ATCC 98650;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:4;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
2 5 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i); and
3 0 (m) a polynucleotide that hybridizes under stringent rnnditions to any
one of the polynucleotides specified in (a~(i) and that has a length that is
at least
25% of the length of SEQ ID N0:3.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 151 to nucleotide 288; the nucleotide sequence of SEQ ID
N0:3 from
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nucleotide 196 to nucleotide 288; the nucleotide sequence of the full-length
protein coding
sequence of clone ya24_1 deposited under accession number ATCC 98650; or the
nucleotide sequence of a mature protein coding sequence of clone ya24_1
deposited under
accession number ATCC 98650. In other preferred embodiments, the
polynucleotide
encodes the full length or a mature protein encoded by the cDNA insert of
clone ya24_1
deposited under accession number ATCC 98650. 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)
contiguous
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 18 to amino acid
27 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 polynudeotide probes that hybridize
2 0 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 5 ya24_i deposited under accession number ATCC 98650;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
3 0 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:
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(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
ya24_i deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 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 151 to nucleotide 288, and
extending
contiguously from a nucleotide sequence rnnresponding to the 5' end of said
sequence of
SEQ ID N0:3 from nucleotide 151 to nucleotide 288, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide
151 to
nucleotide 288. Also preferably the polynucleotide isolated according to the
above
2 0 process comprises a nucleotide sequence corresponding to the cDNA sequence
of SEQ ID
N0:3 from nucleotide 196 to nucleotide 288, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from
nucleotide 196 to nucleotide 288, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:3 from nucleotide 196 to nucleotide 288.
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 N0:4;
(b) a fragment of the amino acid sequence of SEQ ID N0:4, the
3 0 fragment comprising eight contiguous amino aclds of SEQ ID N0:4; and
(c) the amino acid sequence encoded by the cDNA insert of clone
ya24_1 deposited under accession number ATCC 98650;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4. In further preferred
7
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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) contiguous
amino acids
of SEQ ID N0:4, or 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 18 to amino acid 27 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 615 to nucleotide 908;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 774 to nucleotide 908;
{d) a polynucleotide comprising the nucleotide sequence of the full-
~g'~ Pmt Wing sequence of clone yb42_i deposited under accession number
ATCC 98650;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yb42_1 deposited under accession number ATCC 98650;
2 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone yb42_1 deposited under accession number ATCC
98650;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb42_1 deposited under accession number ATCC 98650;
2 5 (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
comprising eight contiguous amino acids of SEQ ID N0:6;
3 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(ar(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or {i) above ;
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(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a~(i); and
(m) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i) and that has a length that is
at least
25% of the length of SEQ ID N0:5.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 615 to nucleotide 908; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 774 to nucleotide 908; the nucleotide sequence of the full-length
protein coding
sequence of clone yb42_1 deposited under accession number ATCC 98650; or the
nucleotide sequence of a mature protein coding sequence of clone yb42_1
deposited under
accession number ATCC 98650. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone yb42_1
deposited under accession number ATCC 9$650. 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)
contiguous
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 44 to amino acid
53 of SEQ
2 0 ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
2 5 (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 N0:5, but excluding the poly(A) tail at the
3 0 3' end of SEQ ID N0:5; and
(ab) the nucleotide sequence of the cDNA insert of clone
yb42_i deposited under accession number ATCC 98650;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
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{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 N0:5, 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
yb42_1 deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X S5C at 50 degrees C;
(iii) amplifying human DNA sequences; and
25 (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
IV0:5 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but
excluding the
2 0 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 615 to nucleotide 908, 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 908, to a nucleotide sequence
2 5 corresponding to the 3' end of said sequence of SEQ ID N0:5 from
nucleotide 615 to
nucleotide 908. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequenre corresponding to the cDNA sequence of
SEQ ID
N0:5 from nucleotide 774 to nucleotide 908, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from
3 0 nucleotide 774 to nucleotide 908, to a nucleotide sequence corresponding
to the 3' end of
said sequence of SEQ ID N0:5 from nucleotide 774 to nucleotide 908.
In other embodiments, the present invention provides a composition comprising
a protein, whex~itt said protein comprises an amino acid sequence selected
from the group
consisting of:
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(a) the amino acid sequence of SEQ ID N4:6;
(b) a fragment of the amino acid sequence of SEQ ID N0:6, the
fragment comprising eight contiguous amino acids of SEQ ID N0:6; and
(c) the amino acid sequence encoded by the cDNA insert of clone
yb42_1 deposited under accession number ATCC 98650;
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
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 Wight (more preferably twenty, most preferably thirty) contiguous
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 acid
sequence from
amino acid 44 to amino acid 53 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 1203 to nucleotide 2327;
2 0 (c) a polynudeotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone yc9 1 deposited under accession number
ATCC 98650;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone y~ 1 deposited under accession number ATCC 98650;
2 5 (e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone y~ 1 deposited under accession number ATCC
98650;
(f) a polynudeotide encoding a mature protein encoded by the cDNA
insert of clone y~ 1 deposited under accession number ATCC 98650;
3 0 (g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:8;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:8;
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(i) a polynucleotide which is an allelic variant of a polynucleotide of
{a)-(f) above;
(j) a polynudeotide which encodes a speaes homologue of the protein
of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (ar(h); and
{1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (ar(h) and that has a length that is
at least
25% of the length of SEQ ID N0:7.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1203 to nucleotide 2327; the nucleotide sequence of the
full-length
protein coding sequence of clone y~ 1 deposited under accession number ATCC
98650;
or the nucleotide sequence of a mature prntein coding sequence of clone y~ 1
deposited
under accession number ATCC 98650. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone y~ 1 deposited under accession number ATCC 98650. 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
2 0 preferably thirty) contiguous amino ands 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 rnmprising the amino acid sequence from
amino acid 182
to amino acid 191 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 5 ID N0:7.
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
3 0 in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
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(ab) the nucleotide sequence of the cDNA insert of clone
yc9_1 deposited under accession number ATCC 98650;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 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 N0:7, 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
y~ 1 deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
2 0 Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and
exfiending
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
2 5 according to the above process comprises a nucleotide sequence
corresponding to the
cDNA sequence of SEQ ID N0:7 from nucleotide 1203 to nucleotide 2327, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:7 from nucleotide 1203 to nucleotide 2327, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide
1203 to
3 0 nucleotide 2327.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
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(b) a fragment of the amino acid sequence of SEQ ID N0:8, the
fragment comprising eight contiguous amino acids of SEQ ID N0:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone
y~ 1 deposited under accession number ATCC 98650;
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) contiguous
amino acids
of SEQ ID N0:8, or a protein comprising a fragment of the amino acid sequence
of SEQ
ID NO:B having biological activity, the fragment comprising the amino acid
sequence from
amino acid 182 to amino acid 191 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 230 to nucleotide 823;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:9 from nucleotide 584 to nucleotide 823;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ycl9_i deposited under accession
number
ATCC 98650;
(e) a polynucleotide encoding the full-length protein encoded by the
2 5 cDNA insert of clone ycl9_1 deposited under accession number ATCC 98650;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ycl9_1 deposited under accession number ATCC
98650;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
3 0 insert of clone ycl9_1 deposited under accession number ATCC 98650;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
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(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 contiguous amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i); and
(m) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a~(i) and that has a length that is
at least
25% of the length of SEQ ID N0:9.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 230 to nucleotide 823; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 584 to nucleotide 823; the nucleotide sequence of the full-length
protein coding
sequence of clone ycl9_1 deposited under accession number ATCC 98650; or the
nucleotide sequence of a mature protein coding sequence of clone ycl9_1
deposited under
accession number ATCC 98650. In other preferred embodiments, the
polynucleotide
encodes the full length or a mature protein encoded by the cDNA insert of
clone ycl9_1
2 0 deposited under accession number ATCC 98650. In further preferred
embodiments, the
present invention provides a polynucleotide encoding a protein comprising a
fragment
of the amino acid sequence of SEQ ID N0:10 having biological activity, the
fragment
preferably comprising eight (more preferably twenty, most preferably thirty)
contiguous
amino acids of SEQ ID N0:10, or a polynucleotide encoding a protein comprising
a
2 5 fragment of the amino acid sequence of SEQ ID NO:10 having biological
activity, the
fragment comprising the amino acid sequence from amino acid 94 to amino acid
103 of
SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequenre of SEQ
ID N0:9.
3 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:
CA 02320618 2000-08-11
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(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 N0:9, 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
ycl9_1 deposited under accession number ATCC 98650;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
(iii) isolating the DNA polynudeotides 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
2 0 ycl9_i deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynudeotide products of step (b)(iii).
2 5 Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence co~p~g to the cDNA sequence of SEQ ID N0:9, and extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:9 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
3 0 according to the above process comprises a nucleotide sequence
corresponding to the
cDNA sequence of SEQ ID N0:9 from nucleotide 230 to nucleotide 823, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:9 from nucleotide 230 to nucleotide 823, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide
230 to
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nucleotide 823. 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 584 to nucleotide 823, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from
nucleotide 584 to nucleotide 823, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:9 from nucleotide 584 to nucleotide 823.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:10;
(b) a fragment of the amino acid sequence of SEQ ID NO:10, the
fragment comprising eight contiguous amino acids of SEQ ID N0:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone
ycl9_1 deposited under accession number ATCC 98650;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10. 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) contiguous
amino acids
2 0 of SEQ ID NO:10, or a protein comprising a fragment of the amino acid
sequence of SEQ
ID N0:10 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 94 to. amino acid 103 of SEQ ID NO:10.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 292 to nucleotide 534;
(c) a polynucleotide comprising the nucleotide sequence of the full-
3 0 length protein coding sequence of clone yc20_1 deposited under accession
number
ATCC 98650;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yc20_1 deposited under accession number ATCC 98650;
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(e) a polynudeotide comprising the nucleotide sequence of a mature
protein coding sequence of clone yc20_1 deposited under accession number ATCC
98650;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yc20_1 deposited under accession number ATCC 98650;
(g) a polynudeotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(h) a polynudeotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:12;
(i) a polynudeotide which is an allelic variant of a polynudeotide of
(a)-(f) above;
(j) a polynudeotide which encodes a species homologue of the protein
of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynudeotides specified in (ar(h); and
(I) a polynucleotide that hybridizes under stringent conditions to any
one of the polynudeotides specified in (a~(h) and that has a length that is at
least
25% of the length of SEQ ID N0:11.
2 0 Preferably, such polynudeotide comprises the nucleotide sequ~ce of SEQ ID
NO:11 from nucleotide 292 to nucleotide 534; the nucleotide sequence of the
full-length
protein coding sequence of clone yc20_1 deposited under accession number ATCC
98650;
or the nucleotide sequence of a mature protein coding sequence of clone yc20_1
deposited
under accession number ATCC 98650. In other preferred embodiments, the
2 5 polynudeotide encodes the full-length or a mature protein encoded by the
cDNA insert
of done yc20_1 deposited under accession number ATCC 98650. In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:12 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
3 0 preferably thirty) contiguous amino acids of SEQ ID N0:12, or a
polynudeotide encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 35
to amino acid 44 of SEQ ID N0:12.
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WO 99/41284 PCTNS99/02898
Other embodiments provide the gene corresponding to the cDNA sequeruce of SEQ
ID N0:11.
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 N0:11, but excluding the poly(A) tail at the
3' end of SEQ ID N0:11; and
(ab) the nucleotide sequence of the cDNA insert of clone
yc20_1 deposited under accession number-ATCC 98650;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 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
2 0 hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
(ba) SEQ ID N0:11, 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
2 5 yc20_1 deposited under accession number ATCC 98650;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
3 0 Fieferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:11, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
ID N0:11 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
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polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 292 to
nucleotide
534, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
of said sequence of SEQ ID N0:11 from nucleotide 292 to nucleotide 534, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from
nucleotide
292 to nucleotide 534.
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) a fragment of the amino acid sequence of SEQ ID N0:12, the
fragment comprising eight contiguous amino acids of SEQ ID N0:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
yc20_1 deposited under accession number ATCC 98650;
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) contiguous
amino acids
2 0 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 35 to amino acid 44 of SEQ ID N0:12.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
2 5 yeast, insect and mannmalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
3 0 (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention.
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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.
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED g ~S('RTPTTC~Iu
Nucleotide and amino acid sequences, as presently determined, are reported
belqw for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
2 0 acid sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 5 As used herein a "secreted" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
profieins secreted wholly (e.g., soluble profieins) or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
3 0 which are transported across the membrane of the endoplasmic reticulum.
Clone ",L 1"
A polynucleotide of the preset invention has been identified as clone
"yal5_1".
yal5_1 was isolated from a human adult testes cDNA library and was identified
as
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WO 99/41284 PCTNS99/02898
encoding a seaeted or transmembrane protein on the basis of computer analysis
of the
amino acid sequence of the encoded protein. yal5_1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"yal5_1 protein").
The nucleotide sequence of yal5_1 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 yal5_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:2. Amino acids 8
to 20 of
SEQ ID N0:2 are a possible leader/signal sequence, with the predicted mature
amino acid
sequence beginning at amino acid 21. Due to the hydrophobic nature of the
predicted
leader/signal sequence, it is likely to act as a transmembrane domain should
the predicfied
leader/signal sequence not be separated from the remainder of the yal5_1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
yal5_1 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for yal5_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. yal5_1 demonstrated at least some sianilarity with
sequences
identified as 294056 (Human DNA sequence from PAC 436M11 on chromosome
Xp22.11-22.2; contains a serine threonine protein phosphatase gene, ESTs and
STSs).
Based upon sequence similarity, yal5_1 proteins and each similar protein or
peptide may
2 0 share at least some activity. The TopPredII computer program predicts a
potential
transmembrane domain within the yal5_1 protein sequence centered around amino
acid
40 of SEQ ID N0:2. The nucleotide sequence of yal5_1 indicates that it may
contain an
Alu repetitive element.
2 5 Clone "3ra24 1"
A polynucleotide of the present invention has been identified as clone "ya24_1
".
ya24_i was isolated from a human adult testes cDNA library and was identified
as
encoding a secreted or transmembrane protein on the basis of rnmputer analysis
of the
amino acid sequence of the encoded protein. ya24_1 is a full-length clone,
including the
3 0 entire coding sequence of a secreted protein (also referred to herein as
"ya24_1 protein").
The nucleotide sequence of ya24_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 ya24_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:4. Amino acids 3
to 15 of
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SEQ ID N0:4 are a predicted leader/signal sequence, with the predicted mature
amino
aced sequence beginning at amino acid 16. 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 ya24_1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ya24_1 should be approximately 750 bp.
The nucleotide sequence disclosed herein for ya24_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLAST1V/BLAST?C and
FASTA search protocols. ya24_1 demonstrated at least some similarity with
sequences
identified as AA537299 (vk46d03.r1 Soares mouse mammary gland NbMMG Mus
musculus cDNA clone 949637 5'). Based upon sequence similarity, ya24_1
proteins and
each similar protein or peptide may share at least some activity.
Clone "; 1"
A polynucleotide of the present invention has been identified as clone
"yb42_1".
yb42_1 was isolated from a human fetal brain cDNA library and was identified
as
encoding a secreted or transmembrane protein on the basis of computer analysis
of the
amino acid sequence of the encoded protein. yb42_1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"yb42_1 protein").
2 0 The nucleotide sequence of yb42_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
reading frame and the predicted amino acid sequence of the yb42_1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino
acids 41 to 53 of SEQ ID N0:6 are a predicted leader/signal sequence, with the
predicted
2 5 mature amino acid sequence beginning at amino acid 54. 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
yb42_1 protein.
Another potential yb42_i reading frame and predicted amino acid sequence is
3 0 encoded by basepairs 1879 to 2220 of SEQ ID N0:5 and is reported in SEQ ID
N0:19;
amino acids 54 to 66 of SEQ ID N0:19 are a predicted leader/signal sequence,
with the
predicted mature amino acid sequence beginning at amino acid 67 of SEQ ID
N0:19. Due
to the hydrophobic nature of this predicted leader/signal sequence, it is
likely to act as a
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WO 99/41284 PCT/US99/02898
transmembrane domain should the predicted leader/signal sequence not be
separated
from the remainder of the protein of SEQ ID N0:19.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
yb42_1 should be approximately 3900 bp.
The nucleotide sequence disclosed herein for yb42_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. yb42_1 demonstrated at least some similarity with
sequences
identified as AA213992 (zn58d08.s1 Stratagene muscle 937209 Homo sapiens cDNA
clone
562383 3'). Based upon sequence similarity, yb42_1 proteins and each similar
protein or
peptide may share at least some activity. The nucleotide sequence of yb42_1
indicates that
it may contain at least one MIR repeat sequence.
Clone ",yes 1"
A poiynucleotide of the present invention has been identified as clone "yes
1".
y~ 1 was isolated from a human fetal kidney (293 cell line) cDNA library and
was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. y~ 1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"yes 1 protein").
2 0 The nucleotide sequence of yc9_1 as presently defiermined is reported in
SEQ ID
N0:7, and includes a poly(A) tail. What applicants presently believe to be the
proper
reading frame and the predicted amino acid sequence of the y~ 1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit rnntaining
clone
2 5 yc9_1 should be approximately 3300 bp.
The nucleotide sequence disclosed herein for y~ 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. y~ 1 demonstrated at least some similarity with
sequences
identified as AA588539 (nm94a07.s1 NCI_CGAP_Co9 Homo sapiens cDNA clone
3 0 IMAGE:10~5860) and N47418 (yy88e12.r1 Homo Sapiens cDNA clone 280654 5').
The
predicted amino acid sequence disclosed herein for yc9_1 was searched against
the
GenPept and GeneSeq amino acid sequence databases using the BLASTx search
protocol.
The predicted y~ 1 protein demonstrated at least some similarity to sequences
identified
as X97196 (D. melanogaster X gene). Based upon sequence similarity, y~ 1
profieins and
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WO 99/d1284 PCT/US99102898
each similar protein or peptide may share at least some activity. The
TopPredil rnmputer
program predicts two potential transmembrane domains within the yc9_1 protein
sequence, centered around amino acids 80 and 200 of SEQ ID N0:8, respectively.
Clone"ycl9 1"
A polynucleotide of the present invention has been identified as clone
"ycl9_1".
ycl9_1 was isolated from a human fetal kidney (293 cell line) cDNA library and
was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ycl9_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ycl9_1 protein").
The nucleotide sequence of ycl9_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 ycl9_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:10. Amino acids
106 to
118 of SEQ ID N0:10 are a predicted leader/signal sequence, with the predicted
mature
amino acid sequence beginning at amino acid 119. 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 ycl9_1
2 0 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ycl9_1 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for ycl9_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. ycl9_l demonstrated at least some similarity with
sequences
identified as AA126002 (z185a08.s1 Stratagene colon (#937204) Homo sapiens
cDNA clone
511382 3') and AA3070~ (EST177917 Colon carcinoma (HCC) cell line Homo Sapiens
cDNA 5' end). The predicted amino acid sequence disclosed herein for ycl9_1
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
3 0 BLASTX search protocol. The predicted ycl9_1 protein demonstrated at least
some
similarity to sequences identified as X83742 (MAP kinase phosphotase [Xenopus
laevis]),
and to several tyrosine phosphatases from other species. Based upon sequence
similarity,
ycl9_1 proteins and each similar protein or peptide may share at least some
activity. The
TopPredII computer program predicts an additional potential transmembrane
domain
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
within the ycl9_1 protein sequence centered around amino acid 43 of SEQ ID
NO:10; this
region (amino acids 30 to 42 of SEQ ID N0:10) may also be a leader/signal
sequence, with
the predicted mature amino acid sequence beginning at amino acid 43.
ycl9_i protein was expressed in a COS cell expression system, and an expressed
protein band of approximately 23 kDa was detected in conditioned medium and
membrane fractions using SDS polyacrylamide gel electrophoresis.
one "3rc20 1"
A polynucleotide of the present invention has been identified as clone
"yc20_1".
yc20_1 was isolated from a human fetal kidney (293 cell line) cDNA library and
was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. yc20_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"yc20_1 protein").
The nucleotide sequence of yc20_1 as presently determined is reported in SEQ
ID
NO: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 yc20_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 yc20_1 should be approximately 1450 bp.
The nucleotide sequence disclosed herein for yc20_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BIrASTN/BLASTX and
FASTA search protocols. yc20_1 demonstrated at least some similarity with
sequences
identified as AA447839 (aa18c12.r1 Soares NhHM1'u Sl Homo Sapiens cDNA clone
2 5 813622 5'), N33405 (yy41e10.s1 Homo Sapiens cDNA clone 273834 3'), and
T19519 (Human
gene signature HLTMGS00580). Based upon sequence similarity, yc20_1 proteins
and each
similar protein or peptide may share at least some activity.
3 0 Clones yal5_l, ya24_l, yb42_l, y~ 1, ycl9_l, and yc20_1 were deposited on
February 11, 1998 with the American Type Culture Collection (10801 University
Boulevard, Mantissas, Virginia 20110-2209 U.S.A.) as an original deposit under
the
Budapest Treaty and were given the accession number ATCC 98650, from which
each
clone comprising a particular polynucleotide is obtainable. All restrictions
on the
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WO 99/41284
PCTNS99/02898
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.$OS(b), and
the term of the deposit will complywith 37 C.F.R. ~ 1.806.
Each clone has been transfected into separate bacterial cells (E. co&~ in this
composite deposit. Each clone can be removed kom the vector in which it was
deposited
bY Pe~o~g an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce
the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector
("pED6") was derived kom pED6dpc1 by insertion of a new polylinker to
facilitate
cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 44g5-4490); the pNOTs
vector
was derived from pMT2 (Kaufman et al.,1989, Mol. Cell. Biol. 9: 946-958) by
deletion of
the DHFR sequences, insertion of a new polylinker, and i~mon 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 kom the
vectors in which they were deposited.
Ba~~ re~ c°ng a P~~ clone can be obtained from the composite
2 0 deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone, This sequence can be derived kom the
sequences
provided herein, or kom a combination of those sequences. The sequence of an
oligonudeotide probe that was used to isolate or to sequence each full-length
clone is
2 5 identified below, and should be most reliable in isolating the clone of
interest.
Probe
SeauencP
yal5_1 _
SEQ ID N0:13
ya24_1 SEQ ID N0:14
3 0 yb42_1 SEQ ID N0:15
Y~'1 SEQ ID N0:16
ycl9_1 SEQ ID N0:17
yc20_1 SEQ ID N0:18
27
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as, for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a T°, 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 ~P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonudeotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration duomatography 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
2 0 be thawed and 100 Nl of the stock used to inoculate a sterile culture
flask containing 25 ml
of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
2 5 colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
pg/ml and agar at 1.5~° in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyre, denature and bake them.
3 0 The filter is then preferably incubated at 65°C for 1 hour with
gentle agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM 1,DTA
(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
28
CA 02320618 2000-08-11
wo ~iaizsa rrrivs~roz898
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with 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/Terhnology ~Q, 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc. ~, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. Far a bivalent form of the protein, such
a fusion
2 0 could be to the Fc portion of an IgG molecule. Other immunoglobulin
isotypes may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
2 5 listing by translation of the nucleotide sequence of each disclosed clone.
The mature
forms) of such protein may be obtained by expression of the disclosed full-
length
polynucleotide (preferably those deposited with ATCC) in a suitable mammalian
cell or
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
3 0 The present invention also provides genes corresponding to the
polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
29
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
The chromosomal location corresponding to the polynucleotide sequences
disclosed herein may also be determined, for example by hybridizing
appropriately
labeled polynucleotides of the present invention to chromosomes in situ. It
may also be
possible to determine the corresponding chromosomal location for a disclosed
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
polynudeotide 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 "UruGene
clusters"
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
2 5 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
gee (Albert and Morris,1994, Trends Pharmacol. Sci.15(7): 250-254; Lavar~ky et
al.,1997,
Biockem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. Mol.
Biol. 58:1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
3 0 multiple copies of the genes) corresponding to the polynucleotide
sequences disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
also provided (see European Patent No. 0 649 464 Bl, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioassays 14(9): 629-633; Zwaal et
nl.,1993, Proc. Natl.
Acad. Sci. LISA 90(16): 7431-7435; Dark et al.,1994, Proc. Natl. Acad. Sci.
LISA 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,
Natscre 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 profiein 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
2 0 or all of the intracellular and transrnembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
aa~ordance 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
2 5 transnnembrane domains in an amino acid sequence, domains which are
described by the
location of the center of the transmsmbrane domain, with at least ten
transmembrane
amino acids on each side of the reported central residue(s).
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
3 0 preferably at least 75%) of the length of a disclosed profiein and have at
least 609'° sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by rnmparing
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
31
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
In particular, sequence identity may be determined using WLT-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
Enzynology
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
2 0 used for commercial, nonprofit, or academic purposes. In all search
programs in the suite
-- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX -- the gapped alignment
routines are integral to the database search itself, and thus yield much
better sensitivity and
selectivity while producing the more easily interpreted output. Gapping can
optionally be
turned off in all of these programs, if desired. The default penalty (Q) for a
gap of length
2 5 one is Q~ for proteins and BLASTP, and X10 for BLASTN, but may be changed
to any
integer value including zero, one through eight, nine, ten, eleven, twelve
through twenty,
twenty-one through fifty, fifty-one through one hundred, etc. The default per-
residue
penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for
BLASTN,
but may be changed to any integer value including zero, one, two, three, four,
five, six,
3 0 seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through
fifty, fifty-one
through one hundred, etc. Any combination of values for Q and R can be used in
order to
align sequences so as to maximize overlap and identity while minimizing
sequence gaps.
32
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
The default amino acid comparison matrix is BLOSUM62, but other amino acid
comparison matrices such as PAM can be utilized.
Species homologues of the disclosed polynucleotides and proteins are also
provided by the present invention. As used herein, a "species homologue" is a
protein or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
the given polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues may be isolated and identified by making suitable probes or primers
from
the sequences provided herein and screening a suitable nucleic acid source
from the
desired species. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hylobates
concolor, Macaca mulatto, Papio papio, Papio hamadryas, Cercopithecus
aethurps, Cebus capucinus,
2 0 Aotus trivirgatus, Sanguinus Oedipus, Microcehus murinus, Mus musculus,
Rattus norvegicus,
Cricetulus griseus, Fells catus, Mustela visor, Cams familiaris, Oryctolagus
cuniculus, Bos taurus,
Ovis aries, Sus scrofa, and Equus cabalIus, for which genetic maps have been
created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
2 5 (O'Brien and Seu~nez, 1988, Ann. Rev. Genet. 22: 323-351; OBrien et al.,
1993, Nature
Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et
al.,1997, Nature
Genetics 15 ;47 56; OBrien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
1997, Genome Research 7:1123-1137; all of which are incorporated by reference
herein).
The invention also encompasses allelic variants of the disclosed
polynucleotides
3 0 or proteins; that is, naturally-occurring alternative forms of the
isolated polynucleotides
which also encode proteins which are identical or have significantly similar
sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
60% sequence identity (more preferably, at least 75% identity; most preferably
at least 90%
33
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
identity) with the given polynucleotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
overlap and identity while minimizing sequence gaps. Allelic variants may be
isolated and
identified by making suitable probes or primers from the sequences provided
herein and
screening a suitable nucleic acid source from individuals of the appropriate
species.
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
34
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
~P)= Buffer' and Buffer'
A DNA:DNA x 50 65C; lxSSC -or- b5C; 0.3xSSC
42C; lxSSC, SO~o
formamide
B DNA:DNA <50 TB*; lxSSC Ts*; lxSSC
C DNA:RNA x 50 67C; ixSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA t50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA x 50 70C; lxSSC -or- 70C; 03xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; ixSSC TF*; lxSSC
G DNA:DNA x 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 509'o
formamide
H DNA:DNA <50 TH*; 4xSSC TH*;
I DNA:RNA x 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50~o
formamide
J DNA:RNA <50 T~*; 4xSSC T~*; ~
K RNA:RNA x 50 70C; 4xSSC -or- 67C; ixSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC T~*; 2~
M DNA:DNA x 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA x 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 TP*; 6xSSC Tp*; 6xSSC
Q RNA:RNA x 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TR*; 4xSSC TR*; 4~
O
1: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
hybridizing a polynucleotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence rnmplementarity.
t: SSPE (ixSSPE is 0.15M NaCI, lOmM NaH2P0" and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "Ta - 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 (T~ of the hybrid, where Tm
is determined aaording to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,o[Na']) + 0.41(~oG+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'j for lxSSC = 0.165 M).
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
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, lnc., 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
ICaufman et al., Nucleic Acids Res. ~, 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.
ICaufman, Methods in Enzymology ~, 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 profiein. 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 j~ vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0. Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heberologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
36
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, '~~xas Agric~tural E
~~riment
Station Bulletin No. 1555 f 19871. incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRXJ. 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).
37
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the mills of transgeruc 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
38
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify cluomosomes or to map related gene positions; to
compare
2 0 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 1?CR primers for genetic fingerprinting; as a probe to
"subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
2 5 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
pofientially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
3 0 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
polynudeotides encoding the other protein with which binding occurs or to
identify
inhibitors of the binding interaction.
39
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
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 au 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.
ICimmel eds.,1987.
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
sourre 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.
d
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 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
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_l, 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, Inununologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., j. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341,1991; Bertagnolli, et al., J. Immunol. 149:3778-3783,1992; Bowman
et al., J.
Immunol.152: 1756-1761,1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
2 0 stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. j.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, john Wiley and Sons,
Toronto.1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
2 5 cells include, without limitation, those described in: Measurement of
Human and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. j.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Whey and Sons,
Toronto. 1991; deVries et al., j. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
3 0 Measurement of mouse and human interleukin 6 - Nordan, R. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.i-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:18571861, 1986; Measurement of
human
Interleuldn 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto.1991;
41
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, IGJ. 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. ICruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411,1981; Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immun_P S 'm ~la 'n~ or ynressi_ng Acti ~it;,
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
2 0 in regulating (up or down) growth and proliferation of T and/or B
lymphocytes, as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesvinuses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
3 0 Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft versus-host disease and autoimmune inflammatory eye
disease.
42
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
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
distinguishable from immunosuppression in that it is generally antigen-
specific and
persists after exposure to the tolerizing agent has ceased. Operationally,
tolerance can be
demonstrated by the lack of a T cell response upon reexposure to specific
antigen in the
absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B~), e.g.,
preventing
2 0 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
2 5 the transplant. The administration of a molecule which inhibits or blocks
interac#ion 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
(eg., B7
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
3 0 molecule to the natural ligand(s) on the immune cells without transmitting
the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
43
CA 02320618 2000-08-11
WO 99/411,$4 PGTNS99/02898
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 GVI3D 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 GVI3D (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 0 Administration of reagents which block costimulation of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
2 5 the disease. The efficacy of blockimg 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 erythmatosis in MRL/lpr/lpr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
3 0 marine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven
Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
44
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (eg., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 0 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7 2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
2 5 transfected tumor reps are returned to the patient to result in expression
of the peptides
on the surface of the transfecbed cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
3 0 costimulation signal to T cells to induce a T cell mediated immune
response against the
trai~sfected 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
MFIC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
~i2
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
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 3,1n Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974,1982; Handa
et al.,
2 0 J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-
3500,1986; Takai et al.,
J. Imrnunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Hemnann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. lmmunol. 137:3494-3500, 1986; Bowmanet
al., j.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
2 5 Cellular Immunology 133:327 341,1991; Brown et al., J. Immunol.153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol.144:3028-3033,1990; and Assays for B cell
function: In vitro
3 0 antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
ICruisbeek,
46
CA 02320618 2000-08-11
wo 99iaizsa rcrnrs~ro2s9s
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
lnterscience (Chapfier 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. hnmunol. 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 lnaba et al.,
journal of
Experimental Medicine 172:631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 0 Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation, those described in: Antica et al.,
Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
HematopoiesL ReeLati-ng A
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
3 0 lines indicafies involvement in regulating heznatopoiesis, e.g. in
supporting the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
47
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/OZ898
cells such as granulocyfies and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 0 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-
151,1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation {which will identify, among
2 5 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Whey-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907 5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
3 0 Briddell, R.A. In Culture of Hemutopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp.1-21, Whey Liss, Inc.., New York,
NY.1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
48
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WO 99/41284 PCTNS99/02898
Allen, T. In Culture of Hemutopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Hliley-hiss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. RI. Freshney, et al. eds. Vol pp.139-
162, Wiley-Liss,
Inc., New York, NY.1994.
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 osteogeruc agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 0 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
3 0 circumstances where such tissue is not normally formed, has application in
the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
49
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in repairing defects to tendon or ligament tissue. De novo tsndon/ligament
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 0 Parkinson s disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovasculai diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
2 5 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
3 0 generation or regeneration of other tissues, such as organs (including,
for example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
CA 02320618 2000-08-11
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of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
Infiernational 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, ~idermal Wound Healing. pps. 71-112 (Maibach, HI and Rovee, DT, eds.),
Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
2 0 ~,ctivin/Inhibin ActiY~
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 (3 group, may be useful as a fertility inducing therapeutic, based upon
the ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
United States Patent 4,798,885. A protein of the invention may also be useful
for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
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WO 99/41284 PCT/US99/02898
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inh~in 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.
Nail. Acad. Sci. USA 83:3091-3095,1986.
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Glemotactic 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 prod ~ ~~o~~c 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. Gin. Invest. 95:1370-1376,1995;
Lind et al.
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WO 99/41284 PCT/US99/02898
APMIS 103:140-146,1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et
al. J. of
Immunol.152:5860-5867,1994; Johnston et al. J. of Immunol.153:1762-1768,1994.
Hemostatic and Thrombolytic Activity
1-
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
rnagulation
disorders (including hereditary disorders, such as hemophiliac) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (I991); Schaub,
Prostaglandins
35:467-474,1988.
2 0 ~tor /Ligand
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonises 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 rellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
3 0 receptor/ligand interaction. A protein of the present invention
(including, without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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WO 99/41284 PC"T/US99/OZ898
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
ICruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Intersrience (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.
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 inNamnnatory 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
fpm over production of cytokines such as TNF or ILrl. Proteins of the
invention may also
be useful to treat anaphylaxis and hypexsensitivity to an antigenic substance
or material.
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
54
CA 02320618 2000-08-11
WO 99/41284 PGT/US99/02898
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 supex~family, 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 cadherirt activity, and
poiynucleotides 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 r~.~r
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 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
Fragments of profieins 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; C~zawa et al. Cell 63:1033-
1038,1990.
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 iWu'biting angiogenesis), by causing production of other factors, agents
or cell types which
inhibit tumor growth, or by suppressing, eliminating or inhibiting factors,
agents or cell
types which promote tumor growth.
2 5 A protein of the invention may also exh~it 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 rnlor, 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, profiein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
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WO 99/41284 PCT/US99/02898
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.
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 Garner)
diluents, fillers,
salts, buffers, stabilizers, solubiiizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non toxic mafierial that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
2 0 Garner will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, ILr3, IL-4, IL-5, ILr6, IL-7, ILr8,
IL.-9, IL~-10, ILrl l,
l:Lrl2, ILrl3, fLrl4, IL-15, TFN, TNFU, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may contain
other
2 5 agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical comp~ition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
3 0 thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to
minimize side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
57
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/OZ898
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 MFiC-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
pharniaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
2 0 in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Pafient No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
2 5 by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an incrnase in rate of
treatment, healing,
3 0 prevention or amelioration of such conditions. When applied to an
individual active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
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WO 99/41284 PGTNS99/02898
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
rytolcines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytolcine(s), lympholcine(s),
other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lympholcine(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 carned
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
2 0 administered orally, protein of the present invention will be in the form
of a tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid Garner such as a
gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90~° protein of the
present invention.
2 5 When administered in liquid form, a liquid carrier such as water,
petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
3 0 form, the pharmaceutical composition contains from about 0.5 to 90% by
weight of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
59
CA 02320618 2000-08-11
wo m4m Pc-riusmoas98
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should rnntain about 0.01 pg
to about 100
2 0 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 pg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
2 5 contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
3 0 polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
~. Methods for synthesizing such peptides are known in the art, for example,
as in
CA 02320618 2000-08-11
WO 99/41284 PCT/US99102898
R.P. Merrifield, J. Amer.Chem.Soc. $~, 2149-2154 (1963); J.L. ICrstenansky, et
al., FEBS Lett.
~1, 10 (198. Monoclonal antibodies binding to the protein of the invention may
be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
2 0 methods of the invention. Preferably for bone and/or cartilage formation,
the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorted into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
2 5 applications.
The choice of matrix material is based on biocompati'bility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
3 0 sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,
polyglycolic acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
61
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or rnllagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
2 0 provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGR~), and insulin like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
3 0 humans, are desired patients for such treatment with proteins of the
present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
62
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynudeotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
63.
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
SEQUENCE LISTING
<110> Wong, Cordon G.
Clark, Hilary
Fechtel, Rim
Agostino, Michael J.
Genetics Institute. Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6300A
<140>
< 141 > "mss MaH' mailirrp I~el numk~: Err~_ ~ fro bra 90 ~ u~
Date of OepoatlL i l 99 9
i hereby certify that this papa or fee is befnQ
<160> 19 depOSis~ with the United States Postal Service
"f=xpress Mail Post Ot'~e to Addn~stse' aervioe
<170> Patentln Ver. 2.0 under 37 CFR 1 10 on the date indtcabd above
end is addrossad to the Assistant Comn~ionar
<210> 1 Fa nts, lfYashin D. . 20231
<211> 1051
<212> DNA
<213> Homo sapiens
<400> 1
taggccatga aggccgcttg ctattattat gcaaataagt gttgtattac acatactaat 60
ttatgcatgg ttagattatg cagatgcatc acaaacatgg ttattgaata ataggatggg 120
gctcattctc tctaccatct ttataagcag ttttaagcaa attctcctgg taccaatgtg 180
gacatttaaa gaccctttac tcaatgaaat gtctctttcg tattcttttg ctttcatagt 240
taaagccatc agataagtgg aagagaaatc gtccaagttg ttaggttcaa gagtgttagt 300
ctcacttttc aaattcgtag acttttttgt ttaaatgtaa tcttttcctt atagagaaaa 360
tctaaaatgc agttgcttgg catgaatgct ggcatttagt gagattttag tgtatatagc 420
cttgctgctt agctctaggt aacccatcaa attaaaatta cattttcagg atttatagct 480
cattagaata tttatcttgg taagcttctt attctgtcag taatttctaa acaattcagc 540
ttggccaatt tgtgaaatcc cctaaaattt tgaaagtgaa ctcacaagcc ctatgcagta 600
tatttctcaa acaaatctta gtagaaaact tataagccat ccagtaaaaa ttccaaaggt 660
tgagaatgta gcaatattct tgagattcct aatgtctaga gtagttaatc agtgagattt 720
gatgggtgat gagtctaaga aatggatttt gccatggcca ggtgcagtgg cttacgcctg 780
taatcccagc actttgggag gccgaggtgg gcggatcacg aggtcaggag attgagacca 840
tcctggctaa catggtgaaa cccygtctct actaaaaata caaaaaaaaa aaattagccg 900
ggcgtggtgg cgggcacctg tagtccctgc tgaggcagga gaatggcttg aacccgggag 960
gtggagcttg cagtgagccg agatcacgcc actgcactcc agcctgggca acagagcaag 1020
attccgtctc aaaaacaaaa aaaaaaaaaa a 1051
<210> 2
<211> 75
<212> PRT
<213> Homo sapiens
<400> 2
Met Gln Ile Ser Val Val Leu His Ile Leu Ile Tyr Ala Trp Leu Aap
1 5 10 15
Tyr Ala Asp Ala Ser Gln Thr Trp Leu Leu Asn Asn Arg Met Gly Leu
20 25 30
Ile Leu Ser Thr Ile Phe Ile Ser Ser Phe Lys Gln Ile Leu Leu Val
35 40 45
Pro Met Trp Thr Phe Lys Asp Pro Leu Leu Asn Glu Met Ser Leu Ser
1
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
50 55 60
Tyr Ser Phe Ala Phe Ile Val Lys Ala Ile Arg
65 70 75
<210> 3
<211> 711
<212> DNA
<213> Homo Sapiens
<400> 3
ttcaggttta gtgctattgt cattgaacac tggtattttc tgtatcatat naaacattaa 60
aattcaaata attataagca tttggcaaaa acaagagaaa agaaacttgc catattttac 120
aagctgcaat tttagaaaag ctttaactta atgatagttt tatcattgtt ttcttgtccc 180
aaacttatcc agggccatag aagtatgaat ctaattaaaa cagaaatggg aattattgca 240
cagaaatggg aaataactaa ttttaaatca gtcmaattgg cttcttatta aatacaataa 300
ttcttatgaa aatcatagta ccctattttc agacacagct gccagtttac acatttctca 360
gtatcctgaa aggaaaaaag tatagcccca cttatactat gtaaaattac caataaaata 420
tttttatgac tacagatttt gcatttttgt ttacaactat ttaaagagtt ttatgttgta 480
tttagaattt caacctagaa accacacagt acttaaattc tcctggggtc tcctgctttc 540
tcttaaccat ttgcttaata tatatctacc taaaggagac ttctgaattg taaatgaact 600
taaaaataga atgtggatgc aaaatatcac ataagacatc atgataacat ttgaagaaaa 660
aataaaactg tagaccctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 711
<210> 4
<211> 46
<212> PRT
<213> Homo sapiens
<220>
<221> UNSURE
<222> (42)
<400> 4
Met Ile Val Leu Ser Leu Phe Ser Cys Pro Lys Leu Ile Gln Gly His
1 5 10 15
Arg Ser Met Asn Leu Ile Lys Thr Glu Met Gly Ile Ile Ala Gln Lys
20 25 30
Trp Glu Ile Thr Asn Phe Lys Ser Val Xaa Leu Ala Ser Tyr
35 40 45
<210> 5
<211> 4529
<212> DaJA
<213> Homo sapiens
<400> 5
cacctgggac ctctcacagg tggcatgccc ttccaagacc aggagagaac accacaactt 60
tgcaggacca gcatctctca gcctgcctcc tccctttctc catatcaata tgatatggat 120
gagtactcat ccagcattgg atgatatgga tgaatggtca tccatcccag agcactgtcc 180
ctactccagt tccagggcac tcttagtctg ttcttggcac atacagcttt gcatctgtca 240
gtgtccatgc aaaagccaac acaagctgtg atggactgtg cattgggtag ccaccagagg 300
taactgccac ttatctatca gtcagccttt gacaggcctc acgtagatac gtacctccca 360
tcacctccaa gctggaatac actttactca tctgtaaaat gggtatgaac atgcctcctt 420
tgcagarctt ttttgaagat tctgcattta ctagtttaac aactgttcag tacctattat 480
gggcctagct accggcacat agtaaacact cagttacaat agctttaaga aaatctgtct 540
2
CA 02320618 2000-08-11
wo ~i4ms4 rcTNS~rozs9s
tagacaaagg agtttcaaaa ctatgtgaag agtatacaag aagtagtagt ccaaactgtt 600
ttttcagttg tttaatgatc tctatgataa agatgttcag ggagaggaat atcttgcgtg 660
ataactggag taaaacccac ttagatttct atgtccccaa acctctggtg tggtcagggt 720
tggcacctgc cctctcaacc atttgtctca ctgtacatgc atcttgtcta tgtcttccac 780
tagagaccat gagggcagag atcagaccta tgtacgttgc acattgctgt ccccagtacc 840
tagcacagca ccttgcccca gtacgcactt catatatatt tcctggacta cggcaggtga 900
gccctttgtg agggttccac ctgcccactg gcctggcctt taatgtcagg ggagcatttc 960
tgatatatta atgcccctct aattgtgctg cattcgttta cataggcagt gcagcagcct 1020
ggctttggtg tttaaagacc ggattgcagt caeagctctg ctgtttgctg gctatgaact 1080
ccaggtaagt tgccaggttc ctctgagcac taatgataat attacctgcc tcacagggtt 1140
gttttgagag cagcttctgc tgctgcccca tgccccagaa ttatggaaaa ccattttgta 1200
aacttcaagt cacttcctct ccatagtaca gatcaatggt agaccagtga tcagggagga 1260
tacaaatcat aaccccaaaa aaggcttctt tgttgtattt tacctcaagt gttttcttag 1320
agaacctcaa caaaccacac tattttctag taatcagcca cttatcccac catttgcagt 1380
aaggaaaact gaggcacgcc ttgatggctt attcagcatc agcagcctca caaaagacag 1440
aagtctcctt tggattagct ttatcatctt tcccttaagc agagatgatg gtgcctttct 1500
cgctgctctg gctcagttaa gaagtcaagt ctgatttatt ggtcatccta actatcgttt 1560
cctgaaggcc cgaagccctg taatgctgtt gtctggcttc ccagatttat gaccatggtt 1620
tctggccccc aacaatatct ggagttcggt ttacatattc acttccacca tccaatccaa 1680
atcctcccag gcagcaatca agctggggct gattcctcat ccatttgtgg gctcactttt 1740
tctggcaagt cagccttcat tctccaggac gcttagaagc cgcatgttgg cggtggattg 1800
tgctacggga aagagtgaat gtgtagcctg cacatttgaa gccttgctgg ggtagagagc 1860
aacgctactg ctcctggaat gtccaggcca ctgttagaaa ggccatctta ttccttagaa 1920
gagtctagcc ctgctgcagg cttcagaaac gtgtgctcta agctcgttcc tgctcatttg 1980
aagcagtctt ctgaaaaggt ctgcagaaac ctgcctttac cctctccctc atcctgcgtg 2040
gcctttgtca catccttcct ccaaggttcc tctggtggtg gtagtaggga ggctgccacc 2100
tggctttgca tgtgggaaag cccagcctct tccctccacc ctcacagcag gacagccaaa 2160
cttggatttt ttcatgagga agcaactctt atgagctctc tggttcagct ctctggttaa 2220
aagaccatac tgtgctgttt ctggggcagc acagacctgg ttgatcgtgt ccacaggaag 2280
aacagccttt gggattcatt agtctggtta agaccagccc tgcagcctcc tctgccagct 2340
atagaactat gtgttagagt tggtgctgaa gccagtcgca aacccatccc agatgcgtct 2400
ggtcttgttg atgttcaaat gccacctcct cctccaggaa gacttcacca cctccccaag 2460
gctgccttat gggctccttc tctgttccct ctcgactctc cccctctatc actatggcca 2520
ttttatagaa ttttaattat tgtctcgggt gtctccacta gactgtaagt atcaagtgtg 2580
tagagacttt tgtttcaact ctgtatcccc aaaattcagc atagttcata gcacatacag 2640
tactctcaat gcctgttgaa tgaataaaga gtctgcttgc atgtaaaact acagtggttt 2700
ggtcttggag acacttgttt tggaattctt ctactgatat ggacagcatc aagatgatgg 2760
gaaatgtatt ggtctgggat ttgttttgcc tacaaagttt cttatgttgg ctcatctcca 2820
actagataaa accctgagtc taggcttccc cttctaacaa agctgctata cctttatgga 2880
cctccatttt cctgtctgta aaatgagagt gtgggctagc tagaaaaatt tctggtgtcc 2940
cccttgctgc tttgtaattc tggagtacgt tcggagaaca ccccaatgtg acasaggatc 3000
tggaaattct tcatasaata ttcagggagg gagacaactc agggaacatg acaactgtct 3060
ggaattattt gaggggttgt catgtagasg atgaaaagga ttagattatc ttgaaaggcc 3120
ccacaggtta taaataagac caattattaa ttcattcaac aaatatttga gtacctgcca 3180
tgtgcaaggc actattctaa gcaataatga tgcagtaatg aatacaacag acaaaaatct 3240
tgttgccttt atggagttga cattctagtg aacagaaaca attcaaaaac aaacacaatg 3300
gagaggagct tcttggaccc tcaaaacgag ctgcctgtga actattgagc tctttgtgtt 3360
caggaatacg tttagtactt agaaatctgc ttccagtggc tgcttagact aaatgacctt 3420
gttaggtttt tttagcccag caatcccatg aatctaaggc tagcgaagat gtgtgatctc 3480
tctattctaa caccactaag ccaaatcatg acetcctctc aatggtaact gtcactctaa 3540
aaagagagat aaggtagatg cagtgggctg ttcttatgaa aaggcaccta aaaaatagta 3600
ctttagaaac atgcattgtg gtgggggtct gggtggaggg gttgcttttt cttgagatcc 3660
ctgtgatatt gaggatgaag aaagagaaac acagggggtg ggcaacttcc ttgaaggacc 3720
agagagagtg agtcatgagg aagagaagga ggagacagag aacaggagag ttggggatga 3780
agcctttgat gaggggagtg aggaggggca gaagtaggag acagacatga ggaaaccacc 3840
aactgaagtc ccagaaaccc agcctccgtc ccctctaggt cctactgccg tcatcctctt 3900
tctgtaaagg tgagagtcta ggtgcagtgt cccaggttcc aaacacaagt tcatactcat 3960
tgactagtgg gatgcccact gtgagagcag gaaggagggc tagagagatc taagagggca 4020
gaagccctgc cctgccagta ttttttgcct ccagccacct gctctgttct gagacatgcc 4080
tgagaccagc tgcctacagg gggaccaggg tctgtttctg ctgttaacct tggttccctc 4140
3
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ctgcattcct gcatgtgggg atgtagacag gaagcctgca ggtatggagc aattgaagca 4200
gagtctagat agagcccaga ctccttactt cccatagtac tattctgcat tttgtagtgc 4260
tgcttatgct tttcagtgtt tccacatcta tttactaata acatcatagt acaacagcaa 4320
gaacatggcc tcagaactca aagttctgga attctaatat tttatttgat tgtcagtatt 4380
tctgtgagat agataaaata ggaattattt cctctgatct gcaggtgagg aaactcagtc 4440
acagggagca catttatcca tacttgtgga gctatagtat aactagtatt aggacaagga 4500
tagccaacat agttaaaaaa aaaaaaaaa 4529
<210> 6
<211> 98
<212> PRT
<213> Homo sapiens
<400> 6
Met Ile Ser Met Ile Lys Met Phe Arg Glu Arg Asn Ile Leu Arg Asp
1 5 10 15
Asn Trp Ser Lys Thr His Leu Asp Phe Tyr Val Pro Lys Pro Leu Val
20 25 ~ 30
Trp Ser Gly Leu Ala Pro Ala Leu Ser Thr Ile Cys Leu Thr Val His
35 40 45
Ala Ser Cys Leu Cys Leu Pro Leu Glu Thr Met Arg Ala Glu Ile Arg
50 55 60
Pro Met Tyr Val Ala His Cys Cys Pro Gln Tyr Leu Ala Gln His Leu
65 70 75 80
Ala Pro Val Arg Thr Ser Tyr Ile Phe Pro Gly Leu Arg Gln Val Ser
85 90 95
Pro Leu
<210> 7
<211> 3537
<212> DNA
<213> Homo sapiens
<400> 7
aacttagatt tcttaaaact gtatacagat aactaatttt tattttgaaa tagcttttat 60
gttcttgtgt ttatgtaata gtcacattaa ttttgctctt ttggctttgt gttgtgttta 120
tttgatgact gttttattcg gtttaatttt ttcttgattt tgcaaattca tttttatcat 180
ctttaagtgg tggagtggcc atttcagctt gaaatatttc actgtcttta taatttcatt 240
ccatgcatgt gttttgtgtt tgtgacggtg tggtggtatt tagtctatag aagaacatgt 300
ggagcaggtc aaccaaaact gcatagcaga agattgccac atcaaggtag tattccaaga 360
tcttgtattt agctcattct ttaaagttca aggagctaac gcctcatctc tgatactaac 420
catgacctgc ccaactggct aactactcag agaactcata gtactcccat accacttcag 480
tggcacaggg gaggagatgc ttcactgtat ctggcttgct ctgtcttgcc cttggttctt 540
ctctttcttg ctccctgtcc catcttcact tgtcactttt tctgttttaa gcatggctgt 600
ttcatcttct tgattctgcc ttttctttcc ttcccatcac caaccctaag ccattgcatc 660
ttcctctcat cagtctcttt tattcctttc gaagtatccc tctttccaac ttttccttat 720
ttctgtctca ccacccttac ttcaaagggt ggtttctttg cggttttcta tggcttttta 780
ctccactctg tgcctcttaa tgtatacaca ttcttattgc agaatattta tgcacttttg 840
gaagagctaa gagagacata ctactgtata aattactaat aaaaatatat tagccatgtt 900
ttataattaa tcagtatctt accatcacag tggaaattgg ggtgattaca ggacatcaga 960
gaatttaagc aaattttcca aagctagttt tcaacacatt aaagttactc ttaccctttt 1020
ttcttgctgc tgctgctgct gctacatttt cttcttatcc tacatttaaa tcttatggga 1080
4
CA 02320618 2000-08-11
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tgtgcataac tctaaataga gctgtgctgc agcaactaaa actgctgatg aatgttttct 1140
aggttgaagc tgatctgggc tatccaggtg gaaaggcgaa agtcatccat aaggaatctg 1200
atatgatcat ggcattttct gttaataagg caaattgtaa tgaaattgtt ttggcttcaa 1260
cacatgatgt tcaagaactt gatgttactt ctctactggc ctgtcagtca tacatatgga 1320
tcggagaaga atatgacaga gaatccaaaa gttcagatga tgttgattat cgtggttcca 1380
ctacaactct ttatcaaccc agtgcaacat cctattcagc aagtcaggtg catccacctt 1440
catctctgcc atggctgggc actggacaga ctagcactgg agctagtgtg cttatgaaaa 1500
ggaatctaca tsatgttaag agaatgactt cacacccagt ccatcsatac tatcttacag 1560
gtgctcagga cggcagtgta cgaatgtttg aatggacgcg gcctcagcaa cttgtctgct 1620
ttcgtcaagc tggcaatgca agagttacta gattatattt taattcacaa ggcaacaagt 1680
gtggtgttgc ggatggagag ggttttctga gtatctggca agttaaccaa actgcatcaa 1740
atcctaaacc ttatatgagt tggcagtgcc acagtaaagc cacaagtgac tttgcattta 1800
ttacctcttc aagtctagtt gccacatctg gacactccaa tgacaataga aatgtttgcc 1860
tctgggacac attaatatca cccggaaaca gcctcattca tggtttcacg tgccacgatc 1920
atggtgccac ggtactgcag tatgcaccca aacagcaact cctaatctcg gggggtagga 1980
aaggacacgt ctgcattttt gacatcaggc aaaggcagct cattcacacg ttccaggccc 2040
atgactcagc tattaaggct ctggccttgg atccctatga ggaatatttt accacaggtt 2100
cagcagaagg taacataaag gtttggagat tgacaggcca tggcctaatt cattcattta 2160
aaagtgaaca tgctaagcag tccatatttc gaaacattgg ggctggagtc atgcagattg 2220
acatcatcca gggcaatcgg ctcttctcct gtggtgcaga tggcacgctg aaaaccaggg 2280
ttttgcccaa tgcttttaac atccctsaca gaattcttga cattctataa agattggggt 2340
tttattttta tatacatttc agttaaaagg cacactacag tcatcactag gcaattctgc 2400
tttctaagca gttgtattga aaacagagaa tctctgtgta gaatttgaat atgacccaag 2460
ctgagtatta tctaaacagg ttggtggaat gaatgcgcat gtaccttatt atgctgacat 2520
actaaaaaaa ataaaaccta gtattgtatg aaggatagct attctttaca gcatttagca 2580
aacctgattc agaaaacatt tgagattagc aaattagtaa cttgaaataa tgaaaaggac 2640
gtttatacca aattaaggaa gaaaatgttg ctgatttggg tttttcttcc tgttcttacc 2700
actgactgaa gcatgcctgc agtctcctcc tctgttgaat gaaggataat cataaggtgt 2760
ttgttaggag cgctagacca cctggaaaac tttcttagct gtggagcagt gcgcagtgac 2820
cagttctctg ctgtgagagg ccgtttccat tctttcctgc tgaatatttt tcctgttagt 2880
gtttatactg agctagtact gtaacttgca aatgagtgca aatttaaatg caatgtttta 2940
ctcacaattt gcacattcac attttttgga ctgctagttt ttctatttaa atatttgcct 3000
tcatgttagg aatgtactat gtgaacatga catatttgta gttaaccaaa cacaccttct 3060
tagtccagtt tagtactttt tcttttcgtg tattcaaggt taaacaccca aacatttaag 3120
gatatgttga aactacacca atagagcatt tcatatcata attaaaatga atgttaggct 3180
tcttgtggcc agttaatagt tgatgagatt ggtgacatta tttattgcca cagcctattg 3240
tataaactat gcagagttaa atatttgctt gtaaaatatt agccaatgtt gtcattattt 3300
tgatgtattt ccttggttat gaccaaaaat atgttgagat actgaaacta atgtctgtgt 3360
gtttaaatgt ttaccagcaa attgtcttat catgttaatg agaatgttca atgcctgtgt 3420
ggtaaatagt aaatacaatg gcataaaagt aactttctct gaagatgtga tgttcaggct 3480
gtgaaatata tatgtaaaag aaaaataaat gttatttgtt agaaasaaaa aaaaaaa 3537
<210> 8
<211> 375
<212> PRT
<213> Homo sapiens
<400> 8
Met Ile Met Ala Phe Ser Val Asn Lys Ala Asn Cys Asn Glu Ile Val
1 5 10 15
Leu Ala Ser Thr His Asp Val Gln Glu Leu Asp Val Thr Ser Leu Leu
20 25 30
Ala Cys Gln Ser Tyr Ile Trp Ile Gly Glu Glu Tyr Asp Arg Glu Ser
35 40 45
Lys Ser Ser Asp Asp Val Asp Tyr Arg Gly Ser Thr Thr Thr Leu Tyr
50 55 60
CA 02320618 2000-08-11
WO 99/41284 PCTIUS99/02898
Gln Pro Ser Ala Thr Ser Tyr Ser Ala Ser Gln Val His Pro Pro Ser
65 70 75 80
Ser Leu Pro Trp Leu Gly Thr Gly Gln Thr Ser Thr Gly Ala Ser Val
85 90 95
Leu Met Lys Arg Asn Leu His Asn Val Lys Arg Met Thr Ser His Pro
100 105 110
Val His Gln Tyr Tyr Leu Thr Gly Ala Gln Asp Gly Ser Val Arg Met
115 120 125
Phe Glu Trp Thr Arg Pro Gln Gln Leu Val Cys Phe Arg Gln Ala Gly
130 135 140
Asn Ala Arg Val Thr Arg Leu Tyr Phe Asn Ser Gln Gly Asn Lys Cys
145 150 155 160
Gly Val Ala Asp Gly Glu Gly Phe Leu Ser Ile Trp Gln Val Asn Gln
165 170 175
Thr Ala Ser Asn Pro Lys Pro Tyr Met Ser Trp Gln Cys His Ser Lys
180 185 190
Ala Thr Ser Asp Phe Ala Phe Ile Thr Ser Ser Ser Leu Val Ala Thr
195 200 205
Ser Gly His Ser Asn Asp Asn Arg Asn Val Cys Leu Trp Asp Thr Leu
210 215 220
Ile Ser Pro Gly Asn Ser Leu Ile His Gly Phe Thr Cys His Asp His
225 230 235 240
Gly Ala Thr Val Leu Gln Tyr Ala Pro Lys Gln Gln Leu Leu Ile Ser
245 250 255
Gly Gly Arg Lys Gly His Val Cys Ile Phe Asp Ile Arg Gln Arg Gln
260 265 270
Leu Ile His Thr Phe Gln Ala His Asp Ser Ala Ile Lys Ala Leu Ala
275 280 285
Leu Asp Pro Tyr Glu Giu Tyr Phe Thr Thr Gly Ser Ala Glu Gly Asn
290 295 300
Ile Lys Val Trp Arg Leu Thr Gly His Gly Leu Ile His Ser Phe Lys
305 310 315 320
Ser Glu His Ala Lys Gln Ser Ile Phe Arg Asn Ile Gly Ala Gly Val
325 330 335
Met Gln Ile Asp Ile Ile Gln Gly Asn Arg Leu Phe Ser Cys Gly Ala
340 345 350
Asp Gly Thr Leu Lys Thr Arg Val Leu Pro Asn Ala Phe Asn Ile Pro
355 360 365
Asn Arg Ile Leu Asp Ile Leu
370 375
6
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
<210> 9
<211> 1466
<212> DNA
<213> Homo sapiens
<400> 9
ccgcgcagga ggacggagcc ctaaccgcaa cccgcgccgc gccgcgccga tttgatttgt 60
atccactgtc accagcactg ctcacttagg actttctgga tccggaccca ggcagcgcac 120
actggactct tgaggaagaa ggagactcta attttggatt ccttggtgga ggaaaataaa 180
acactctggt cttgccgcca acgatgcaag tgtgactgct ggcgtcttca tgagctccag 240
aggtcacagc acgctaccaa ggactctcat ggcccctcgg atgatttccg agggagacat 300
argaggcatt gctcaaatca cctcctctct attcctgggc agaggcagtg tggcctccaa 360
tcggcacctc ctccaggctc gtggcatcac ctgcattgtt aatgctacca ttgagatccc 420
taatttcaac tggccccaat ttgagtatgt taaagtgcct ctggctgaca tgccgcatgc 480
ccccattgga ctgtactttg acaccgtggc tgacaagatc cacagtgtga gcaggaagca 540
cggggccacc ttggtgcact gtgctgcagg ggtgagccgc tcagccacgc tgtgtatcgc 600
gtacctgatg aaattccaca acgtgtgcct gctggaggcg tacaactggg tgaaagcccg 660
gcgacctgtc atcaggccca acgtaggctt ctggaggcaa ctgatagact acgagcgcca 720
gctctttggg aagtcgacag ttaaaatggt acagacacct tatggcatag ttcccgacgt 780
ctatgagaag gagtcccgac acctgatgcc ttactggggg atttagtgcc actgaagcct 840
gcgtcagcag cccgagcggg gccggcatct gctccccgcc gtctgctccc tctccactct 900
cttctcaaat ggctgacttc tggttctccc tcaagtgttt tttacactgg gtgttcaaat 960
ttattttaag agatagggag ggaggggaca taaagggaat gcatacattg ctagtcacat 1020
ttttaaaatt aacattttgg aatagtgttt atggaaatct ttagctttta atcattttta 1080
ccaatttgaa cagtttaata aactggttct gctctcttct gaatctcakg ccttkggcac 1140
cttggtaggt gcaggaggag ctcagtgcaa aaatcacttt ggggcctcat taacccttta 1200
gagacaagyt ttgccccagg ytgcggacca gacagatgyt tagggaaggt tgataccagc 1260
ttcagtctct astggattag ccctactctt tcctttcccc tccattattt agtgactctg 1320
taagtaagtt aaatacaccc ttattattta gctgttaagt aactataatg aaatctgctg 1380
caaaatctct cttggaatcc atgtgcccag gattatatta gcattatttt taataaatct 1440
atatgcttaa caaaaaaaaa aaaaaa 1466
<210> 10
<211> 198
<212> PRT
<213> Homo sapiens
<220>
<221> UNSURE
<222> (25)
<400> 10
Met Ser Ser Arg Gly His Ser Thr Leu Pro Arg Thr Leu Met Ala Pro
1 5 10 15
Arg Met Ile Ser Glu Gly Asp .Ile Xaa Gly Ile Ala Gln Ile Thr Ser
20 25 30
Ser Leu Phe Leu Gly Arg Gly Ser Val Ala Ser Asn Arg His Leu Leu
35 40 45
Gln Ala Arg Gly Ile Thr Cys Ile Val Asn Ala Thr Ile Glu Ile Pro
50 55 60
Asn Phe Asn Trp Pro Gln Phe Glu Tyr Val Lys Val Pro Leu Ala Asp
65 70 75 80
Met Pro His Ala Pro Ile Gly Leu Tyr Phe Asp Thr Val Ala Asp Lys
85 90 95
7
CA 02320618 2000-08-11
WO 99/41284 PCTNS99/02898
Ile His Ser Val Ser Arg Lys His Gly Als Thr Leu Val His Cys Ala
100 105 110
Ala Gly VaI Ser Arg Ser Ala Thr Leu Cys Ile Ala Tyr Leu Met Lys
115 120 125
Phe His Asn Val Cys Leu Leu Glu Ala Tyr Asn Trp Val Lys Ala Arg
130 135 140
Arg Pro Val Ile Arg Pro Asn Val Gly Phe Trp Arg Gln Leu Ile Asp
145 150 155 160
Tyr Glu Arg Gln Leu Phe Gly Lys Ser Thr Val Lys Met Val Gln Thr
165 170 175
Pro Tyr Gly Ile Val Pro Asp Val Tyr Glu Lys Glu Ser Arg His Leu
180 185 190
Met Pro Tyr Trp Gly Ile
195
<210> 11
<211> 1521
<212> DNA
<213> Homo eapiens
<400> 11
aatttcttgg gcatttaccc atgccagaag gctaacctgg ggggaggggg gcgcttgtgc 60
tggtgaggca cttggataca tactgatgct gcaagttcag gggatttttc ttactcttag 120
gtttaaccaa gaacactgag cagggaaaaa ccctgccttt cctaactgca tgtatttttt 180
cctttttgga aaggtggtag agactcagaa gctttccttg ttttcttcag gcctgctccc 240
agttttctta acagtttctt ttgttgcttt ctctctccct tgttgctttc catggcagta 300
atcctcctag agtccaagca gtctgttgta tggagcaggg tgtgtgggtt ttctgggccc 360
atcattatgg ctgcttcaga gtcagaagaa agccataggg cagtagggga gctcctattg 420
cctarcccct ctccctttgt ggctcccact ctagctgcct atttttgctc atcagctggt 480
gagtcagtat gggccagcag ttctccctcc ctaagccctt gctactttat gggttagctt 540
tgcaggtttg gtggcttgag gggtgggggc aactcaccac tgccaggtaa ctccctgaag 600
ggtgggagtg gattatcttc taggctctta cccgcggtag ggaagggcat caacactgtc 660
ttccttccat tctcctttcc cccatcccat ttagtgctgc cacagggcag aagcacacaa 720
accaaccaca cagtctctga cttctcctaa gcactttgag ttgttgaatg gggctcaggg 780
gcaagagttt ttgctgccct ccccagcgtg gtcacagggt tattgaactg cctgcacttg 840
tttctcatgc aactccagca ttttccccag aagttgaact atggatagca gcttggtatg 900
gatttcctaa atcttaacat ttgaagcagc ttcttgaggc tggcaactat cctggtttct 960
gtcttggagg gggtggtttg tttgctgggg cccaacgtct gtcccaagtg gtggggtgag 1020
agtaagttaa ctttggtgcc aggtgagagg tgggggctct ttgcttagac tccctatcat 1080
ggaaagattg gagttttcta tgcagggcac tggggaaaag gattgctgat tctgactgac 1140
cctgatcaga gagattagga ttgtattttg acataggatt tggaacccat ctaaatgttg 1200
aagttccctg agacrgctct ccagctgctg agcctgcgcc aggggytaag cagcccctaa 1260
tgagaggctc tgctcccttt cccacctcgc caatgttgtt gttgctgcct ttttgatttg 1320
tatcctctgt tatagacatt ttttraaaac gatttcctct ttcattgtgc acaagtgctg 1380
agagtctgag gccccatttc tgctgtgtat atatatcctg actcggggct tttattcagc 1440
aaactgttca ttcttctgtc agacaatgtc atattcaact ctgttcatat taaaccactg 1500
tgaagcaaaa aaaaaaaaaa a 1521
<210> 12
<211> 81
<212> PRT
<213> Homo sapiens
g
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
<220>
<221> UNSURE
<222> (45)
<400> 12
Met Ala Val Ile Leu Leu Glu Ser Lys Gln Ser Val Val Trp Ser Arg
1 5 10 15
Val C)rs Gly Phe Ser Gly Pro Ile Ile Met Ala Ala Ser Glu Ser Glu
20 25 30
Glu Ser His Arg Ala Val Gly Glu Leu Leu Leu Pro Xaa Pro Ser Pro
35 40 45
Phe Val Ala Pro Thr Leu Ala Ala Tyr Phe Cys Ser Ser Ala Gly Glu
50 55 60
Ser Val Trp Ala Ser Ser Ser Pro Ser Leu Ser Pro Cys Tyr Phe Met
65 70 75 gp
Gly
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 13
ggatggggct cattctctc 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 14
gaaatgtgta aactggcagc 20
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 15
gaatgcagca caattagagg g 21
<210> 16
<211> 21
9
CA 02320618 2000-08-11
WO 99/41284 PCT/US99/02898
<212> DT1A
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 16
ctgcttagaa agcagaattg c 21
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotfde
<400> 17
tgtgactagc aatgtatgc 19
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 18
tcagaagaaa gccatagggc 20
<210> 19
<211> 113
<212> PRT
<213> Homo sapiens
<400> 19
Met Ser Arg pro Leu Leu Glu Arg pro Ser Tyr Ser Leu Glu Glu Ser
1 5 10 15
Ser Pro Ala Ala Gly Phe Arg Asn Val Cys Ser Lya Leu Val Pro Ala
20 25 30
His Leu Lys Gln Ser Ser Glu Lys Val Cys Arg Asn Leu Pro Leu Pro
35 40 45
Ser Pro Ser Ser Cys Val Ala Phe Val Thr Ser Phe Leu Gln Gly Ser
50 55 60
Ser Gly Gly Gly Ser Arg Glu Ala Ala Thr Trp Leu Cys Met Trp Glu
65 70 75 80
Ser Pro Ala Ser Ser Leu His Pro His Ser Arg Thr Ala Lys Leu Gly
85 90 95
Phe Phe His Glu Glu Ala Thr Leu Met Ser Ser Leu Val Gln Leu Ser
100 105 110
Gly
