Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING'THEM
This application is a continuation-in-part of provisional application Ser. No.
60/080,969, filed April 7,1998, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polvnucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
past decade. The now routine hybridization cloning and expression cloning
techniques
2 5 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
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
3 0 sequence motif, as well as various PCR-based or low stringency
hybridization cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
3 5 polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 45 to nucleotide 590;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 126 to nucleotide 590;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ya9_1 deposited under accession number
ATCC 98724;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ya9_1 deposited under accession number ATCC 98724;
{f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ya9_1 deposited under accession number ATCC
98724;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ya9_1 deposited under accession number ATCC 98724;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ TD N0:2;
2 5 (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any
3 0 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 N0:1.
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:1 from nucleotide 45 to nucleotide 590; the nucleotide sequence of SEQ ID
NO:1 from
nucleotide 126 to nucleotide 590; the nucleotide sequence of the full-length
protein coding
sequence of clone ya9_1 deposited under accession number ATCC 98724; or the
nucleotide sequence of a mature protein coding sequence of clone ya9_1
deposited under
accession number ATCC 98724. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone ya9_1
deposited under accession number ATCC 98724. 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 86 to amino acid
95 of SEQ
ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
2 0 (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 N0:1, but excluding the poly(A) tail at the
2 5 3' end of SEQ ID NO:1; and
(ab) the nucleotide sequence of the cDNA insert of clone
ya9_2 deposited under accession number ATCC 98724;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
3 0 (iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
3
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(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 N0:1, but excluding the poly(A) tail at the
3' end of SEQ ID N0:1; and
(bb) the nucleotide sequence of the cDNA insert of clone
ya9_1 deposited under accession number ATCC 98724;
(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 N0:1, 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 NO:1. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID NO:1 from nucleotide 45 to nucleotide 590, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
2 0 SEQ ID NO:1 from nucleotide 45 to nucleotide 590, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:1 from nucleotide 45
to
nucleotide 590. Also preferably the polynucleotide isolated according to the
above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:1 from nucleotide 126 to nucleotide 590, and extending contiguously from a
2 5 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from
nucleotide 126 to nucleotide 590, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID NO:1 from nucleotide 126 to nucleotide 590.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 consisting of:
(a) the amino acid sequence of SEQ ID NO: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
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(c} the amino acid sequence encoded by the cDNA insert of clone
ya9_1 deposited under accession number ATCC 98724;
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
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 86 to amino acid 95 of SEQ ID N0:2.
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 194 to nucleotide 466;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 338 to nucleotide 466;
(d) a polynucleotide comprising the nucleotide sequence of the full-
2 0 length protein coding sequence of clone yall 1 deposited under accession
number
ATCC 98724;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yall 1 deposited under accession number ATCC 98724;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 5 protein coding sequence of clone yal l 1 deposited under accession number
ATCC
98724;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yall 1 deposited under accession number ATCC 98724;
(h) a polynucleotide encoding a protein comprising the amino acid
3 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;
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(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 N0:3.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 194 to nucleotide 466; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 338 to nucleotide 466; the nucleotide sequence of the full-length
protein coding
sequence of clone yall_1 deposited under accession number ATCC 98724; or the
nucleotide sequence of a mature protein coding sequence of clone yall_1
deposited under
accession number ATCC 98724. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone yal l_1
deposited under accession number ATCC 98724. 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
2 0 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 40 to amino acid
49 of SEQ
ID N0:4.
2 5 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:
3 0 (i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group
consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the
3' end of SEQ ID N0:3; and
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(ab) the nucleotide sequence of the cDNA insert of clone
yall 1 deposited under accession number ATCC 98724;
(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 poiynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
{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
yall_1 deposited under accession number ATCC 98724;
(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 N0:3, 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
2 5 according to the above process comprises a nucleotide sequence
corresponding to the
cDNA sequence of SEQ ID N0:3 from nucleotide 194 to nucleotide 466, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:3 from nucleotide 194 to nucleotide 466, tc~ a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide
194 to
3 0 nucleotide 466. Also preferabiy the polynucleotide isolated according to
the above
process comprises a nucleotide sequence corresponding to the cDNA sequence of
SEQ ID
N0:3 from nucleotide 338 to nucleotide 466, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from
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nucleotide 338 to nucleotide 466, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:3 from nucleotide 338 to nucleotide 466.
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
fragment comprising eight contiguous amino acids of SEQ ID N0:4; and
(c) the amino acid sequence encoded by the cDNA insert of clone
yall 1 deposited under accession number ATCC 98724;
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
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 40 to amino acid 49 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an
2 0 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 15 to nucleotide 233;
2 5 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 174 to nucleotide 233;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ya28_1 deposited under accession
number
ATCC 98724;
3 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ya28_1 deposited under accession number ATCC 98724;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ya28_1 deposited under accession number ATCC
98724;
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(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ya28_1 deposited under accession number ATCC 98724;
{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;
(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 N0:5.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 15 to nucleotide 233; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 174 to nucleotide 233; the nucleotide sequence of the full-length
protein coding
2 0 sequence of clone ya28_1 deposited under accession number ATCC 98724; or
the
nucleotide sequence of a mature protein coding sequence of clone ya28_1
deposited under
accession number ATCC 98724. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone ya28_1
deposited under accession number ATCC 98724. In further preferred embodiments,
the
2 5 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
3 0 fragment comprising the amino acid sequence from amino acid 31 to amino
acid 40 of SEQ
ID N0:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
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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 N0:5, but excluding the poly(A} tail at the
3' end of SEQ ID N0:5; and
(ab) the nucleotide sequence of the cDNA insert of clone
ya28_1 deposited under accession number ATCC 98724;
{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 bX SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
2 0 (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
ya28_1 deposited under accession number ATCC 98724;
(ii) hybridizing said primers) to human genomic DNA in
2 5 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 N0:5, and
extending
3 0 contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:5 to
a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID NO:5 from nucleotide 15 to nucleotide 233, and
extending
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contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:5 from nucleotide 15 to nucleotide 233, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 15
to
nucleotide 233. 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 174 to nucleotide 233, and extending contiguously from a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from
nucleotide 174 to nucleotide 233, to a nucleotide sequence corresponding to
the 3' end of
said sequence of SEQ ID N0:5 from nucleotide I74 to nucleotide 233.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) a fragment of the amino acid sequence of SEQ ID N0:6, the
fragment comprising eight contiguous amino acids of SEQ ID NO:6; and
{c) the amino acid sequence encoded by the cDNA insert of clone
ya28_1 deposited under accession number ATCC 98724;
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
2 0 embodiments, the present invention provides a protein comprising a
fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
preferably
comprising eight (more preferably twenty, most preferably thirty) 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
2 5 amino acid 31 to amino acid 40 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 102 to nucleotide 461;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone yb81 1 deposited under accession
number
ATCC 98724;
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(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone yb81 1 deposited under accession number ATCC 98724;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone yb81 1 deposited under accession number ATCC
98724;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb81 1 deposited under accession number ATCC 98724;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:8;
(i) a polynucieotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(h); and
(i) a polynucleotide that hybridizes under stringent conditions to any
2 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 N0:7.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 102 to nucleotide 461; the nucleotide sequence of the
full-length
protein coding sequence of clone yb81 1 deposited under accession number ATCC
98724;
2 5 or the nucleotide sequence of a mature protein coding sequence of clone
yb81 1 deposited
under accession number ATCC 98724. In other preferred embodiments, the
poiynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone yb81 1 deposited under accession number ATCC 98724. In further
preferred
embodiments, the present invention provides a poiynucleotide encoding a
protein
3 0 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 polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID NO:B
having
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biological activity, the fragment comprising the amino acid sequence from
amino acid 55
to amino acid 64 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
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 N0:7, but excluding the poly(A) tail at the
3' end of SEQ ID N0:7; and
(ab) the nucleotide sequence of the cDNA insert of clone
yb81 1 deposited under accession number ATCC 98724;
(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
2 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from
the group consisting of:
{ba) SEQ ID N0:7, but excluding the poly{A) tail at the
2 5 3' end of SEQ ID N0:7; and
(bb) the nucleotide sequence of the cDNA insert of clone
yb81 1 deposited under accession number ATCC 98724;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C;
3 0 (iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:7 to
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a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but
excluding the
poly(A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide
isolated
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:7 from nucleotide 102 to nucleotide 461, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:7 from nucleotide I02 to nucleotide 461, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide
102 to
nucleotide 461.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) 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
yb81_1 deposited under accession number ATCC 98724;
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
2 0 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 N0:8 having biological activity, the fragment comprising the amino acid
sequence from
amino acid 55 to amino acid 64 of SEQ ID N0:8.
2 5 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucieotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:9 from nucleotide 170 to nucleotide 2968;
{c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 1370 to nucleotide 2968;
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WO 99/51732 PCT/US99/07643
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ycl4_1 deposited under accession
number
ATCC 98724;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ycl4_1 deposited under accession number ATCC 98724;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ycl4_1 deposited under accession number ATCC
98724;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ycl4_1 deposited under accession number ATCC 98724;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:10 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 ;
2 0 {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.
2 5 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:9 from nucleotide 170 to nucleotide 2968; the nucleotide sequence of SEQ ID
N0:9
from nucleotide 1370 to nucleotide 2968; the nucleotide sequence of the full-
length
protein coding sequence of clone ycl4_1 deposited under accession number ATCC
98724;
or the nucleotide sequence of a mature protein coding sequence of clone ycl4_1
deposited
3 0 under accession number ATCC 98724. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ycl4_1 deposited under accession number ATCC 98724. 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
CA 02324984 2000-10-06
WO 99151732 PCT/US99/07643
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) contiguous amino acids of SEQ ID NO:10, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 461
to amino acid 470 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced
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 N0:9, 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
ycl4_1 deposited under accession number ATCC 98724;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
2 0 (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 5 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
3 0 ycl4_1 deposited under accession number ATCC 98724;
(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).
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WO 99/51732 PCT/US99/07643
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and
extending
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
according to the above process comprises a nucleotide sequence corresponding
to the
cDNA sequence of SEQ ID N0:9 from nucleotide 170 to nucleotide 2968, and
extending
contiguously from a nucleotide sequence corresponding to the 5' end of said
sequence of
SEQ ID N0:9 from nucleotide 170 to nucleotide 2968, to a nucleotide sequence
corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide
170 to
nucleotide 2968. 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 1370 to nucleotide 2968, and extending contiguously from
a
nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from
25 nucleotide 1370 to nucleotide 2968, to a nucleotide sequence corresponding
to the 3' end
of said sequence of SEQ ID N0:9 from nucleotide 1370 to nucleotide 2968.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
2 p (a) the amino acid sequence of SEQ ID NO:10;
(b) a fragment of the amino acid sequence of SEQ ID NO:10, the
fragment comprising eight contiguous amino acids of SEQ ID NO:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone
ycl4_1 deposited under accession number ATCC 98724;
2 5 the protein being substantially free from other iriammalian 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
3 0 of SEQ ID N0:10, or a protein comprising a fragment of the amino acid
sequence of SEQ
ID NO:10 having biological activity, the fragment comprising the amino acid
sequence
from amino acid 461 to amino acid 470 of SEQ ID N0:10.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
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WO 99/51732 PCTIUS99107643
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with , such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable earner. 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 earner.
BRIEF DESCRIPTION OF THE DRAWINGS
2 0 Figures lA and 1B are schematic representations of the pED6 and pNOTs
vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
2 5 Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
3 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.
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WO 99/51732 PCTIUS99/07643
As used herein a "secreted" protein is one which, when expressed in a suitable
host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins} or partially (e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
Clone"ya9 1"
A polynucleotide of the present invention has been identified as clone
"ya9_I".
ya9_1 was isolated from a human adult testes 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. ya9_1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as
"ya9_1 protein").
The nucleotide sequence of ya9_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 ya9_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:2. Amino acids
15 to 27
of SEQ ID N0:2 are a predicted leader/signal sequence, with the predicted
mature amino
2 0 acid sequence beginning at amino acid 28. 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 ya9_1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ya9_1 should be approximately 950 bp.
2 5 The nucleotide sequence disclosed herein for ya9_1 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ya9_1 demonstrated at least some similarity with
sequences
identified as AA442366 (zv62c04.r1 Soares testis NHT Homo Sapiens cDNA clone
758214
5') and AA609166 (af12a08.s1 Soares testis NHT Homo sapiens cDNA clone 1031414
3').
3 0 Based upon sequence similarity, ya9_1 proteins and each similar protein or
peptide may
share at least some activity. The TopPredII computer program predicts an
additional
potential transmembrane domain within the ya9_1 protein sequence, centered
around
amino acid 62 of SEQ ID N0:2.
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Clon ",xal1 1"
A polynucleotide of the present invention has been identified as clone
"yall_1".
yall_1 was isolated from a human adult testes 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. yall 1 is a full-length clone,
including the
entire coding sequence of a secreted protein {also referred to herein as "yall
1 protein").
The nucleotide sequence of yal l_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 yal l_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID N0:4. Amino acids
36 to 48
of SEQ ID N0:4 are a predicted Ieader/signal sequence, with the predicted
mature amino
and sequence beginning at amino acid 49. 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 yall 1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
yal2 1 should be approximately 500 bp.
The nucleotide sequence disclosed herein for yall 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. yall 1 demonstrated at least some similarity with
sequences
2 0 identified as 268274 (Human DNA sequence from cosmid L129H7, Huntingtori s
Disease
Region, chromosome 4p16.3 contains Pseudogene and CpG island). Based upon
sequence
similarity, yall 1 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts an additional potential
transmembrane domain within the yall 1 protein sequence, centered around amino
acid
2 5 81 of SEQ ID N0:4.
Clone ";~a28 1"
A polynucleotide of the present invention has been identified as clone
"ya28_1".
ya28_1 was isolated from a human adult testes cDNA library and was identified
as
3 0 encoding a secreted or transmembrane protein on the basis of computer
analysis of the
amino acid sequence of the encoded protein. ya28_1 is a full-length clone,
including the
entire coding sequence of a secreted protein {also referred to herein as
"ya28_1 protein').
The nucleotide sequence of ya28_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
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WO 99/51732 PCTIUS99107643
reading frame and the predicted amino acid sequence of the ya28_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino acids
41 to 53
of SEQ ID N0:6 are a predicted leader/signal sequence, with the predicted
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 ya28_1
protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
done
ya28_1 should be approximately 300 bp.
The nucleotide sequence disclosed herein for ya28_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ya28_1 demonstrated at least some similarity with
sequences
identified as AA576255 (nm62b09.s1 NCI_CGAP_Br3 Homo Sapiens cDNA clone
IMAGE:1072793). Based upon sequence similarity, ya28_1 proteins and each
similar
protein or peptide may share at least some activity.
ya28_1 protein was expressed in a COS cell expression system, and an expressed
protein band of approximately 23 kDa was detected in membrane fractions using
SDS
polyacrylamide gel electrophoresis.
Clone "yb81 1"
2 0 A polynudeotide of the present invention has been identified as clone
"yb81 1".
yb81_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. yb81 1 is a full-length clone,
including the
entire coding sequence of a secreted protein (also referred to herein as "yb81
1 protein').
2 5 The nucleotide sequence of yb81 1 as presently determined is reported in
SEQ ID
N0:7, and includes a poly(A} tail. What applicants presently believe to be the
proper
reading frame and the predicted amino acid sequence of the yb81 1 protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
3 0 yb81 1 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for yb81 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. yb81 1 demonstrated at least some similarity with
sequences
identified as T67164 (Human alpha-N-acetylglucosamirudase gene). Based upon
sequence
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WO 99151732 PCTNS99/07643
similarity, yb81 1 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts a potential transmembrane
domain
within the yb81 1 protein sequence centered around amino acid 73 of SEQ ID
N0:8.
Clone _ycl4 1"
A polynucleotide of the present invention has been identified as clone
"ycl4_2".
ycl4_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. ycl4_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ycl4_1 protein').
The nucleotide sequence of ycl4_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 ycl4_1 protein
corresponding
to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Amino acids
388 to
400 of SEQ ID NO:10 are a predicted leader/signal sequence, with the predicted
mature
amino acid sequence beginning at amino acid 401. 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 ycl4_1
2 0 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ycl4_1 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for ycl4_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. ycl4_1 demonstrated at least some similarity with
sequences
identified as AA007392 (zh99a08.r1 Soares fetal liver spleen 1NFLS S1 Homo
sapiens
cDNA clone 429398 5'), AA573120 {nj41e10.s1 NCI_CGAP_AA1 Homo Sapiens cDNA
clone IMAGE 995082 similar to TR 6285999 6285999 ORF, COMPLETE CDS), and
D13b42
(Human mRNA for KIAA0017 gene, complete cds). The predicted amino acid
sequence
3 0 disclosed herein for ycl4_1 was searched against the GenPept and GeneSeq
amino acid
sequence databases using the BLASTX search protocol. The predicted ycl4_1
protein
demonstrated at least some similarity to sequences identified as D13642
(ICIAA0017
[Homo sapiens]) and 247816 (unknown [Saccharomyces cerevisiaeJ (S. cerevisiae
chromosome XIII cosmid 9827, and translated products)). The ycl4_1 protein
contains the
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WO 99/51732 PCT/US99/07643
immunoglobulin and major histocompatibility complex protein signature at its
extreme
N-terminus (starting at amino acid 3 of SEQ ID NO:20). Based upon sequence
similarity,
ycl4_1 proteins and each similar protein or peptide may share at least some
activity. The
TopPredII computer program predicts four additional potential transmembrane
domains
within the ycl4_1 protein sequence, centered around amino acids 50, 200, 210,
and 830 of
SEQ ID N0:10, respectively.
ycl4_1 protein was expressed in a COS cell expression system, and an expressed
protein band of approximately 92 kDa was detected in membrane fractions using
SDS
polyacrylamide gel electrophoresis.
DgQosit of Clones
Clones ya9_1, yall 1, ya28_l, yb81 1, and ycl4_1 were deposited on April
7,1998
with the American Type Culture Collection {10801 University Boulevard,
Manassas,
Virginia 20110-2209 U.S.A.) as an original deposit under the Budapest Treaty
and were
given the accession number ATCC 98724, from which each clone comprising a
particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
deposited material will be irrevocably removed upon the granting of the
patent, except
for the requirements specified in 37 C.F.R. f 1.808(b), and the term of the
deposit will
comply with 37 C.F.R. ~ 1.806.
2 0 Each clone has been transfected into separate bacterial cells (E. coh~ in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Figures lA and iB, respectively. The pED6dpc2 vector
2 5 ("pED6") was derived from pED6dpc1 by insertion of a new polylinker to
facilitate
cDNA cloning {Kaufman et al.,1991, Nucleic Acids Res. 19: 4485-4490); the
pNOTs vector
was derived from pMT2 (Kaufman et al.,1989, Mol. Cell. Biol. 9: 946-958) by
deletion of
the DHFR sequences, insertion of a new polylinker, and insertion of the M13
origin of
replication in the CIaI site. In some instances, the deposited clone can
become "flipped"
3 0 (i.e., in the reverse orientation) in the deposited isolate. In such
instances, the cDNA insert
can still be isolated by digestion with EcoRI and NotI. However, NotI will
then produce
the 5' site and EcoRI will produce the 3' site for placement of the cDNA in
proper
orientation for expression in a suitable vector. The cDNA may also be
expressed from the
vectors in which they were deposited.
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WO 99151732 PCT/US99/07643
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of an
oligonucleotide probe that was used to isolate or to sequence each full-length
clone is
identified below, and should be most reliable in isolating the clone of
interest.
Clone Probe Seduence
ya9_1 SEQ ID N0:11
ya28_1 SEQ ID N0:12
ycl4_1 SEQ ID N0:13
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
2 0 parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("Ns"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
2 5 The oligonucleotide should preferably be labeled with y 3~P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
3 0 by measurement in a scintillation counter. Preferably, specific activity
of the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 pl 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
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WO 99/51732 PCT/US99/07643
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ug/ml and agar at 1.5% in a 150 rnm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 lzg/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation 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 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 2 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
2 0 be employed.
The positive colonies are picked, grown in culture, and plasmid D:VTA 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
2 5 biological activity are also encompassed by the present invention.
Fragments of the
protein may be in linear form or they may be ryclized using known methods, for
example,
as described in H.U. Saragovi, et al., Bio/Technology Q 773-778 (1992) and in
R.S.
McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments rnay be fused to carrier molecules such as
3 0 immunoglobulins for many purposes, including increasing the valency of
protein binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature
forms) of such protein may be obtained by expression of the disclosed full-
length
polynucleotide (preferably those deposited with ATCC) in a suitable mammalian
cell or
other host cell. The sequences) of the mature forms) of the protein may also
be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
2 0 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
2 5 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
3 0 disclosed herein, public database sequences having at least some
similarity to the
polynucleotide of the present invention have been listed by database accession
number.
Searches using the GenBank accession numbers of these public database
sequences can
then be performed at an Internet site provided by the National Center for
Biotechnology
Information having the address http://www.ncbi.nlm.nih.gov/UniGene/, in order
to
26
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
identify "UruGene clusters" of overlapping sequences. Many of the "UniGene
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 poiynucleotide sequences disclosed herein are provided.
The
desired change in gene expression can be achieved through the use of antisense
polvnucleotides or ribozymes that bind and/or cleave the mRNA transcribed from
the
gene {Albert and Morris,1994, Trends Pharmacol. Sci.15(7): 250-254; Lavarosky
et al.,1997,
Biochem. MoI. Med. 62(1): 11-22; and Hampel,1998, Prog. Nucleic Acid Res. MoI.
BioI. 58:1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 B1, incorporated by reference
herein).
In addition, organisms are provided in which the gene{s) corresponding to the
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene{s). Partial or complete gene
inactivation
2 0 can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. 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,
2 5 Nature 336: 34&352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
3 0 assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms, part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
27
CA 02324984 2000-10-06
WO 99/51732 PCTNS99/07643
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
For
example, the TopPredII computer program can be used to predict the location of
transmembrane domains in an amino acid sequence, domains which are described
by the
location of the center of the transmsmbrane domain, with at least ten
transmembrane
amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
In particular, sequence identity may be determined using WU-BLAST
2 0 (Washington University BLAST) version 2.0 software, which builds upon WU-
BLAST
version 1.4, which in turn is based on the public domain NCBI-BLAST version
1.4
(Altschul and Gish,1996, Local alignment statistics, Doolittle ed., Methods in
Enzymology
266: 460-480; Altschul et al., /990, Basic local alignment search tool,
Journal of Molecular
Biology 215: 403-410; Gish and States, 1993, Identification of protein coding
regions by
2 5 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
3 0 programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at
that
site, in addition to several support programs. WU-BLAST 2.0 is copyrighted and
may not
be sold or redistributed in any form or manner without the express written
consent of the
author; but the posted executables may otherwise be freely used for
commercial,
nonprofit, or academic purposes. In all search programs in the suite - BLASTP,
BLASTN,
28
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
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
one is Q=9
for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any
integer
value including zero, one through eight, rune, ten, eleven, twelve through
twenty, twenty-
one through fifty, fifty-one through one hundred, etc. The default per-residue
penalty for
extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but
may be
changed to any integer value including zero, one, two, three, four, five, six,
seven, eight,
nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one
through one
hundred, etc. Any combination of values for Q and R can be used in order to
align
sequences so as to maximize overlap and identity while minimizing sequence
gaps. The
default amino and 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
polynucieotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60% sequence
2 0 identity (more preferably, at least 75% identity; most preferably at least
90% identity) with
the given polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
2 5 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
3 0 species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
pygmaeus, Hyiobates
concalor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus marinas, Mus musculus, Ruttus
norvegicus,
Cricetulus griseus, Fells catus, Mustela vison, Canis familinris, Oryctolagus
cuniculus, Bos taurus,
29
CA 02324984 2000-10-06
WO 99151732 PCTNS99/07643
Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been
created
allowing the identification of syntenic relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
(O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al.,
1993, Nature
Genetics 3:103-112; Johansson et al., 1995, Genomics 25: 682-690; Lyons et
al.,1997, Nature
Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
1997, Genome Research 7:1123-1137; all of which are incorporated by reference
herein).
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurnng alternative forms of the isolated
polynucleotides
which also encode proteins which are identical or have significantly similar
sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
60% sequence identity {more preferably, at least 75% identity; most preferably
at least 90%
identity) with the given polynucleotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
overlap and identity while minimizing sequence gaps. Allelic variants may be
isolated and
identified by making suitable probes or primers from the sequences provided
herein and
screening a suitable nucleic acid source from individuals of the appropriate
species.
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
2 0 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
2 5 stringent as, for example, conditions G-L; and reduced stringency
conditions are at least
as stringent as, for example, conditions M-R.
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)t Buffer' and Buffer'
A DNA:DNA 2 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50%
formamide
B DNA:DNA <50 TB*; lxSSC TH*; lxSSC
C DNA:RNA Z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50%
formamide
D DNA:RNA <50 Tp*; IxSSC To*; lxSSC
E RNA:RNA Z 50 70C; IxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50%
formamide
F RNA:RNA <50 TF*; lxSSC TF*; IxSSC
G DNA:DNA 2 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50%
formamide
H DNA:DNA <SO T"*; 4xSSC TH*; 4xSSC
I DNA:RNA 2 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, SO%
formamide
j DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:ItNA Z 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50%
formamide
L RNA:IZ1VA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA x 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50%
formamide
N DNA:DNA <50 T~*; 6x5SC Ty*; 6xSSC
O DNA:RNA 2 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50%
formamide
P DNA:RNA <50 TP*; 6xSSC TP*; 6xSSC
Q RNA:RNA z 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50%
formamide
2 R RNA:IZ1VA <50 TR*; 4xSSC TR*; 4xSSC
0
t: The hybrid length is that antiapated for the hybridized regions) of the
hybridizing poiynucleotides. When
hybridizing a polynucleotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE {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 lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "'TH - 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 according to the
following equations. For hybrids less than 18 base pairs in length,
Tm{°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,°[Na']) + 0.41(%G+C}
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na*] for lxSSC = 0.165 M).
31
CA 02324984 2000-10-06
WO 99/51732 PCTNS99/07643
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity} with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res.19 4485-4490 (1991), in order to produce the
protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 1~ 537-566 (1990). As defined herein "operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in 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 cerevisiue, Schizosaccharomyces pombe, Ktuyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
32
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 155 l1987Z 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 immunoaffiruty
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 Invitrogen Corporation (Carlsbad, CA), respectively. The protein can also
be tagged with
an epitope and subsequently purified by using a specific antibody directed to
such
epitope. One such epitope ("Flag") is commercially available from the Eastman
Kodak
Company {New Haven, CT).
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CA 02324984 2000-10-06
WO 99/51732 PCTNS99/07643
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
2 0 The proteins provided herein also include proteins characterized by amino
acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberateiy 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
34
CA 02324984 2000-10-06
WO 99/51732 PCTNS99/07643
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states}; as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes 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 PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
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
potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
3 0 described in Gyuris et al., 1993, CeII 75: 791-803 and in Rossi et
aL,1997, Proc. NatI. Acad.
Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to
identify
polynucleotides encoding the other protein with which binding occurs or to
identify
inhibitors of the binding interaction.
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
Kimmel eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 5 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
3 0 can be added to the medium in or on which the microorganism is cultured.
C t~okin~,and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
36
CA 02324984 2000-10-06
WO 99!51732 PCT/US99/07643
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor-dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+}, 2E8, RBS, DAl, 123, T1165, HT2, CTLL2, TF-1, Mo7e and
CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Knusbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol.152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
2 0 stimulation, Kruisbeek, A.M, and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
2 5 cells include, without limitation, those described in: Measurement of
Human and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
3 0 Measurement of mouse and human interleukin 6 - Nordan, R. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
in IrnmunoIogy. J.E.e.a. Coligan eds. VoI 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
3?
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Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 19$0; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulatin off' r Suppressing Acttvitv
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
2 0 in regulating (up or down) growth and proliferation of T and/or B
lymphocytes, as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
2 5 protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
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.
38
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WO 99/51732 PCT/US99/07643
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
S 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, B7)), e.g.,
preventing
2 0 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
2 5 the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s} on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
3 0 molecule to the natural ligand(s) on the immune cells without transmitting
the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
39
CA 02324984 2000-10-06
WO 99/51732 PCT/1JS99107643
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 {/992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
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 blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr 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
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 0 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
2 5 transfected tumor cells are returned to the patient to result in
expression of the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
3 0 costimulation signal to T cells to induce a T cell mediated immune
response against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
X32
41
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Hemnann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974,1982; Handa
et al.,
2 0 J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol.137:3494-
3500,1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
2 5 Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell
function: In vitro
3 0 antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. VoI 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses} include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
42
CA 02324984 2000-10-06
WO 99/51732 PCTIUS99/07643
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783,1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:96/-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzvnkiewicz
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 I55:1I1-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematovoiesis Re zg~aring Activity
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
3 0 lines indicates involvement in regulating hematopoiesis, e.g. in
supporting the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production or
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
43
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WO 99/51732 PCT/US99/07643
cells such as granuiocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myeio-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 far 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
ai., Molecular and Cellular Biology 13:473-486, 1993; McCianahan 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 a1. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
3 0 Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
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WO 99/51732 PCTNS99/07643
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al, eds. Vol pp.139-
162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic piastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 0 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
2 5 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
3 0 circumstances where such tissue is not normally formed, has application in
the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligarnent 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, Huntingtori s disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
2 S invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
3 0 generation or regeneration of other tissues, such as organs {including,
for example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
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WO 99/51732 PCT/US99/07643
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing. pps. 71-112 (Maibach, HI and Rovee, DT,
eds.}, Year
Book Medical Publishers, l:nc., Chicago, as modified by Eaglstein and Mertz,
J. Invest.
Dermatol 71:382-84 (1978).
Activin/InhibinActivitsr
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 99151732 PCT/US99/07643
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-
782,1986; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et
al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chgmokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 0 population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
of cells can be readily determined by employing such protein or peptide in any
known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
2 5 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
3 0 include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober,. Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. invest. 95:1370-
1376,1995; Lind et al.
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APMIS 103:140-146, 1995; Muller et al Eur. J. Irnmunol. 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 Thrombalytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
{e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Li~and Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
2 5 receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
development of cellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
3 0 receptor/ligand interaction. A protein of the present invention
(including, without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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WO 99/51732 PCTNS99107643
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med.168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med.169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
2 0 inflammatory response syndrome (SIRS)), ischemia-reperfusion injury,
endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
_ from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss
or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
3 0 tumor growth and metastasis. Cadherin malfunction is also implicated in
other human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
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CA 02324984 2000-10-06
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extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polvnucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
2 0 in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
2 5 inappropriately expressed cadherins, restoring normal cell adhesive
properties and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
3 0 the adhesion of inappropriately expressed tumor-cell cadherins, preventing
the cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
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Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-
1038,1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
antibody-dependent cell-mediated cytotoxicity (ADCC)). A protein may exhibit
its tumor
inhibitory activity by acting on tumor tissue or tumor precursor tissue, by
inhibiting
formation of tissues necessary to support tumor growth (such as, for example,
by
2 0 inhibiting angiogenesis), by causing production of other factors, agents
or cell types which
inhibit tumor growth, or by suppressing, eliminating or inhibiting factors,
agents or cell
types which promote tumor growth.
Other Activities
2 5 A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
3 0 or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
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WO 99/51732 PCT/US99/07643
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
Garner.
Such a composition may also contain (in addition to protein and a Garner)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
2 0 carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may further
contain other
2 5 agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
ar 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
53
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
compositions of the invention may comprise a protein of the invention in such
muitimeric
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 antigen{s) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
1 S invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with ampMpathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lameliar layers
2 0 in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, digiycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
2 5 by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
3 0 prevention or amelioration of such conditions. When applied to an
individual active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
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WO 99/51732 PCT/US99/07643
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factors}, thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
2 0 administered orally, protein of the present invention will be in the form
of a tablet,
capsule, powder, solution ar elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
2 5 When administered in liquid form, a liquid Garner such as water,
petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
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
CA 02324984 2000-10-06
WO 99/51732 PCT/US99/07643
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, isotorucity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringei s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
2 0 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 lZg
to about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
2 5 contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
3 0 polyclonal and monoclonal antibodies which specifically react with the
protein. As used
herein, the term "antibody" includes without limitation a polyclonal antibody,
a
monoclonal antibody, a chimeric antibody, a single-chain antibody, a CDR-
grafted
antibody, a humanized antibody, or fragments thereof which bind to the
indicated protein.
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WO 99151732 PCT/US99/07643
Such term also includes any other species derived from an antibody or antibody
sequence
which is capable of binding the indicated protein.
Antibodies to a particular protein can be produced by methods well known to
those
skilled in the art. For example, monoclonal antibodies can be produced by
generation of
antibody-producing hybridomas in accordance with known methods (see for
example,
Goding, 1983, Monoclonal antibodies: principles and practice, Academic Press
Inc., New
York; and Yokoyama, 1992, "Production of Monoclonal Antibodies" in Current
Protocols
in Immunology, Unit 2.5, Greene Publishing Assoc. and John Wiley & Sons).
Polyclonal
sera and antibodies can be produced by inoculation of a mammalian subject with
the
relevant protein or fragments thereof in accordance with known methods.
Fragments of
antibodies, receptors, or other reactive peptides can be produced from the
corresponding
antibodies by cleavage of and collection of the desired fragments in
accordance with
known methods (see for example, Goding, supra; and Andrew et al., 1992,
"Fragmentation
of Immunoglobulins" in Current Protocols in Immunology, Unit 2.8, Greene
Publishing
Assoc. and John Wiley & Sons). Chimeric antibodies and single chain antibodies
can also
be produced in accordance with known recombinant methods (see for example,
5,169,939,
5,194,594, and 5,576,184). Humanized antibodies can also be made from
corresponding
marine antibodies in accordance with well known methods (see for example, U.S.
Patent
Nos. 5,530,101, 5,585,089, and 5,693,762). Additionally, human antibodies may
be
2 0 produced in non-human animals such as mice that have been genetically
altered to express
human antibody molecules (see for example Fishwild et al., 1996, Nature
Biotechnology
14: 845-851; Mendez et al., 1997, Nature Genetics 15: 146-156 (erratum Nature
Genetics
16: 410); and U.S. Patents 5,877,397 and 5,625,126). Such antibodies may be
obtained
using either the entire protein or fragments thereof as an immunogen. The
peptide
2 5 immunogens additionally may contain a cysteine residue at the carboxyl
terminus, and
are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods
for
synthesizing such peptides are known in the art, for example, as in R.P.
Merrifield, J.
Amer.Chem.Soc. ~5, 2149-2154 (1963); J.L. Krstenansky, et aL, FEBS Lett.
211,10 (1987).
Monoclonal antibodies binding to the protein of the invention may be useful
3 0 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
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abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bane and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
2 0 applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
2 5 sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,
polyglycolic acid and
polyanhydrides. Other potential rnaterials 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
3 0 ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
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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
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
2 0 question. These agents include various growth factors such as epidermal
growth factor
(EGF), platelet derived growth factor (PDGF}, transforming growth factors (TGF-
a and
TGF-(i), 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
2 5 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
3 0 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
59
CA 02324984 2000-10-06
WO 99/51732 PCTNS99/07643
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
CA 02324984 2000-10-06
WO 99/51732 PGT/US99/07643
SEQUENCE LISTING
<110> Wong, Gordon G.
Clark, Hilary
Fechtel, Kim
Agostino, Michael J.
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6301A
<140>
<141>
<160> 13
<170> PatentIn Ver. 2.0
<210> 1
<211> 697
<212> DNA
<213> Homo Sapiens
<400> 1
tgaagcttct gcacatgtag ttcctagagc tgctgcttat taaaatgtca acatcttcat 60
cttctagctg ggacaacctc ttagagtctc tctctctcag cacagtatgg aattggatac 120
aagcaagttt tttgggagag actagtgcac ctcagcaaac aagtttggga ctattatata 180
atcttgctcc agctgtgcaa atcatcttga ggatttcttt cttgatttta ttgggaatag 240
gaatatatgc cttatggaaa cgaagtattc agtcaattca gaaaacattg ttgtttgtaa 300
tcacactcta caaactttac aagaagggct cacatatttt tgaggctttg ctagccaacc 360
cagaaggaag tggtctccga attcaagaca ataataatct tttcctgtcc ttgggtctgc 420
aagagaaaat tttgaaaaaa cttaagacag tggaaaacaa aatgaagaac ctagaaggga 480
taatcgttgc tcaaaaacct gccacgaaga gggattgctc ctctgagccc tactgcagct 540
gctctgactg ccagagtccc ttgtccacat cagggtttac ttcccccatt tgaaatgtga 600
tggactccaa tcttttccag gaaagcactg tttccctcat gtgtgcagtg gtgtatcaat 660
aaagatagag aacgctattg aaaaaaaaaa aaaaaaa 697
<210> 2
<211> 182
<212> PRT
<213> Homo Sapiens
<400> 2
Met Ser Thr Ser Ser Ser Ser Ser Trp Asp Asn Leu Leu Glu Ser Leu
1 5 10 15
Ser Leu Ser Thr Val Trp Asn Trp Ile Gln Ala Ser Phe Leu Gly Glu
20 25 30
Thr Ser Ala Pro Gln Gln Thr Ser Leu Gly Leu Leu Tyr Asn Leu Ala
35 40 45
Pro Ala Val Gln Ile Ile Leu Arg Ile Ser Phe Leu Ile Leu Leu Gly
50 55 60
Ile Gly Ile Tyr Ala Leu Trp Lys Arg Ser Ile Gln Ser Ile Gln Lys
65 70 75 80
Thr Leu Leu Phe Val Ile Thr Leu Tyr Lys Leu Tyr Lys Lys Gly Ser
1
CA 02324984 2000-10-06
WO 99151732 PCT/US99/07643
85 90 95
His Ile Phe Glu Ala Leu Leu Ala Asn Pro Glu Gly Ser GIy Leu Arg
100 105 110
Ile Gln Asp Asn Asn Asn Leu Phe Leu Ser Leu Gly Leu Gln Glu Lys
115 120 125
Ile Leu Lys Lys Leu Lys Thr Val Glu Asn Lys Met Lys Asn Leu Glu
130 135 140
Gly Ile Ile Val Ala Gln Lys Pro Ala Thr Lys Arg Asp Cys Ser Ser
145 150 155 160
Glu Pro Tyr Cys Ser Cys Ser Asp Cys Gln Ser Pro Leu Ser Thr Ser
165 170 175
Gly Phe Thr Ser Pro Ile
lao
<210> 3
<211> 983
<212> DNA
<213> Homo Sapiens
<400> 3
gggcgtggtg tcttgctgtc tcaggccctt cctctgtgca tccacgtaag tgtgggtaag 60
ccaagaatag cctgggttca aatccacctt tgccgttaat tggctgtgtg ctggtagaca 120
agttacttag cttctctgtg cccccactcc ctcatctgca cgcgagaatt gtaacagagc 180
cttcctcaga gcgatgatgg tgtttaggat gacatgcgct gcacagcaag tcctggccgt 240
ggcggctgcc attgcagccc gtggggtcac cctggaggcc gtgattgctg atgttgtgct 300
gttcttgtgc cttgctctct tcttctcggt gcccccagac tctctctgcc ttcggaggac 360
ggcgtctctc tcgcctcaca ctgtgtgcac gggcagtgcg gacgggtgct ggcttggtct 420
ttccagccct gcctcgctcg gggcctgctg catcgtagct caggcctagg acccatctct 480
gtacctgcag gtcttgggtg ctgcccggca tgagtggagg agtttatcag sacaggacct 540
tttataggag gttttaactt tagaagggaa tagaaaagtg tcatggcagc aatatttatt 600
tctagatcac cctgagtttt ttttctttgt ttkgtttwat tgtcctcttt acaccatgag 660
tttttaatga tgaatgagtg aaggagtgac agtgcgggtt gagcatccct tatccagatg 720
ctccagaatc ggaaaccctc tgacaccgat gtggcacctc aggcatagct gagctagtga 780
cacctttgct ttctcatggg tcagtgtaca caaaccttgt ttcatgcaca aaattatcaa 840
aagtaccgca caaaattacc ctcaggctgt gtgtataagg tgtatatgaa acataaatga 900
atttcctgtt cagacctggg tcctatccaa acatatctca ttacgtatat acagatgttc 960
cgaatcagaa aaaaaaaaaa aaa 983
<210> 4
<211> 91
<212> PRT
<213> Homo Sapiens
<400> 4
Met Met Val Phe Arg Met Thr Cys Ala Ala Gln Gln Val Leu Ala Val
1 5 10 15
Ala Ala Ala Ile Ala Ala Arg Gly Val Thr Leu Glu Ala Val Ile Ala
20 25 30
Asp Val Val Leu Phe Leu Cys Leu Ala Leu Phe Phe Ser Val Pro Pro
35 40 45
z
CA 02324984 2000-10-06
WO 99151732 PCTNS99/07643
Asp Ser Leu Cys Leu Arg Arg Thr Ala Ser Leu Ser Pro His Thr Val
50 55 60
Cys Thr Gly Ser Ala Asp Gly Cys Trp Leu Gly Leu Ser Ser Pro Ala
65 70 75 BO
Ser Leu Gly Ala Cys Cys Ile Val Ala Gln Ala
85 90
<210> 5
<211> 267
<212> DNA
<213> Homo sapiens
<400> 5
ggaaaacacc ctaaatgtgc atgcatctcc aagtcctcca ggagccacgc gtgctccaca 60
ttctttcttt tcctggcaca tttttggctg ccaggatgac tgagaaaata atcaaccttc 120
ccatcagtca cctgtttctc ttcctgagca gtttccttct gccactcagt caaaaggccc 180
acaaacatgt tcaccaagtc ctaacctcta gaagggagag agacttcaga gactgatttt 240
tagctgcaac caaaaaaaaa aaaaaaa 267
<210> 6
<211> 73
<212> PRT
<213> Homo sapiens
<400> 6
Met Cys Met His Leu Gln Val Leu Gln Glu Pro Arg Val Leu His Ile
1 5 10 15
Leu Ser Phe Pro Gly Thr Phe Leu Ala Ala Arg Met Thr Glu Lys Ile
20 25 30
Ile Asn Leu Pro Ile Ser His Leu Phe Leu Phe Leu Ser Ser Phe Leu
35 40 45
Leu Pro Leu Ser Gln Lys Ala His Lys His Val His Gln Val Leu Thr
50 55 60
Ser Arg Arg Glu Arg Asp Phe Arg Asp
65 70
<210> 7
<211> 679
<212> DNA
<213> Homo sapiens
<400> 7
aaacttctgg gatcacaggc atgagccacc gtgcctggcc ctgatctggg atttaaaggg 60
gcagttgctg agcaaataga agccagggca agaaaggatt tatgtgcttg ggattcttga 120
ggggttttcc agagtcagtc cccaattctg atgtttttat acgcaacact aggggtcctt 180
ctgaagttat cccggcccct gggaaactca cgtggcatgg ggcccccttc tgccctggtc 240
tctttcatcc catctgtccc ggccccagag cctgtccagt ctggtcctct ctgtcctgct 300
tggtttgtca ccatgtgatg ttgggatggc ttgtctttgc cgcctcactt cctaagctga 360
cagacgtgtc tgctagggac caccagctcc caagccgcat gacggtgctt tccttcaagg 420
ttcagaggct tccctttcag tctggcccat ctcccacatc ctastggctc tgctgtctca 480
ggggcagctc tcctttttaa cttattggca gagctgggat gacttatagg tccctggctc 540
agtgagtaag caagttcaga gacttggctt tggccatttt gttctcttag gctcatcctt 600
3
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ggatgccaac agggaaataa cctgccagat ttcagtcact actttttaga agttaaaaaa 660
aaaaaaaaaa aaaaaaaaa 679
<210> B
<211> 120
<212> PRT
<213> Homo sapiens
<400> 8
Met Cys Leu Gly Phe Leu Arg Gly Phe Pro Glu Ser Val Pro Asn Ser
1 5 10 15
Asp Val Phe Ile Arg Asn Thr Arg Gly Pro Ser Glu Val Ile Pro Ala
20 25 30
Pro Gly Lys Leu Thr Trp His Gly Ala Pro Phe Cys Pro Gly Leu Phe
35 40 45
His Pro Ile Cys Pro Gly Pro Arg Ala Cys Pro Val Trp Ser Ser Leu
50 55 60
Ser Cys Leu Val Cys His His Val Met Leu Gly Trp Leu Val Phe Ala
65 70 75 80
Ala Ser Leu Pro Lys Leu Thr Asp Val Ser Ala Arg Asp His Gln Leu
85 90 95
Pro Ser Arg Met Thr Val Leu Ser Phe Lys Val Gln Arg Leu Pro Phe
100 105 110
Gln Ser Gly Pro Ser Pro Thr Ser
115 120
<210> 9
<211> 3340
<212> DNA
<213> Homo sapiens
<400> 9
ggaggaacac ggcaacttcc ttattacagt tcctggaggg tcagatggtc caagtggagt 60
actgatctgc tctgaaaact atattactta caagsacttt ggtgaccagc cagatatccg 120
ctgtccaatt cccaggaggc ggaatgacct ggatgaccct gaaagaggaa tgatttttgt 180
ctgctctgca acccataaaa ccaaatcgat gttcttcttt ttggctcaaa ctgagcaggg 240
agatatcttt aagatcactt tggagacaga tgaagatatg gttactgaga tccggctcaa 300
atattttgat actgtacccg ttgctgctgc catgtgtgtg cttaaaacag ggttcctttt 360
tgtagcatca gaatttggaa accattactt atatcaaatt gcacatcttg gagatgatga 420
tgaagaacct gagttttcat cagccatgcc tctggaagaa ggagacacat tcttttttca 480
gccmagacca cttaaaaacc ttgtgctggt tgatgagttg gacagcctct ctcccattct 540
gttttgccag atagctgatc tggccaatga agatactcca cagttgtatg tggcctgtgg 600
taggggaccc cgatcatctc tgagagtcct aagacatgga cttgaggtgt cagaaatggc 660
tgtttctgag ctacctggta accccaacgc tgtctggaca gtgcgtcgac acattgaaga ?20
tgagtttgat gcctacatca ttgtgtcttt cgtgaatgcc accctagtgt tgtccattgg 780
agaaactgta gaagaagtga ctgactctgg gttcctgggg accaccccga ccttgtcctg 840
ctccttatta ggagatgatg ccttggtgca ggtctatcca gatggcattc ggcacatacg 900
agcagacaag agagtcaatg agtggaagac ccctggaaag aaaacaattg tgaagtgtgc 960
agtgaaccag cgacaagtgg tgattgccct gacaggagga gagctggtct atttcgagat 1020
ggatccttca ggacagctga atgagtacac agaacggaag gagatgtcag cagatgtggt 1080
gtgcatgagt ctggccaatg taccccctgg agagcagcgg tctcgcttcc tggctgtggg 1140
gcttgtggac aacactgtca gaatcatctc cctggatccc tcagactgtt tgcaacctct 1200
4
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aagcatgcag gctctcccag cccagcctga gtccttgtgt atcgtggaaa tgggtgggac 1260
tgagaagcag gatgagctgg gtgagagggg ctcgattggc ttcctatacc tgaatattgg 1320
gctacagaac ggtgtgctgc tgaggactgt cttggaccct gtcactgggg atttgtctga 1380
tactcgcact cggtacctgg ggtcccgtcc tgtgaagctc ttccgagtcc gaatgcaagg 1440
ccaggaggca gtattggcca tgtcaagccg ctcatggttg agctattctt accaatctcg 1500
cttccatctc accccactgt cttacgagac actggaattt gcatcgggtt ttgcctcgga 1560
acagtgtccc gagggcattg tggccatctc caccaacacc ctacggattt tggcattaga 1620
gaagctcggt gctgtcttca atcaagtagc cttcccactg cagtacacac ccaggaaatt 1680
tgtcatccac cctgagagta acaaccttat tatcattgaa acggaccaca atgcctacac 1740
tgaggccacg aaagctcaga gaaagcagca gatggcagag gaaatggtgg aagcagcagg 1800
ggaggatgag cgggagctgg ccgcagagat ggcagcagca ttcctcaatg aaaacctccc 1860
tgaatccatc tttggagctc ccaaggctgg caatgggcag tgggcctctg tgatccgagt 1920
gatgaatccc attcaaggga acacactgga ccttgtccag ctggaacaga atgaggcagc 1980
ttttagtgtg gctgtgtgca ggttttccaa cactggtgaa gactggtatg tgctggtggg 2040
tgtggccaag gacctgatac taaacccccg atctgtggca gggggcttcg tctatactta 2100
caagcttgtg aacaatgggg aaaaactgga gtttttgcac aagactcctg tggaagaggt 2160
ccctgctgct attgccccat tccaggggag ggtgttgatt ggtgtgggga agctgttgcg 2220
tgtctatgac ctgggaaaga agaagttact ccgaaaatgt gagaataagc atattgccaa 2280
ttatatctct gggatccaga ctattggaca tagggtaatt gtatctgatg tccaagaaag 2340
tttcatctgg gttcgctaca agcgtaatga aaaccagctt atcatctttg ctgatgatac 2400
ctacccccga tgggtcacta cagccagcct cttggactat gacactgtgg ctggggcaga 2460
caagtttggc aacatatgtg tggtgaggct cccacctaac accaatgatg aagtagatga 2520
ggatcctaca ggaaacaaag ccctgtggga ccgtggcttg ctcaatgggg cctcccagaa 2580
ggcagaggtg atcatgaatt accatgtcgg ggagacggtg ctgtccttgc agaagaccac 2640
gctgatccct ggaggctcag aatcacttgt ctataccacc ttgtctggag gaattggcat 2700
ccttgtgcca ttcacgtccc atgaggacca tgacttcttc cagcatgtgg aaatgcacct 2760
gcggtctgaa catccccctc tctgtgggcg ggaccacctc agctttcgct cctactactt 2820
ccctgtgaag aatgtgattg atggagacct ctgtgagcag ttcaattcca tggaacccaa 2880
caaacaaaag aacgtctctg aagaactgga ccgaacccca cccgaagtgt ccaagaaact 2940
cgaggatatc cggacccgct acgccttctg agccctcctt tcccggtggg gcttgccaga 3000
gactgtgtgt tttgtttccc ccaccaccat castgccacc tggcttctgc catgtggcag 3060
gagggtgact ggataattaa gastgcatta tgaaagtcaa cagctctttc ccctcagctc 3120
ttctcstgga atgactggct tcccctcaaa ttggcactga gatttgctac acttctcccc 3180
acctggtaca tgatacatga ccccaggttc cagtgtagaa cctgagtccc ccattcccca 3240
aagccatccc tgcattgata tgtcttgact ctcctgtcta cttttgcaca cacccttaat 3300
ttttaattgg ttttcttgta aatacaaaaa aaaaaaaaaa 3340
<210> 10
<211> 933
<212> PRT
<213> Homo Sapiens
<400> 10
Met Ile Phe Val Cys Ser Ala Thr His Lys Thr Lys Ser Met Phe Phe
1 5 10 15
Phe Leu Ala Gln Thr Glu Gln Gly Asp Ile Phe Lys Ile Thr Leu Glu
20 25 30
Thr Asp Glu Asp Met Val Thr Glu Ile Arg Leu Lys Tyr Phe Asp Thr
35 40 45
Val Pro Val Ala Ala Ala Met Cys Val Leu Lys Thr Gly Phe Leu Phe
50 55 60
Val Ala Ser Glu Phe Gly Asn His Tyr Leu Tyr Gln Ile Ala His Leu
65 70 75 80
Gly Asp Asp Asp Glu Glu Pro Glu Phe Ser Ser Ala Met Pro Leu Glu
85 90 95
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Glu Gly Asp Thr Phe Phe Phe Gln Pro Arg Pro Leu Lys Asn Leu Val
100 105 110
Leu Val Asp Glu Leu Asp Ser Leu Ser Pro Ile Leu Phe Cys Gln Ile
115 120 125
Ala Asp Leu Ala Asn Glu Asp Thr Pro Gln Leu Tyr Val Ala Cys Gly
130 135 140
Arg Gly Pro Arg Ser Ser Leu Arg Val Leu Arg His Gly Leu Glu Val
145 150 155 160
Ser Glu Met Ala Val Ser Glu Leu Pro Gly Asn Pro Asn Ala Val Trp
165 170 I75
Thr Val Arg Arg His Ile Glu Asp Glu Phe Asp Ala Tyr Ile Ile Val
180 185 190
Ser Phe Val Asn Ala Thr Leu Val Leu Ser Ile Gly Glu Thr Val Glu
195 200 205
Glu Val Thr Asp Ser Gly Phe Leu Gly Thr Thr Pro Thr Leu Ser Cys
210 215 220
Ser Leu Leu Gly Asp Asp Ala Leu Val Gln Val Tyr Pro Asp Gly Ile
225 230 235 240
Arg His Ile Arg Ala Asp Lys Arg Val Asn Glu Trp Lys Thr Pro Gly
245 250 255
Lys Lys Thr Ile Val Lys Cys Ala Val Asn Gln Arg Gln Val Val Ile
260 265 270
Ala Leu Thr Gly Gly Glu Leu Val Tyr Phe Glu Met Asp Pro Ser Gly
275 280 285
Gln Leu Asn Glu Tyr Thr Glu Arg Lys Glu Met Ser Ala Asp Val Val
290 295 300
Cys Met Ser Leu Ala Asn Val Pro Pro Gly Glu Gln Arg Ser Arg Phe
305 310 315 320
Leu Ala Val Gly Leu Val Asp Asn Thr Val Arg Ile Ile Ser Leu Asp
325 330 335
Pro Ser Asp Cys Leu Gln Pro Leu Ser Met Gln Ala Leu Pro Ala Gln
340 345 350
Pro Glu Ser Leu Cys Ile Val Glu Met Gly Gly Thr Glu Lys Gln Asp
355 360 365
Glu Leu Gly Glu Arg Gly Ser Ile Gly Phe Leu Tyr Leu Asn Ile Gly
370 , 375 380
Leu Gln Asn Gly Val Leu Leu Arg Thr Val Leu Asp Pro Val Thr Gly
385 390 395 400
Asp Leu Ser Asp Thr Arg Thr Arg Tyr Leu Gly Ser Arg Pro Val Lys
405 410 415
6
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Leu Phe Arg Val Arg Met Gln Gly Gln Glu Ala Val Leu Ala Met Ser
420 425 430
Ser Arg Ser Trp Leu Ser Tyr Ser Tyr Gln Ser Arg Phe His Leu Thr
435 440 445
Pro Leu Ser Tyr Glu Thr Leu Glu Phe Ala Ser Gly Phe Ala Ser Glu
450 455 460
Gln Cys Pro Glu Gly Ile Val Ala Ile Ser Thr Asn Thr Leu Arg Ile
465 470 475 480
Leu Ala Leu Glu Lys Leu Gly Ala VaI Phe Asn Gln Val Ala Phe Pro
485 490 495
Leu Gln Tyr Thr Pro Arg Lys Phe Val Ile His Pro Glu Ser Asn Asn
500 505 510
Leu Ile Ile Ile Glu Thr Asp His Asn Ala Tyr Thr Glu Ala Thr Lys
515 520 525
Ala Gln Arg Lys Gln Gln Met Ala Glu Glu Met Val Glu Ala Ala Gly
530 535 540
Glu Asp Glu Arg Glu Leu Ala Ala Glu Met Ala Ala Ala Phe Leu Asn
545 550 555 560
Glu Asn Leu Pro Glu Ser Ile Phe Gly Ala Pro Lys Ala Gly Asn Gly
565 570 575
Gln Trp Ala Ser Val Ile Arg Val Met Asn Pro Ile Gln Gly Asn Thr
580 585 590
Leu Asp Leu Val Gln Leu Glu Gln Asn Glu Ala Ala Phe Ser Val Ala
595 600 605
Val Cys Arg Phe Ser Asn Thr Gly Glu Asp Trp Tyr Val Leu Val Gly
610 615 620
Val Ala Lys Asp Leu Ile Leu Asn Pro Arg Ser Val Ala Gly Gly Phe
625 630 635 640
Val Tyr Thr Tyr Lys Leu Val Asn Asn Gly Glu Lys Leu Glu Phe Leu
645 650 655
His Lys Thr Pro Val Glu Glu Val Pro Ala Ala Ile Ala Pro Phe Gln
660 665 670
Gly Arg Val Leu Ile Gly Val Gly Lys Leu Leu Arg Val Tyr Asp Leu
675 680 685
Gly Lys Lys Lys Leu Leu Arg Lys Cys Glu Asn Lys His Ile Ala Asn
690 695 700
Tyr Ile Ser Gly Ile Gln Thr Ile Gly His Arg Val Ile Val Ser Asp
705 710 715 720
Val Gln Glu Ser Phe Ile Trp Val Arg Tyr Lys Arg Asn Glu Asn Gln
725 730 735
7
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Leu Ile Ile Phe Ala Asp Asp Thr Tyr Pro Arg Trp Val Thr Thr Ala
740 745 750
Ser Leu Leu Asp Tyr Asp Thr Val Ala Gly Ala Asp Lys Phe Gly Asn
755 760 765
Ile Cys Val Val Arg Leu Pro Pro Asn Thr Asn Asp Glu Val Asp Glu
?70 775 780
Asp Pro Thr Gly Asn Lys Ala Leu Trp Asp Arg Gly Leu Leu Asn Gly
785 790 795 800
Ala Ser Gln Lys Ala Glu Val Ile Met Asn Tyr His Val Gly Glu Thr
8os alo 81s
Val Leu Ser Leu Gln Lys Thr Thr Leu Ile Pro Gly Gly Ser Glu Ser
820 825 830
Leu Val Tyr Thr Thr Leu Ser Gly Gly Ile Gly Ile Leu Val Pro Phe
835 840 845
Thr Ser His Glu Asp His Asp Phe Phe Gln His Val Glu Met His Leu
850 855 B60
Arg Ser Glu His Pro Pro Leu Cys Gly Arg Asp His Leu Ser Phe Arg
s65 870 875 sso
Ser Tyr Tyr Phe Pro Val Lys Asn Val Ile Asp Gly Asp Leu Cys Glu
885 890 895
Gln Phe Asn Ser Met Glu Pro Asn Lys Gln Lys Asn Val Ser Glu Glu
900 905 910
Leu Asp Arg Thr Pro Pro Glu Val Ser Lys Lys Leu Glu Asp Tle Arg
915 920 925
Thr Arg Tyr Ala Phe
930
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 11
cctcaagatg atttgcacag c 21
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
g
CA 02324984 2000-10-06
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<400> 12
ggagagcacg ttcagaggg 19
<220> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 13
gactgactct gggttcctgg 20
9
