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 the following applications: Ser.
No.
08/701,931, filed August 23,1996, which is a continuation-in-part of
application Ser. No.
08/702,420, filed August 14, 1996 and now abandoned; Ser. No. 08/721,925,
filed
September 27, 1996, which is a continuation-in-part of application Ser. No.
08/701,931;
and Ser. No. 08/743,690, filed November 6, 1996, which is a continuation-in-
part of
application Ser. No. 08/702,420; all of which are incorporated by reference
herein.
Although the '420 application was filed before the '931 application, the '420
application
was inexplicably asigned a higher serial number than the '931 application.
However, the
'420 application was correctly afforded a priority date of August 14,1996.
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
2 0 polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
2 5 past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein {i.e., partial DNA/amino acid sequence of
the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
3 0 isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA/amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
3 5 cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION -
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 27 to nucleotide 1652;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 129 to nucleotide 1652;
{d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 1 to nucleotide 413;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AM349 2 deposited under accession
number ATCC 98155;
{f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AM349 2 deposited under accession number ATCC 98155;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AM349 2 deposited under accession number
ATCC 98155;
2 0 (h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AM349 2 deposited under accession number ATCC 98155;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
{j) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:2 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
{a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
3 0 (m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:1 from nucleotide 27 to nucleotide 1652; the nucleotide sequence of SEQ ID
NO:1 from
nucleotide 129 to nucleotide 1652; the nucleotide sequence of SEQ ID NO:1 from
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nucleotide 1 to nucleotide 413; the nucleotide sequence of the full-length
protein coding
sequence of clone AM349_2 deposited under accession number ATCC 98155; or the
nucleotide sequence of the mature protein coding sequence of clone AM349 2
deposited
under accession number ATCC 98155. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone AM349_2 deposited under accession number ATCC 98155. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 1 to amino
acid 129.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 1 to
amino acid 129;
(c) fragments of the amino acid sequence of SEQ ID N0:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AM349_2 deposited under accession number ATCC 98155;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 1 to amino acid 129.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 788 to nucleotide 1153;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:3 from nucleotide 1759 to nucleotide 2146;
(d} a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AR310 3 deposited under accession
number ATCC 98155;
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(e) a polynucleotide encoding the full-length protein encoded by the -
cDNA insert of clone AR310 3 deposited under accession number ATCC 98155;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AR310_3 deposited under accession number
ATCC 98155;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AR310 3 deposited under accession number ATCC 98155;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity;
(j} a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 788 to nucleotide 1153; the nucleotide sequence of SEQ ID
N0:3
2 0 from nucleotide 1759 to nucleotide 2146; the nucleotide sequence of the
full-length protein
coding sequence of clone AR310 3 deposited under accession number ATCC 98155;
or the
nucleotide sequence of the mature protein coding sequence of clone AR310_3
deposited
under accession number ATCC 98155. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 5 clone AR310 3 deposited under accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) fragments of the amino acid sequence of SEQ ID N0:4; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AR310_3 deposited under accession number ATCC 98155;
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the protein being substantially free from other mammalian proteins. Preferably
such -
protein comprises the amino acid sequence of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
{b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 448 to nucleotide 663;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 547 to nucleotide 663;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 321 to nucleotide 603;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AS186_3 deposited under accession
number ATCC 98155;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AS186_3 deposited under accession number ATCC 98155;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AS186_3 deposited under accession number
2 0 ATCC 98155;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AS186_3 deposited under accession number ATCC 98155;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
2 5 (j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity;
(k) . a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
{1) a polynucleotide which encodes a species homologue of the protein
3 0 of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 448 to nucleotide 663; the nucleotide sequence of SEQ ID
N0:5 from
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nucleotide 547 to nucleotide 663; the nucleotide sequence of SEQ ID N0:5 from
nucleotide 321 to nucleotide 603; the nucleotide sequence of the full-length
protein coding
sequence of clone AS186_3 deposited under accession number ATCC 98155; or the
nucleotide sequence of the mature protein coding sequence of clone AS186_3
deposited
under accession number ATCC 98155. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone AS186_3 deposited under accession number ATCC 98155. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:6 from amino acid 1 to amino
acid 52.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ ID N0:6 from amino acid 1 to
amino acid 52;
(c) fragments of the amino acid sequence of SEQ ID N0:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 0 AS186_3 deposited under accession number ATCC 98155;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6 or the amino acid
sequence
of SEQ ID N0:6 from amino acid 1 to amino acid 52.
In one embodiment, the present invention provides a composition comprising an
2 5 isolated polynucleotide selected from the group consisting of:
{a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 140 to nucleotide 1498;
3 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 185 to nucleotide 1498;
{d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 132 to nucleotide 457;
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(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AY160 2 deposited under accession
number ATCC 98155;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AY160_2 deposited under accession number ATCC 98155;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AY160 2 deposited under accession number
ATCC 98155;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AY160 2 deposited under accession number ATCC 98155;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
{j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
2 0 to any one of the polynucleotides specified in (a}-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 140 to nucleotide 1498; the nucleotide sequence of SEQ ID
N0:7
from nucleotide 185 to nucleotide 1498; the nucleotide sequence of SEQ ID N0:7
from
nucleotide 132 to nucleotide 457; the nucleotide sequence of the full-length
protein coding
2 5 sequence of clone AY160_2 deposited under accession number ATCC 98155; or
the
nucleotide sequence of the mature protein coding sequence of clone AY160_2
deposited
under accession number ATCC 98155. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone AY160_2 deposited under accession number ATCC 98155. In yet other
preferred
3 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino
acid 106.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to
amino acid 106;
(c) fragments of the amino acid sequence of SEQ ID N0:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AY160 2 deposited under accession number ATCC 98155;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:B or the amino acid
sequence
of SEQ ID N0:8 from amino acid 1 to amino acid 106:
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 84 to nucleotide 548;
(c) a polynucleotide comprising the nucleotide sequence of the full-
2 0 length protein coding sequence of clone BD127 16 deposited under accession
number ATCC 98155;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BD127 16 deposited under accession number ATCC 98155;
(e) a polynucleotide comprising the nucleotide sequence of the mature
2 5 protein coding sequence of clone BD127 16 deposited under accession number
ATCC 98155;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BD127 16 deposited under accession number ATCC 98155;
(g) a polynucleotide encoding a protein comprising the amino acid
3 0 sequence of SEQ ID N0:10;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
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{j) a polynucleotide which encodes a species homologue of the protein -
of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(h).
S Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 84 to nucleotide 548; the nucleotide sequence of the full-
length
protein coding sequence of clone BD127_16 deposited under accession number
ATCC
98155; or the nucleotide sequence of the mature protein coding sequence of
clone
BD127 16 deposited under accession number ATCC 98155. In other preferred
embodiments, the polynucleotide encodes the full-length or mature protein
encoded by
the cDNA insert of clone BD127 16 deposited under accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9 or SEQ ID N0:11.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID N0:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone
2 0 BD127 16 deposited under accession number ATCC 98155;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 236 to nucleotide 1480;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:12 from nucleotide 450 to nucleotide 800;
(d} a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BL205_14 deposited under accession
number ATCC 98155;
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{e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BL205_14 deposited under accession number ATCC 98155;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BL205_14 deposited under accession number
ATCC 98155;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BL205_14 deposited under accession number ATCC 98155;
(h} a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:13;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:13 having biological activity;
(j} a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:12 from nucleotide 236 to nucleotide 1480; the nucleotide sequence of SEQ
ID N0:12
2 0 from nucleotide 450 to nucleotide 800; the nucleotide sequence of the full-
length protein
coding sequence of clone BL205_14 deposited under accession number ATCC 98155;
or
the nucleotide sequence of the mature protein coding sequence of clone BL205
14
deposited under accession number ATCC 98155. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 5 clone BL205_14 deposited under accession number ATCC 98155. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:13 from amino acid 89 to amino
acid
188.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
3 0 ID N0:12.
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 N0:13;
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(b) the amino acid sequence of SEQ ID N0:13 from amino acid 89 to
amino acid 188;
(c) fragments of the amino acid sequence of SEQ ID N0:13; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BL205_14 deposited under accession number ATCC 98155;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:13 or the amino acid
sequence
of SEQ ID N0:13 from amino acid 89 to amino acid 188.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 69 to nucleotide 371;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 109 to nucleotide 350;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone H438_1 deposited under accession
number ATCC 98140;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone H438_1 deposited under accession number ATCC 98140;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone H438_1 deposited under accession number
ATCC 98140;
2 5 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone H438_1 deposited under accession number ATCC 98140;
(h} a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:15;
(i) a polynucleotide encoding a protein comprising a fragment of the
3 0 amino acid sequence of SEQ ID N0:15 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
{k) a polynucleotide which encodes a species homologue of the protein
of (h} or (i) above ; and
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(1) a polynucleotide capable of hybridizing under stringent conditions -
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:14 from nucleotide 69 to nucleotide 371; the nucleotide sequence of SEQ ID
N0:14
from nucleotide 109 to nucleotide 350; the nucleotide sequence of the full-
length protein
coding sequence of clone H438_1 deposited under accession number ATCC 98140;
or the
nucleotide sequence of the mature protein coding sequence of clone H438_1
deposited
under accession number ATCC 98140. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone H438_1 deposited under accession number ATCC 98140. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:15 from amino acid 27 to amino
acid
94.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
TD N0:14.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a} the amino acid sequence of SEQ ID N0:15;
2 0 (b) the amino acid sequence of SEQ ID N0:15 from amino acid 17 to
amino acid 94;
(c) fragments of the amino acid sequence of SEQ ID N0:15; and
(d) the amino acid sequence encoded by the cDNA insert of clone
H438_1 deposited under accession number ATCC 98140;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein cornprises the amino acid sequence of SEQ ID N0:15 or the amino acid
sequence
of SEQ ID N0:15 from amino acid 17 to amino acid 94.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
3 0 yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
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The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
Fig. 2 is an autoradiograph evidencing the expression of the following clone
disclosed herein, H438_l, in COS cells.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
2 0 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) can then be determined from such nucleotide sequence. The amino
acid
2 5 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.
3 0 As used herein a "secreted" protein is one which, when expressed in a
suitable host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins} or partially (e.g. ,
receptors) from the cell
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in which they are expressed. "Secreted" proteins also include without
limitation proteins -
which are transported across the membrane of the endoplasmic reticulum.
Clone "AM349 2"
A polynucleotide of the present invention has been identified as clone "AM349
2".
AM349 2 was isolated from a human fetal kidney cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. AM349 2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "AM349 2 protein").
The nucleotide sequence of AM349 2 as presently determined is reported in SEQ
ID N0:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AM349 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:2. Amino acids 22 to 34 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 35, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AM349 2 should be approximately 3400 bp.
2 0 The nucleotide sequence disclosed herein for AM349 2 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AM349_2 demonstrated at least some similarity with
sequences
identified as AA078927 (zm92a08.s1 Stratagene ovarian cancer (#937219) Homo
sapiens
cDNA clone 545366 3'), H06061 (y172e10.s1 Homo sapiens cDNA clone 43276 3'),
U46493
2 5 (Cloning vector pFlp recombinase gene, complete cds), W81648 (zd84d09.r1
Soares fetal
heart NbHHI9W Homo Sapiens cDNA clone 347345 5'), and W81649 (zd84d09.s1
Soares
fetal heart NbHHI9W Homo sapiens cDNA clone 347345 3'). The predicted amino
acid
sequence disclosed herein for AM349 2 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
3 0 AM349_2 protein demonstrated at least some similarity to sequences
identified as J01969
(DNA polymerase [Human adenovirus type 5]) and X59599 (protein-tyrosine
phosphatase). Based upon sequence similarity, AM349 2 proteins and each
similar
protein or peptide may share at least some activity.
14
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WO 99127079 PCT/US98124808
Clone "AR310- 3" -
A polynucleotide of the present invention has been identified as clone
"AR310_3".
AR310 3 was isolated from a human adult retina cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. AR310_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "AR310 3 protein').
The nucleotide sequence of AR310 3 as presently determined is reported in SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AR310_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AR310 3 should be approximately 3800 bp.
The nucleotide sequence disclosed herein for AR310 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AR310 3 demonstrated at least some similarity with
sequences
identified as AA313755 (EST185840 Colon carcinoma (HCC) cell line II Homo
sapiens
cDNA 5' end), N35123 (yy20bOl.s1 Homo sapiens cDNA clone 271753 3'), N36408
2 0 (yy33f03.s1 Homo Sapiens cDNA clone 273053 3'), W61057 (zc54a11.r1 Soares
senescent
fibroblasts NbHSF Homo sapiens cDNA clone 326108 5' similar to contains
element MSR1
repetitive element), and X16706 (Human fra-2 mRNA). Based upon sequence
similarity,
AR310 3 proteins and each similar protein or peptide may share at least some
activity.
The TopPredII computer program predicts a potential transmembrane domain
within the
2 5 AR310_3 protein sequence centered around amino acid b6 of SEQ ID N0:4;
this region is
also a possible signal sequence.
Clone "AS186 3"
A polynucleotide of the present invention has been identified as clone "AS186
3".
3 0 AS186 3 was isolated from a human fetal brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins {see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. AS186_3 is a full-
length clone,
CA 02309624 2000-OS-18
WO 99127079 PCTIUS98/24808
including the entire coding sequence of a secreted protein (also referred to
herein as -
"AS186 3 protein').
The nucleotide sequence of AS186_3 as presently determined is reported in SEQ
ID N0:5. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AS186_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6. Amino acids 21 to 33 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 34, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AS186_3 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for AS186_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AS186_3 demonstrated at least some similarity with
sequences
identified as J00083 (Human Alu family interspersed repeat; clone BLUR11} and
U14574
(***ALU WARNING Human Alu-Sx subfamily consensus sequence). The predicted
amino
acid sequence disclosed herein for AS186_3 was searched against the GenPept
and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted AS186_3 protein demonstrated at least some similarity to sequences
identified
as S58722 (X-linked retinopathy protein tC-terminal, clone XEH.Bc} [human]).
Based upon
2 0 sequence similarity, AS186_3 proteins and each similar protein or peptide
may share at
least some activity. The nucleotide sequence of AS186_3 indicates that it may
contain an
Alu repetitive element.
Clone "AY160 2"
A polynucleotide of the present invention has been identified as clone "AY160
2".
AY160_2 was isolated from a human adult retina cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
3 0 identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. AY160 2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "AY160 2 protein').
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The nucleotide sequence of AY160 2 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AY160 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:8. Amino acids 3 to 15 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 16, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AY160_2 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for AY160_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AY160 2 demonstrated at least some similarity with
sequences
identified as D12485 (Human mRNA for nucleotide pyrophosphatase, complete
cds),
D30649 (Rat mRNA for phosphodiesterase I, complete cds), N77069 (yz84h12.r1
Homo
Sapiens cDNA clone 289799 5'), and 247987 (R.norvegicus mRNA for RB13-6
antigen).
The predicted amino acid sequence disclosed herein for AY160 2 was searched
against
the GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol. The predicted AY160 2 protein demonstrated at least some similarity
to
sequences identified as D12485 (HUMNPP_2 NPPase [Homo sapiens]), D30649
(phosphodiesterase I [Rattus rattus]), and 247987 (RNRB13X6_1 RB13-6 antigen
[Rattus
2 0 norvegicus]). The AY160 2 protein also has some domains of sequence
similarity to a
variety of integral membrane proteins including glycoprotein PC-1. Based upon
sequence
similarity, AY160_2 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts a potential transmembrane
domain
near the carboxy terminus of the AY160_2 protein, around amino acid 418 of SEQ
ID
2 5 N0:8.
Clone "BD127 16"
A polynucleotide of the present invention has been identified as clone
"BD127_16".
BD127_16 was isolated from a human fetal kidney cDNA library using methods
which are
3 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BD127_16 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BD127_16 protein').
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The nucleotide sequence of the 5' portion of BD127 16 as presently determined
is
reported in SEQ TD N0:9. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:10. The predicted amino acid
sequence
of the BD127_16 protein corresponding to the foregoing nucleotide sequence is
reported
in SEQ ID N0:10. Additional nucleotide sequence from the 3' portion of
BD127_16,
including the polyA tail, is reported in SEQ ID N0:11.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BD127_16 should be approximately 1080 bp.
The nucleotide sequence disclosed herein for BD127_16 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BD127 16 demonstrated at least some similarity with
sequences
identified as M55683 (Human cartilage matrix protein). The predicted amino
acid
sequence disclosed herein for BD127 16 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
BD127 16 protein demonstrated at least some similarity to sequences identified
as U03272
(HSU03272_1 fibrillin-2 [Homo sapiens]) and X04571 (HSEGFRER_1 Human mRNA for
kidney epidermal growth factor (EGF) precursor [Homo sapiens]). Based upon
sequence
similarity, BD127_16 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of BD127_16 indicates that it may contain a
PTR7
2 0 repetitive element.
Clone "BL205 14"
A polynucleotide of the present invention has been identified as clone
"BL205_14".
BL205_14 was isolated from a human adult testes cDNA library using methods
which are
2 5 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BL205 14 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BL205_14 protein').
3 0 The nucleotide sequence of BL205_14 as presently determined is reported in
SEQ
ID N0:12. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BL205_14 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:13.
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WO 99/Z7079 PCT/US98/24808
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BL205_14 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for BL205_14 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BL205_14 demonstrated at least some similarity with
sequences
identified as T08683 (EST06575 Homo sapiens cDNA clone HIBBI30 5' end) and
U55178
(Mus musculus TIL mRNA from progressing tumor site, clone NFB#3). Based upon
sequence similarity, BL205_14 proteins and each similar protein or peptide may
share at
least some activity.
Clone "H438 1"
A polynucleotide of the present invention has been identified as clone
"H438_1".
H438_1 was isolated from a human adult blood {peripheral blood mononuclear
cells
treated with phytohemagglutinin and phorbol myristate acetate and mixed
lymphocyte
reaction) cDNA library using methods which are selective for cDNAs encoding
secreted
proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a
secreted or
transmembrane protein on the basis of computer analysis of the amino acid
sequence of
the encoded protein. H438_1 is a full-length clone, including the entire
coding sequence
of a secreted protein (also referred to herein as "H438_1 protein").
2 0 The nucleotide sequence of H438_1 as presently determined is reported in
SEQ ID
N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the H438_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 H438_1 should be approximately 2100 bp.
The nucleotide sequence disclosed herein for H438_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. H438_1 demonstrated at least some similarity with
sequences
identified as H06234 (y178e09.r1 Homo sapiens cDNA clone 44074 5') and 856040
3 0 (yg91a04.s1 Homo Sapiens cDNA clone 40669 3'). Based upon sequence
similarity,
H438_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
H438_1 protein sequence, centered around amino acid 25 of SEQ ID N0:15.
Fig. 2 is an autoradiograph evidencing expression of clone H438_1 in COS
cells.
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WO 99/27079 PCT/US98/24808
Deposit of Clones
Clones AM349_l, AR310_2, AS186_2, AY160_l, BD127_l l, BL205 7, and H438_1
were deposited on August 14, 1996 with the American Type Culture Collection as
an
original deposit under the Budapest Treaty and were given the accession number
ATCC
98140, from which each clone comprising a particular polynucleotide is
obtainable. An
additional isolate of each of clones AM349_1, AR310_2, AS186_2, AY160_l,
BD127_l l,
BL205 7 (namely AM349 2, AR310 3, AS186_3, AY160 2, BD127_16, and BL205_14,
respectively) was deposited on August 23, 1996 with the American Type Culture
Collection as an original deposit under the Budapest Treaty under accession
number
ATCC 98155. All restrictions on the availability to the public of the
deposited material will
be irrevocably removed upon the granting of the patent, except for the
requirements
specified in 37 C.F.R. ~ 1.808(b).
Each clone has been transfected into separate bacterial cells (E. coli) in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman
et al.,
1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from
pMT2
2 0 (Kaufman et al., 1989, Mol. CeII. BioI. 9: 946-958) by deletion of the
DHFR sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
orientation) in the deposited isolate. In such instances, the cDNA insert can
still be
isolated by digestion with EcoRI and NotI. However, NotI will then produce the
5' site
2 5 and EcoRI will produce the 3' site for placement of the cDNA in proper
orientation for
expression in a suitable vector. The cDNA may also be expressed from the
vectors in
which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
3 0 An oligonucleotide probe or probes should be designed to the sequence that
is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of the
oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
CA 02309624 2000-OS-18
WO 99127079 PCTIUS98/24808
Clone Probe Sequence -
AM349 2 SEQ ID N0:16
AR310_3 SEQ ID N0:17
AS186_3 SEQ ID N0:18
AY160 2 SEQ ID N0:19
BD127 16 SEQ ID NO:20
BL205_14 SEQ ID N0:21
H438_1 SEQ ID N0:22
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-{N-biotinyi-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
{a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's'"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
2 0 A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-32P ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
2 5 methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 pl of the stock used to inoculate a sterile culture flask
containing 25 ml
3 0 of sterile L-broth containing ampicillin at 100 pg/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
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CA 02309624 2000-OS-18
WO 99127079 PCT/US98l24808
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other -
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 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
mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to le+6 dpm/mL. The filter is then
preferably
10 incubated at 65°C with gentle agitation overnight. The filter is
then preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
2 0 Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, et al., Bio/Technology U 773-778 (1992) and in
R.S.
McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
2 5 herein by reference. Such fragments may be fused to carrier molecules such
as
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. 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
3 0 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 form
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WO 99/27079 PCT/US98/24808
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 sequence of the mature form 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 cDNA sequences
disclosed herein. "Corresponding genes" are the regions of the genome that are
transcribed to produce the mlZlVAs from which the cDNA sequences are derived
and any
contiguous regions of the genome necessary for the regulated expression of
such genes,
including but not limited to coding sequences, 5' and 3' untranslated regions,
alternatively
spliced exons, introns, promoters, enhancers, and silencer or suppressor
elements. The
corresponding genes can be isolated in accordance with known methods using the
sequence information disclosed herein. Such methods include the preparation of
probes
or primers from the disclosed sequence information for identification and/or
amplification of genes in appropriate genomic libraries or other sources of
genomic
materials.
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
2 0 transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
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
2 5 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 $
or more
3 0 (more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least $5% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
Species homologs of the disclosed poiynucleotides and proteins are also
provided
by the present invention. As used herein, a "species homologue" is a protein
or
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WO 99/27079 PCT/US98/24808
polynucleotide with a different species of origin from that of a given protein
or w
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide, as determined by those of skill in the art. Species homologs
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.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
which also encode proteins which are identical, homologous, or related to that
encoded
by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
under reduced stringency conditions, more preferably stringent conditions, and
most
preferably highly stringent conditions, to polynucleotides described herein.
Examples of
stringency conditions are shown in the table below: highly stringent
conditions are those
that are at least as stringent as, for example, conditions A-F; stringent
conditions are at
least as stringent as, for example, conditions G-L; and reduced stringency
conditions are
at least as stringent as, for example, conditions M-R.
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WO 99/27079 PCT/US98I24808
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)t Buffer' and Buffers
A DNA:DNA x 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC Ta*; ixSSC
C DNA:RNA x 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E - RNA:RNA x 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA c50 TF*; lxSSC TF*; IxSSC
G DNA:DNA x 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA t50 TH*; 4xSSC TH*; 4xSSC
I DNA:RNA x 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50~ formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA x 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA Z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA x 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 TP*; 6xSSC Tp*; 6xSSC
Q RNA:RNA x 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
0
_: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. Whan
hybridizing a polynucleotide to a target polynudeotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynudeotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaHZP04, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log~°[Na']) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
CA 02309624 2000-OS-18
WO 99/27079 PCTNS98/Z4808
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity {more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 9 4485-4490 (1991), in order to produce the
protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology ~ 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 cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
26
CA 02309624 2000-OS-18
WO 99127079 PCTIUS98I24808
strain capable of expressing heterologous proteins. If the protein is made in
yeast or -
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 0987), incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TR7C). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
27
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98/24$08
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.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or 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,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
28
CA 02309624 2000-OS-18
WO 99/Z7079 PCT/US98/24808
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 fox proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" ar other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction}, the
polynucleotide can also be used in interaction trap assays (such as, far
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
3 0 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
29
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98124808
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. Maruatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kixnmel
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 0 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
2 5 can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferatlon/Differentiatipn ~tiyity
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
3 0 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
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98/24808
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3, -
MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Tmmunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et aL, J. Immunol. 149:3778-3783,1992; Bowman
et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, 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.
2 0 Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
2 5 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. j. In
Current Protocols
3 0 in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
31
CA 02309624 2000-OS-18
WO 99/27079 PGT/US98/24808
Assays for T-cell clone responses to antigens (which will identify, among
others, -
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et aL,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411,1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Suppressing Activitt
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.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
2 0 infections, or may result from autoimrnune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which 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,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention 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
32
CA 02309624 2000-OS-18
WO 997079 PCT/US98/24808
example, organ transplantation), may also be treatable using a protein of the
present -
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
33
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98/24808
tolerance in a subject, it may also be necessary to block the function of a
combination of -
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et aL, Science 257:789-792 (1992) and Turka et aL, Proc.
Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
34
CA 02309624 2000-OS-18
WO 99127079 PCT/US98/24808
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 au or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivo with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfecHon in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
~iZ
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.
CA 02309624 2000-OS-18
WO 99/27079 PCTNS98I24808
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having -
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrrnann et al., J. Immunol.128:1968-1974, 1982; Handa
et al.,
J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-3500,1986;
Takai et al.,
J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Th1/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol.144:3028-3033,1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Currenf Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
36
CA 02309624 2000-OS-18
wo 99n~o~9 PcTius9sna8oa
7, Immunologic studies in Humans); Takai et al., J. Immunol.137:3494-
3500,1986; Takai -
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783,1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells} include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; MacatQnia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Regulating Activitv
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity}
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
37
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rnyelo-suppression; in supporting the growth and proliferation of
megakaryocytes and -
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-
151,1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
2 5 Hematopoietic Cells. R.I. Freshney, et al. eds. VoI pp. 265-268, Wiley-
Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sri. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp.1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et at. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
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WO 99127079 PCT/US98/24808
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.139-
162, Wiley-Liss, -
Inc., New York, NY. 1994.
Tissue Growth ActivitX
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regeneration activity that may be attributable
to the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
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congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and 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,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
3 0 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
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogeruc activity.
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A protein of the present invention may also be useful for gut protection or -
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing. pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
(3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
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.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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WO 99/27079 PCTNS98/24808
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-
782,1986; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et
al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by 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
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.li. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et aI.
APMIS 103:140-146,1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et
aI. j. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-
1768,1994.
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Hemostatic and Thrombolvtic 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 thrornbolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474,1988.
Receptor/Ligand ActivitX
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their Iigands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their Iigands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/Iigand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
43
CA 02309624 2000-OS-18
WO 99127079 PCT/US98IZ4808
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-Inflammator3r Achvitv
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
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
2 0 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 Su~~ressor ActivitX
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pernphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
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
44
CA 02309624 2000-OS-18
WO 99127079 PCT/US98/24808
first cadherin domain provide the basis for homophilic adhesion; modification
of this -
recogrution site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 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
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
CA 02309624 2000-OS-18
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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
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hernatopoietic
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WO 99/Z7079 PCT/US98/24808
Iineages; 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 carrier)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, T1VF, IL-1, IL-2, IL-3, ILr4, IL-5, ILr6, IL-7, IL-8,
IL-9, IL-10, ILrl l,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
2 0 cell factor, and erythropoietin. The pharmaceutical composition may
further contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
2 5 in formulations of the particular cytokine, lymphokine, other
hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
3 0 or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
47
CA 02309624 2000-OS-18
WO 99127079 PCTNS98/24808
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 MI~C proteins. M~iC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable earners, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,$71; U.S. Patent No.
4,501,728; U.S.
2 0 Patent No. 4,$37,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
2 5 amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
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.
3 0 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
48
CA 02309624 2000-OS-18
WO 99127079 PCT/US98/24808
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine{s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application ox
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
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
2 0 When administered in liquid form, a liquid carrier such as water,
petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
2 5 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
3 0 invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
49
CA 02309624 2000-OS-18
WO 99IZ7079 PCT/US98I24808
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride -
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
mg (preferably about 0.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 0 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
2 5 polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
3 0 R.P. Merrifield, J. Amer.Chem.Sac. 85 2149-2154 (1963); j.L. ICrstenansky,
et al., FEBS Lett.
211, 10 {1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
CA 02309624 2000-OS-18
WO 99127079 PGT/US98lZ4808
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, rnay
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
2 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 materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
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.
S1
CA 02309624 2000-OS-18
WO 99127079 PCTIUS98/24808
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
hyaluroruc
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-(3), 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
52
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98I24808
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.
53
CA 02309624 2000-OS-18
WO 99/27079 PCTIUS98/24808
SEQUENCE LISTING
<110> Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Evans, Cheryl
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> 60088-PCT
<140>
< "Exprosa Maii"
141 mailing abel
> nun ber:~
9~7 9 a c..r
~
Gate of Deposit
~
-o. 1 44
<160>22 I hereby certify
i~at this
paper or
fee is bein
g
deposited
with the
Un'tted States
Postal Service
"
Express Mail
<170>Patentln Ver. 2 .0 Post OIhCe
to ,qddmasee"
servke
under 37 CFR
1 10 on the
date lndicatsd
above
and is addressed
<210>1 to the Assistant
Commissioner
For P nta, , . 20231
Washing
<211>3423
< DNA ~.-...
212
>
<213>Homo sapiens
<400> 1
tgttaggcaa atacacatta ataagaatgc ctagaagagg actgattctt cacacccgga 60
cccactggtt gctgttgggc cttgctttgc tctgcagttt ggtattattt atgtacctcc 120
tggaatgtgc cccccagact gatggaaatg catctcttcc tggtgttgtt ggggaaaatt 180
atggtaaaga gtattatcaa gccctcctac aggaacaaga agaacattat cagaccaggg 240
caaccagtct gaaacgccaa attgcccaac taaaacaaga attacaagaa atgagtgaga 300
agatgcggtc actgcaagaa agaaggaatg taggggctaa tggcataggc tatcagagca 360
acaaagagca agcacctagt gatcttttag agtttcttca ttcccaaatt gacaaagctg 420
aagttagcat aggggccaaa ctacccagtg agtatggggt cattcccttt gaaagtttta 480
ccttaatgaa agtatttcaa ttggaaatgg gtctcactcg ccatcctgaa gaaaagccag 540
ttagaaaaga caaacgagat gaattggtgg aagttattga agcgggcttg gaggtcatta 600
ataatcctga tgaagatgat gaacaagaag atgaggaggg tccccttgga gagaaactga 660
tatttaatga aaatgacttc gtagaaggtt attatcgcac tgagagagat aagggcacac 720
agtatgaact cttttttaag aaagcagacc ttacggaata tagacttgtg accctcttcc 780
gcccttttgg acctctcatg aaagtgaaga gtgagatgat tgacatcact agatcaatta 840
ttaatatcat tgtgccactt gctgaaagaa ctgaagcatt tgtacaattt atgcagaact 900
tcagggatgt ttgtattcat caagacaaga agattcatct cacagtggtg tattttggta 960
aagaaggact gtctaaagtc aattctatcc tagaatctgt caccagtgag tctaattttc 1020
acaattacac cttggtctca ttgaatgaag aatttaatcg tggacgagga ctaattgtgg 1080
gtgcccgagc ttgggacaag ggagaggtct tgatgttttt ctgtgatgtt gatatctatt 1140
tctcagccga attccttaac agctgccggt taaatgctga gccaggtaaa aaggtgtttt 1200
accctgtggt gttcagtctt tacaatcctg ccattgttta tgccaaccag aaattgccac 1260
cacctgtgga gcagcagctg gttcacaaaa aggattctgg cttttggcaa aattttggct 1320
ttggaatgac ttgtcagtat cgtccaaatt tcctgaccat tggtggattt gacatggaat 1380
tgaaaggttg gggtggaaaa aattttcatc tttatcaaaa atacttacat ggtgacctca 1440
ttgtgattcg gactccggtt cctggtcttt tccacctctg gcatgaaaag cgctgtgctg 1500
atgagctgac ccccgagcag taccgcatgt gcatccagtc taaagccatg aatgaggcct 1560
ctcactccca cctgggaatg ctggtcttca gggaggaaat agagacgcat cttcataaac 1620
aggcatacag gacaaacagt gaagctgttg gttgaaatca taattaatgc gttactgtat 1680
gaaccacaaa acagcactat ttatttagcc ttacttctac ttccagatgc agtgcctctt 1740
ttggagaaga catgtttatt tttcatgttc tttctgacat tactttagca attcaacttg 1800
atgtgagaag aaaaaacaaa tgtttcaaca caaaatctct gttttgtgag aatactgcac 1860
1
CA 02309624 2000-OS-18
WO 99127079 PCT/US98/24808
tatggaataa ttgacaaatt gaaatctcat atttgtccca aaagttgttt tgagttagtt 1920
ctacctggtg cccatgttct gattgtgtgt gggattgcat ggtgtcctga ttgcatctag 1980
gtggaccgga tggaatgtgc tgggccactg ttgggtggag agcagcacat tcttacagag 2040
gagatggagc gttatgagca tagtatgtgg ataggtatct tcacctgccc gcccctgagt 2100
cagcctcctt gacttgatag cttgaagaat ccttttccac tgaaatagag gataattaat 2160
tgacacatct gaaatcccca atcaatcaat caagagaaag gtagaactaa aaactcctta 2220
acttactgtt gcttacaccc ctgaaagtct gtttttaagc aaatgggtaa tagtagaaaa 2280
taggttagaa tctatggctt gattaaaaat atgttattac attatcatgt tcaggattag 2340
gattagtagt cagttgctgt aaactatttt gaacaaacag aaaagaacac ggaaacattt 2400
ttaacagagc atttaattat gttggaatac aggatcctag ctctgtctgg gaacattagt 2460
ttatttgagc cagctctatc agggtcttcc catggtggtt cagaatagat gagcatagca 2520
tggttttgtt tgtttttgct ttcaattttc taatttggca tggatccata tgtatttact 2580
atcctttttc taatatatta atatatgcta catttgtatt tgcattacta taatactttg 2640
agttgaaaaa gagtttcatt gtggagagaa aaagcaaatg gtatgccaca agatcactct 2700
gatttgagaa aagggaggag gggaagatag tctgaatgga aatctgaaat acggaatgtt 2760
ttagagaaat atgtcacttg catatagaat gttttaattg aggtataaat taatgagaca 2820
aagtgaaaaa gaaattatat tcagatagga ctgcactaca ttatttgtca cacatggatc 2880
tgttaccatc aggtcaattc ctagtatgca taaatttttt aaccctttta aaagagacct 2940
atgttgaaaa cccctgaaaa ttcactgaag aaaaatcatt actctttttc tcagtaaatc 3000
atatcatctg aaatattaca aatttcaaat ttctaggtgc tatattaatt caatattaca 3060
ataactctta cctaattatt cttacaagtt ttaagttgtg gtagtttagt gattttttta 3120
aaagatgtgt gaaatgttct ctgcaaaata attcaggcca ctgtctcctt ttatatatta 3180
ttataattat ttattatgaa gaccagtgaa ttacgatatt taaagtgaga gaacttaatt 3240
atttgcaaag gtaagttaca gcttgttttt tgagagaatc aaatgagttt acttttgttc 3300
ttgttgtttt taactagctt taagtttaaa gatggaagct aagcaatgga aatgctatac 3360
gtttttgaca tttattaaat ggtaccaata aagtatttta ttaccaaaaa aaaaaaaaaa 3920
aaa 3423
<210> 2
<211> 542
<212> PRT
<213> Homo Sapiens
<400> 2
Met Pro Arg Arg Gly Leu Ile Leu His Thr Arg Thr His Trp Leu Leu
1 5 10 15
Leu Gly Leu Ala Leu Leu Cys Ser Leu Val Leu Phe Met Tyr Leu Leu
20 25 30
Glu Cys Ala Pro Gln Thr Asp Gly Asn Ala Ser Leu Pro Gly Val Val
35 40 45
Gly Glu Asn Tyr Gly Lys Glu Tyr Tyr Gln Ala Leu Leu Gln Glu Gln
50 55 60
Glu Glu His Tyr Gln Thr Arg Ala Thr Ser Leu Lys Arg Gln Ile Ala
65 70 75 80
Gln Leu Lys Gln Glu Leu Gln Glu Met Ser Glu Lys Met Arg Ser Leu
85 90 95
Gln Glu Arg Arg Asn Val Gly Ala Asn Gly Ile Gly Tyr Gln Ser Asn
100 105 110
Lys Glu Gln Ala Pro Ser Asp Leu Leu Glu Phe Leu His Ser Gln Ile
115 120 125
Asp Lys Ala Glu Val Ser Ile Gly Ala Lys Leu Pro Ser Glu Tyr Gly
130 135 140
2
CA 02309624 2000-OS-18
WO 99127079 PCT/US98/24808
Val Ile Pro Phe Glu Ser Phe Thr Leu Met Lys Val Phe Gln Leu Glu
145 150 155 160
Met Gly Leu Thr Arg His Pro Glu Glu Lys Pro Val Arg Lys Asp Lys
165 170 175
Arg Asp Glu Leu Val Glu Val Ile Glu Ala Gly Leu Glu Val Ile Asn
180 185 190
Asn Pro Asp Glu Asp Asp Glu Gln Glu Asp Glu Glu Gly Pro Leu Gly
195 200 205
Glu Lys Leu Ile Phe Asn Glu Asn Asp Phe Val Glu Gly Tyr Tyr Arg
210 215 220
Thr Glu Arg Asp Lys Gly Thr Gln Tyr Glu Leu Phe Phe Lys Lys Ala
225 230 235 240
Asp Leu Thr Glu Tyr Arg Leu Val Thr Leu Phe Arg Pro Phe Gly Pro
245 250 255
Leu Met Lys Val Lys Ser Glu Met Ile Asp Ile Thr Arg Ser Ile Ile
260 265 270
Asn Ile Ile Val Pro Leu Ala Glu Arg Thr Glu Ala Phe Val Gln Phe
275 280 285
Met Gln Asn Phe Arg Asp Val Cys Ile His Gln Asp Lys Lys Ile His
290 295 300
Leu Thr Val Val Tyr Phe Gly Lys Glu Gly Leu Ser Lys Val Asn 5er
305 310 315 320
Ile Leu Glu Ser Val Thr Ser Glu Ser Asn Phe His Asn Tyr Thr Leu
325 330 335
Val Ser Leu Asn Glu Glu Phe Asn Arg Gly Arg Gly Leu Ile Val Gly
340 345 350
Ala Arg Ala Trp Asp Lys Gly Glu Val Leu Met Phe Phe Cys Asp Val
355 360 365
Asp Ile Tyr Phe Ser Ala Glu Phe Leu Asn Ser Cys Arg Leu Asn Ala
370 375 380
Glu Pro Gly Lys Lys Val Phe Tyr Pro Val Val Phe Ser Leu Tyr Asn
385 390 395 400
Pro Ala Ile Val Tyr Ala Asn Gln Lys Leu Pro Pro Pro Val Glu Gln
405 410 415
Gln Leu Val His Lys Lys Asp Ser Gly Phe Trp Gln Asn Phe Gly Phe
420 425 430
Gly Met Thr Cys Gln Tyr Arg Pro Asn Phe Leu Thr Ile Gly Gly Phe
435 440 445
Asp Met Glu Leu Lys Gly Trp Gly Gly Lys Asn Phe His Leu Tyr Gln
450 455 460
3
CA 02309624 2000-OS-18
WO 99127079 PCT/US98124808
Lys Tyr Leu His Gly Asp Leu Ile Val Ile Arg Thr Pro Val Pro Gly
465 470 475 480
Leu Phe His Leu Trp His Glu Lys Arg Cys Ala Asp Glu Leu Thr Pro
485 490 495
Glu Gln Tyr Arg Met Cys Ile Gln Ser Lys Ala Met Asn Glu Ala Ser
500 505 510
His Ser His Leu Gly Met Leu Val Phe Arg Glu Glu Ile Glu Thr His
515 520 525
Leu His Lys Gln Ala Tyr Arg Thr Asn Ser Glu Ala Val Gly
530 535 540
<210> 3
<211> 3803
<212> DNA
<213> Homo sapiens
<400> 3
gagaactcgg tttggtagac ttggacatct ctctggcttc tgaagagcct gaagctggcc 60
tggaccattc ctgtcccttt gttaccatac tgtctctgga gtgatggtgt ccttccctgc 120
cccaccacgc atgctcagtg ccttttggtt tcaccttccc tcgacttgac ccttccctcc 180
cccagcgtca gtttcactcc ctcttggttt ttatcaaatt tgccatgaca tttcatctgg 240
gtggtctgaa tattaaagct cttcatttct ggagatgggg cagcaggtgg ctcttctgct 300
ggggctgact tgtccagaag gggacaaagt gcaatacaga gccttcccta ccctgacgcc 360
tcccagtcat catctccaga actcccagcg gggctccctg agctctcaag gagatgctgc 420
catcactggg aggctcagag gacccttcct gcccaccttc ggagacggct tctggaggaa 480
cggcttggcc agaagacagg gtgtgagtga gacagtgggg cacaggttgg gtttgccaaa 540
cgcctaatta ccaggccagg aagcatgcca acaaagccac acgggtgtcc tagccagctt 600
cccttcacct ggtgtcttga gtagggcgtc tcctgtaatt actgccttgc cattctgccc 660
ctggaccctt ctctccggac cagggaggcg tccctcccta ggagccacac attatactcc 720
aagtccctgc cgggctccgc ctttccccca ccctggctct cagggtgacg ccacccacag 780
agatttaatg agcgtgggcc tggaccttcc ccagatgctg ccaggcagcc cctccccaag 840
cctcaaagaa gcatttgctg aggatggaga ggcaggggag ggaggcggga ggccgtcact 900
ggagtggcgt ctgcagcagc tgctgcccca gcacccgctc agcctgtcct ggctgctcac 960
ctccccgcag ggcaccgggc ctttcctgcc ctctgtggtc atctgccacc tgctggatca 1020
agtgctttct cttttacact cccctgtccc caccccagtg cactcttctg gcccaggcgg 1080
caagcaagct gtgaacagct ggcctgagct gtcgctgtgg cttgtggctc atgcgccatt 1140
cctggttgtc tgttgaatct ttctggctgc tggagttgga gataggatgt tttgcttccc 1200
actgcaggag agctgccccc tttcacgggg ttggggaagg gtccccctgg cctccagcag 1260
gagcacagct cagcagggtc cctgctgccc acccctctga gccttttctc cccagggtat 1320
ggctcctgct gagtttcttg tccagcaggg ccttgacagg aatccaggga gtagctcctg 1380
gccagaacca gcctctgcgg ggcttgtgct ctgcaaagac tctgctgctg gggattcagc 1440
tctagaggtc acagtatcct cgtttgaaag ataattaaga tcccccgtgg agaaagcagt 1500
gacacattca cacagctgtt ccctcgcatg ttatttcatg aacatgacct gttttcgtgc 1560
actagacaca cagagtggaa cagccgtatg cttaaagtac atgggccagt gggactggaa 1620
gtgacctgta caagtgatgc agaaaggagg gtttcaaaga aaaaggattt tgtttaaaat 1680
actttaaaaa tgttatttcc tgcatccctt ggctgtgatg cccctctccc gatttcccag 1740
gggctctggg agggaccctt ctaagaagat tgggcagttg ggtttctggc ttgagatgaa 1800
tccaagcagc agaatgagcc aggagtagca ggagatgggc aaagaaaact ggggtgcact 1860
cagctctcac aggggtaatc atctcaagtg gtatttgtag ccaagtggga gctattttct 1920
tttttgtgca tatagatatt tcttaaatga agctgctttc ttgtctttta tttctaaaag 1980
cccccttata ccccactttg tgcagcaaag atccccgtgc aggtcacagc ctgatttgtg 2040
gccaggctgg acaaattcct gaggcacaac ttggcttcag ttcagatttc aagctgtgtt 2100
ggtgttggga ccagcagaag gcaaacgtcc agccaacaca caggactgta agaggactct 2160
gagctacgtg ccctgtgaag acccccaggc tttgtcatag gaggtcgttc agcttcccca 2220
4
CA 02309624 2000-OS-18
WO 99/27079 PGTIUS98/24808
aagtcagagg tgatttgatt tggggaagac tgaatattca cacctaagtc gtgagcatat 2280
cctgagtttt acttccttat ggcttgccct ccaagttctc tctctcatac acacacacac 2340
ccttgctcca gaatcaccag acacctccat ggctccagct atgggaacag ctgcattggg 2400
gctgcctttc tgtttggctt aggaacttct gtgcttcttg tggctccact cgcgaggcag 2460
ctcggaggtg tggactccga ttgggctgca ggcagctctg ggacggcaca gggcgggcgc 2520
tctgatcagc tcgtgtaaaa cacaccgtct tcttggcctc ctggccagtc tttctgcgaa 2580
tagtcctctc cctggccagt tgaatggggg aagctgctgg cacaggaagg agaggcgatc 2640
ccggctgagg cttaggaaat tgctggagcc ggctccaagc agataattca ctggggaggt 2700
tttcagagtc aaacatcatt ctgcctgtgt tgggggccag gtgtgtcaca caagcatctc 2760
aaagtcaaaa gccatctggg gctgctgctt ctctttctca ggctctgggg aaaggaatct 2820
ccctctcctc tcacttgatt ccaagtgtgg ttgaattgtc tggagcactg ggactttttt 2880
tctcttttcc ttgatggacc aacagtgcaa atgcaatctc gccatttaac tttcaggtcg 2940
atttcctttc ctgatcagac atctttgtgc cccctttagg aaggaaaaga atacacctac 3000
gatgtgccag gcactgtgtt aggcgctttt atatagatcc tcgttaggat gagactaagg 3060
gatgaggaca tctctttata aaaggcccct aagtaatgga taaacagaaa cacttagagg 3120
tgagaaggtc tgtcttcaag atccaaggta agattgcctt cagtctgatg tttgttctca 3180
aggacttatc ccctacaata ttctcccact ccatacttct ccttctaccc caccatgtgc 3240
tcccgtgcac tcctcagatg gtcagagggg taacccaagt ccttagagaa tttggggacc 3300
aatagaatat gtgatgtgtg aattttcttt aaaaaactta aggagtcttt gctaccttct 3360
gcttgttgag ttgttttggc attcatatta aaagccagca tctcactatt tattgacagg 3420
ttgggctgtg tgtgtgcgca tgtgtgtata catttccagg cgtgcctgtg tcctgtagct 3480
ttttaaaagg aaacccagtc atcccactat gaatctggca tcttcttatg cttctagtgt 3540
tttggccata catcaaccaa ggggtttaat ttatccaatg cttgacgaca tgttcaggag 3600
gggctggatc aaattttgag agggttatgg gaaagggagg gggagaagaa attgacattt 3660
attttattat ttattttaaa tgtttacatc ttctttatgt tgtatcaagc ctgaatagaa 3720
actgatagca ttaaaatact ccgttcctct ctctaaaaaa aaaaaaaaaa aaaaaaaaaa 3780
aaaaaaaaaa aaaaaaaaaa aaa 3803
<210> 4
<211> 122
<212> PRT
<213> Homo sapiens
<400> 4
Met Ser Val Gly Leu Asp Leu Pro Gln Met Leu Pro Gly Ser Pzo Ser
1 5 10 15
Pro Ser Leu Lys Glu Ala Phe Ala Glu Asp Gly Glu Ala Gly Glu Gly
20 25 30
Gly Gly Arg Pro Ser Leu Glu Trp Arg Leu Gln Gln Leu Leu Pro Gln
35 40 45
His Pro Leu Ser Leu Ser Trp Leu Leu Thr Ser Pro Gln Gly Thr Gly
50 55 60
Pro Phe Leu Pro Ser Val Val Ile Cys His Leu Leu Asp Gln Val Leu
65 70 75 g0
Ser Leu Leu His Ser Pro Val Pro Thr Pro Val His Ser Ser Gly Pro
85 90 95
Gly Gly Lys Gln Ala Val Asn Ser Trp Pro Glu Leu Ser Leu Trp Leu
100 105 110
Val Ala His Ala Pro Phe Leu Val Val Cys
115 120
<210> 5
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98/24808
<211> 1700 _
<212> DNA
<213> Homo Sapiens
<400> 5
cggccaaaga ggcctactta ggtagatggt gcaaccagtg gttccagatg gagaaaaggt 60
gaaaacaagt tggcattttt ttgtgccctt caagatctga cttgctttat tttttaattt 120
ttatgtcttc tagcacattt gaaagtgtga acatttaaac tcttattctg tttcagtttg 180
catatgaaga tgttttaagt aagttctgga attatataaa aaaaaaatag agagagtgag 240
gatgcccaga tgacaacaag cagaaaaatt catcctttaa ataaaagcct ctattctcat 300
ttggaaagca aaatgttctc tcttaaaagt agcagctgta aaaaaagcag gaaggcagac 360
cacactaatc taagttgtaa aatatgtttt ggtagcttaa cagagattta gctgtttctg 420
agaaaaaaaa tcaaatctaa ttttaaaatg aaggtattta aaaccatggc acaagggagc 480
cttatttatg gagctggtgg gaagccagga tgtttccaat ccgctgctct tacaggagcc 540
tgtgcctcgc cagttctgtg ctgcagtggg cagccaactg aagtgcatga gtcaaatgca 600
cgaagcagca gacacctgtc ctttcagaag gcaagaggtg atgaaatgag tgaattccag 660
aactagtgga aagaaaacgt aatgattacc ccagattttt ttcttctatt tatttatttt 720
tattgatgca taatagctgc acatagtttt cgggtacgca tgataattta atacattcat 780
aaaccttgta aagatcaaat cagtgtacct gagatttcca tcaccttaaa gatttatctt 840
ctttttatac tagaaccatt caaattcttc tcttctagct attttgcagt atatattatt 900
ataaaccata gtcaccatac tgatctaaca ctgtctcttt tttctatcac atttcagatt 960
ttgtttttct taaaattgat ctgtacaaac taatatttct ttctaaaaac aaggtgaaaa 1020
gtgtttgggt ttttttcccc ctacatggaa ttcaagcttt gaacttgtgt gtttctttca 1080
atgtcataca tactgatctt tatagaatac tgaaggtgct gtttcttttc taaatggtat 1140
gcctgatctt tgaatgaaag gtatatcatg gtgccaaaaa ccttcaacat ttagccttgg 1200
gaccccattg cttgcacagt ggacctattt tctaatttgg aaaatataag cctgtatacc 1260
aagaatatat tttttgaggc tggggagtat atttttttgg ataagttcac taattaccat 1320
aataaatagt aaatacaaat tttcttgtga tctagtacat aagctgggcc ctgtggctca 1380
tgcctgtaat ctcagcattt tgggaggcca aagttgatgg attactggag gccaggagtt 1440
caagaccagc ctggccaaca tggagaaacc tcatctatat taaaaataca aaaattagct 1500
gggtgcagtg gtgcacgcct gtagtcccaa ttgcaattgc tagggaggct gaggtataag 1560
aaatgagaat tgcttgagat gttgtaagtt ttaagcttgg gcccaggagg cagagagtgt 1620
agtgagccga gattacccca ctgtactcca gcctgggcga cagagtgaga ctccatctca 1680
aaaaaaaaaa aaaaaaaaaa 1700
<210> 6
<211> 72
<212> PRT
<213> Homo Sapiens
<400> 6
Met Lys Val Phe Lys Thr Met Ala Gln Gly Ser Leu Ile Tyr Gly Ala
1 5 10 15
Gly Gly Lys Pro Gly Cys Phe Gln Ser Ala Ala Leu Thr Gly Ala Cys
20 25 30
Ala Ser Pro Val Leu Cys Cys Ser Gly Gln Pro Thr Glu Val His Glu
35 40 45
Ser Asn Ala Arg Ser Ser Arg His Leu Ser Phe Gln Lys Ala Arg Gly
50 55 60
Asp Glu Met Ser Glu Phe Gln Asn
65 70
<210> 7
<211> 1915
<212> DNA
6
CA 02309624 2000-OS-18
WO 99127079 PCTNS98124808
<213> Homo Sapiens
<400> 7
catcgcccct cttcctccag gtcccccttc cccgcaactt cccacgagtg ccaggtgccg 60
cgagcgccga gttccgcgca ttggaaagaa gcgaccgcgg cggctggaac cctgattgct 120
gtccttcaac gtgttcatta tgaagttatt agtaatactt ttgttttctg gacttataac 180
tggttttaga agtgactctt cctctagttt gccacctaag ttactactag tatcctttga 240
tggcttcaga gctgattatc tgaagaacta tgaatttcct catctccaga attttatcaa 300
agaaggtgtt ttggtagagc atgttaaaaa tgtttttatc acaaaaacat ttccaaacca 360
ctacagtatt gtgacaggct tgtatgaaga aagccatggc attgtggcta attccatgta 420
tgatgcagtc acaaagaaac acttttctga ctctaatgac aaggatcctt tttggtggaa 480
tgaggcagta cctatttggg tgaccaatca gcttcaggaa aacagatcaa gtgctgctgc 540
tatgtggcct ggtactgatg tacccattca cgataccatc tcttcctatt ttatgaatta 600
caactcctca gtgtcatttg aggaaagact aaataatatt actatgtggc taaacaattc 660
gaacccacca gtcacctttg caacactata ttgggaagaa ccagatgcaa gtggccacaa 720
atacggacct gaagataaag aaaacatgag cagagtgttg aaaaaaatag atgatcttat 780
cggtgactta gtccaaagac tcaagatgtt agggctatgg gaaaatctta atgtgatcat 840
tacaagtgat catgggatga cccagtgttc tcaggacaga ctgataaacc tggattcctg 900
catcgatcat tcatactaca ctcttataga tttgagccca gttgctgcaa tacttcccaa 960
aataaataga acagaggttt ataacaaact gaaaaactgt agccctcata tgaatgttta 1020
tctcaaagaa gacattccta acagatttta ttaccaacat aatgatcgaa ttcagcccat 1080
tattttggtt gccgatgaag gctggacaat tgtgctaaat gaatcatcac aaaaattagg 1140
tgaccatggt tatgataatt ctttgcctag tatgcatcca tttctagctg cccacggacc 1200
tgcatttcac aaaggctaca agcatagcac aattaacatt gtggatattt atccaatgat 1260
gtgccacatc ctgggattaa aaccacatcc caataatggg acctttggtc atactaagtg 1320
cttgttagtt gaccagtggt gcattaatct cccagaagcc atcgcgattg ttatcggttc 1380
actcttggtg ttaaccatgc taacatgcct cataataatc atgcagaata gactttctgt 1440
acctcgtcca ttttctcgac ttcagctaca agaagatgat gatgatcctt taattgggtg 1500
acatgtgcta gggcttatac aaagtgtctt tgattaatca caaaactaag aatacatcca 1560
aagaatagtg ttgtaactat gaaaaagaat actttgaaag acaaagaact tagactaagc 1620
atgttaaaat tattactttg ttttccttgt gttttgtttc ggtgcatttg ctaataagat 1680
aacgctgacc atagtaaaat tgttagtaaa tcattaggta acatcttgtg gtaggaaatc 1740
attaggtaat atcaatccta actagaaata ctaaaaatgg cttttgagaa aaatacttcc 1800
tctgcttgta ttttgcgatg aagatgtgat acatctttaa atgaaaatat accaaaattt 1860
agtaggcatg tttttctaat aaatttatat atttgtaaag aaaaaaaaaa aaaaa 1915
<210> 8
<211> 453
<212> PRT
<213> Homo Sapiens
<400> 8
Met Lys Leu Leu Val Ile Leu Leu Phe Ser Gly Leu Ile Thr Gly Phe
1 5 10 15
Arg Ser Asp Ser Ser Ser Ser Leu Pro Pro Lys Leu Leu Leu Val Ser
20 25 30
Phe Asp Gly Phe Arg Ala Asp Tyr Leu Lys Asn Tyr Glu Phe Pro His
35 40 45
Leu Gln Asn Phe Ile Lys Glu Gly Val Leu Val Glu His Val Lys Asn
50 55 60
Val Phe Ile Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly
65 70 75 BO
Leu Tyr Glu Glu Ser His Gly Ile Val Ala Asn Ser Met Tyr Asp Ala
85 90 95
7
CA 02309624 2000-OS-18
wo 99mo~9
PCTNS98/Z48pg
Val Thr Lys Lys His Phe Ser Asp Ser Asn Asp Lys Asp Pro Phe Trp
100 105
110
Trp Asn Glu Ala Val Pro Ile Trp Val Thr Asn Gln Leu Gln Glu Asn
115 120
125
Arg Ser Ser Ala Ala Ala Met Trp Pro Gly Thr Asp Val Pro Ile His
130 135
140
Asp Thr Ile Ser Ser Tyr Phe Met Asn Tyr Asn Ser Ser Val Ser Phe
145 150
155 160
Glu Glu Arg Leu Asn Asn Ile Thr Met Trp Leu Asn Asn Ser Asn Pro
165 170
175
Pro Val Thr Phe Ala Thr Leu Tyr Trp Glu Glu Pro Asp Ala Ser Gly
180 185
190
His Lys Tyr Gly Pro Glu Asp Lys Glu Asn Met Ser Arg Val Leu Lys
195 200
205
Lys Ile Asp Asp Leu Ile Gly Asp Leu Val Gln Arg Leu Lys Met Leu
210 215
220
Gly Leu Trp Glu Asn Leu Asn Val Ile Ile Thr Ser Asp His Gly Met
225 230
235 240
Thr Gln Cys Ser Gln Asp Arg Leu Ile Asn Leu Asp Ser Cys Ile Asp
245 250
255
His Ser Tyr Tyr Thr Leu Ile Asp Leu Ser Pro Val Ala Ala Ile Leu
260 265
270
Pro Lys Ile Asn Arg Thr Glu Val Tyr Asn Lys Leu Lys Asn Cys Ser
275 280
285
Pro His Met Asn Val Tyr Leu Lys Glu Asp Ile Pro Asn Arg Phe Tyr
290 295
300
Tyr Gln His Asn Asp Arg Ile Gln Pro Ile Ile Leu Val Ala Asp Glu
305 310
315 320
Gly Trp Thr Ile Val Leu Asn Glu Ser Ser Gln Lys Leu Gly Asp His
325 330
335
Gly Tyr Asp Asn Ser Leu Pro Ser Met His Pro Phe Leu Ala Ala His
340 345
350
Gly Pro Ala Phe His Lys Gly Tyr Lys His Ser Thr Ile Asn Ile Val
355 360
365
Asp Ile Tyr Pro Met Met Cys His Ile Leu Gly Leu Lys Pro His Pro
370 375
380
Asn Asn Gly Thr Phe Gly His Thr Lys Cys Leu Leu Val Asp Gln Trp
385 390
395 400
Cys Ile Asn Leu Pro Glu Ala Ile Ala Ile Val Ile Gly Ser Leu Leu
405 410
415
CA 02309624 2000-OS-18
WO 99127079
PCT/US98/2480$
Val Leu Thr Met Leu Thr Cys Leu Ile Ile Ile Met Gln Asn Arg Leu _
420 425 430
Ser Val Pro Arg Pro Phe Ser Arg Leu Gln Leu Gln Glu Asp Asp Asp
435 440 445
Asp Pro Leu Ile Gly
450
<210> 9
<211> 54B
<212> DNA
<213> Homo Sapiens
<400> 9
ccgagatctc gccactgcac tccagcctgg gtgaaaaggg aaagaaacca acaagccagg 60
ctgattttct agagggatca gtgatgtggg gtacaatgac accttccctg tggcttgtta 120
tgcctccggt tttgtttttg aatcttggtt gctggtgggg tattgccccc tcggctcctc 1$0
tatgctttcg cgtgtgtgaa aatgcaggag tggaccactg tgcacagcag gaccatggct 240
gtgagcagct gtgtctgaac acggaggatt ccttcgtctg ccagtgctca gaaggcttcc 300
tcatcaacga ggacctcaag acctgctccc gggtggatta ctgcctgctg agtgaccatg 360
gttgtgaata ctcctgtgtc aacatggaca gatcctttgc ctgtcagtgt cctgagggac 420
acgtgctccg cagcgatggg aagacgtgtg caaaattgga ctcttgtgct ctgggggacc 480
acggttgtga acattcgtgt gtaagcagtg aagattcgtt tgtgtgccag tgctttgaag 540
gttatata
548
<210> 10
<211> 155
<212> PRT
<213> Homo Sapiens
<400> 10
Met Trp Gly Thr Met Thr Pro Ser Leu Trp Leu Val Met Pro Pro Val
1 5 10 15
Leu Phe Leu Asn Leu Gly Cys Trp Trp Gly Ile Ala Pro Ser Ala Pro
20 25 30
Leu Cys Phe Arg Val Cys Glu Asn Ala Gly Val Asp His Cys Ala Gln
35 40 45
Gln Asp His Gly Cys Glu Gln Leu Cys Leu Asn Thr Glu Asp Ser Phe
50 55 60
Val Cys Gln Cys Ser Glu Gly Phe Leu Ile Asn Glu Asp Leu Lys Thr
65 70 75 BO
Cys Ser Arg Val Asp Tyr Cys Leu Leu Ser Asp His Gly Cys Glu Tyr
85 90 95
Ser Cys Val Asn Met Asp Arg Ser Phe Ala Cys Gln Cys Pro Glu Gly
100 105 110
His Val Leu Arg Ser Asp Gly Lys Thr Cys Ala Lys Leu Asp Ser Cys
115 120 125
Ala Leu Gly Asp His Gly Cys Glu His Ser Cys Val Ser Ser Glu Asp
130 135 140
9
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98124808
Ser Phe Val Cys Gln Cys Phe Glu Gly Tyr Ile _
145 150 155
<210> 11
<211> 269
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (67)
<220>
<221> unsure
<222> (71)
<220>
<221> unsure
<222> (88)
<220>
<221> unsure
<222> (153)
<220>
<221> unsure
<222> (156)
<220>
<221> unsure
<222> (214)
<220>
<221> unsure
<222> (243)
<400> 11
ttgcattcag gggacagatg aggaaaaact gtagctcaga ttaagtaaag agcctaaggt 60
caccagntag ngagtggtgg gttcaaantc tgctggctgt gattccaaag tcagtcctgg 120
gagaagagga tgctcctgag aaacgctttg ccntgntttc tgaccccggt gatctcacag 180
cacatggtga ggctggcagt gatgtgtcct gggnacat~r_ ttc;t;gcag ctggtcccaa 240
atnttgtctc aattaaaaaa aaaaaaaaa 269
<210> 12
<211> 1604
<212> DNA
<213> Homo Sapiens
<400> 12
aggggcgagc tggctggact cggagcgcgg tcgaggcttt ctgcgttcgc ggcggcggaa 60
tggcccgtgc gcggctcgcc gcgtcgcggc tctgtggtcc ctagacgtcg gctcccgccc 120
tcggcgctga tctccggcgc gggcactgct ttccactcgg ctcctgtcgt ccgttctctc 180
aggctcccgt tcaggatttt tagactctga ggagcagttg gagctaatcc acattatgga 240
aatggaaacc accgaacctg agccagactg tgtagtgcag cctccctctc ctcctgatga 300
cttttcatgc caaatgagac tctctgagaa gatcactcca ttgaagactt gttttaagaa 360
aaaggatcag aaaagattgg gaactggaac cctgaggtct ttgaggccaa tattaaacac 420
tcttctagaa tctggctcac ttgatggggt ttttagatct aggaaccaga gtacagatga 480
gaacagctta catgaaccta tgatgaagaa agccatggaa atcaattcat catgcccacc 540
agcagaaaat aatatgtctg ttctgattcc tgataggaca aatgttgggg accagatacc 600
1~
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98124808
ggaagcccat ccttccactg aagctccaga acgagtggtt ccaatccaag atcacagctt 660 _
tccatcagaa accctcagtg ggacggtggc agattccaca ccagctcact tccagactga 720
tcttttgcca gtttcaagtg atgttcctac tagtcctgac tgcttagaca aagtcataga 780
ttatgttcca ggcattttcc aagaaaacag ttttacaatc caatacattc tggacaccag 840
tgataagctg agtactgagc tctttcagga caaaagtgaa gaggcttccc ttgacctcgt 900
gtttgagctg gtgaaccagt tgcagtacca cactcaccaa gagaacggaa ttgaaatttg 960
catggacttt ctgcaaggca cttgtattta tggcagggat tgtttgaagc accacactgt 1020
cttgccatat cattggcaga tcaaaaggac aactactcaa aagtggcaga gtgtattcaa 1080
tgattctcag gagcacttgg aaagatttta ctgtaaccca gaaaatgata gaatgagaat 1140
gaagtatgga ggacaagaat tttgggcaga tttgaatgcc atgaacgtgt atgaaacaac 1200
tgaatttgac caactacgaa ggctgtccac accaccctct agcaatgtca actctattta 1260
ccacacagtc tggaaattct tctgtaggga ccactttgga tggagagagt atcccgagtc 1320
tgtcattcga ttgattgaag aagccaactc tcggggtctg aaagaggttc gatttatgat 1380
gtggaataac cactacatcc tccacaattc attcttcagg agagagataa aaaggagacc 1440
cctcttccgc tcctgtttta tactgcttcc atatttacag taagtgtcga gtatgaagtt 1500
gcaatattta ctctcatttt atgtaaatgc attcctgaat actagagata aaaaataaat 1560
aagagtctac cttggttagt acccctaaaa aaaaaaaaaa aaaa 1604
<210> 13
<211> 415
<212> PRT
<213> Homo sapiens
<400> 13
Met Glu Met Glu Thr Thr Glu Pro Glu Pro Asp Cys Val Val Gln Pro
1 5 10 15
Pro Ser Pro Pro Asp Asp Phe Ser Cys Gln Met Arg Leu Ser Glu Lys
20 25 30
Ile Thr Pro Leu Lys Thr Cys Phe Lys Lys Lys Asp Gln Lys Arg Leu
35 40 45
Gly Thr Gly Thr Leu Arg Ser Leu Arg Pro Ile Leu Asn Thr Leu Leu
50 55 60
Glu Ser Gly Ser Leu Asp Gly Val Phe Arg Ser Arg Asn Gln Ser Thr
65 70 75 80
Asp Glu Asn Ser Leu His Glu Pro Met Met Lys Lys Ala Met Glu Ile
85 90 95
Asn Ser Ser Cys Pro Pro Ala Glu Asn Asn Met Ser Val Leu Ile Pro
100 105 110
Asp Arg Thr Asn Val Gly Asp Gln Ile Pro Glu Ala His Pro Ser Thr
115 120 125
Glu Ala Pro Glu Arg Val Val Pro Ile Gln Asp His Ser Phe Pro Ser
130 135 140
Glu Thr Leu Ser Gly Thr Val Ala Asp Ser Thr Pro Ala His Phe Gln
145 150 155 260
Thr Asp Leu Leu Pro Val Ser Ser Asp Val Pro Thr Ser Pro Asp Cys
165 170 175
Leu Asp Lys Val Ile Asp Tyr Val Pro Gly Ile Phe Gln Glu Asn Ser
180 185 190
11
CA 02309624 2000-OS-18
WO 99IZ7079 PCT/US98/24808
Phe Thr Ile Gln Tyr Ile Leu Asp Thr Ser Asp Lys Leu Ser Thr Glu
195 200 205
Leu Phe Gln Asp Lys Ser Glu Glu Ala Ser Leu Asp Leu Val Phe Glu
210 215 220
Leu Val Asn Gln Leu Gln Tyr His Thr His Gln Glu Asn Gly Ile Glu
225 230 235 240
Ile Cys Met Asp Phe Leu Gln Gly Thr Cys Ile Tyr Gly Arg Asp Cys
245 250 255
Leu Lys His His Thr Val Leu Pro Tyr His Trp Gln Ile Lys Arg Thr
260 265 270
Thr Thr Gln Lys Trp Gln Ser Val Phe Asn Asp Ser Gln Glu His Leu
275 280 285
Glu Arg Phe Tyr Cys Asn Pro Glu Asn Asp Arg Met Arg Met Lys Tyr
290 295 300
Gly Gly Gln Glu Phe Trp Ala Asp Leu Asn Ala Met Asn Val Tyr Glu
305 310 315 320
Thr Thr Glu Phe Asp Gln Leu Arg Arg Leu Ser Thr Pro Pro Ser Ser
325 330 335
Asn Val Asn Ser Ile Tyr His Thr Val Trp Lys Phe Phe Cys Arg Asp
340 345 350
His Phe Gly Trp Arg Glu Tyr Pro Glu Ser Val Ile Arg Leu Ile Glu
355 360 365
Glu Ala Asn Ser Arg Gly Leu Lys Glu Val Arg Phe Met Met Trp Asn
370 375 380
Asn His Tyr Ile Leu His Asn Ser Phe Phe Arg Arg Glu Ile Lys Arg
385 390 395 400
Arg Pro Leu Phe Arg Ser Cys Phe Ile Leu Leu Pro Tyr Leu Gln
405 410 415
<210> 14
<211> 3196
<212> DNA
<213> Homo sapiens
<400> 14
gcgagcctgc aggggatgac tgctgggtga acctaagtta cacagtggtt gctttcacca 60
aacagaccat gggcttcttg gaagaggccc tgaagctgta tttcccagag ctgcacatgg 120
tacttttgga gagcctggtg gaaatcattt tggttgctgt tcagcatgtg gattatagtc 180
ttcgatgtga gcaggatcca gagaagaaag cttttatcag acagaatgca tcctttttat 240
atgaaacagt cctccctgtg gtggagaaaa ggtttgaaga aggtgtgggg aaacctgcca 300
agcaactcca agatctgagg aatgcatcta gacttattcg tgtgaatcct gaaagtacaa 360
catcagtggt ctaatgcttg ggtctgttta tatgtgtata tatgcagaga gagagcttat 420
atattattta tatttatatt aagttgtatt agcatactct atagtttcaa acacaacttg 480
aaaattaaaa gtgccctctt aaaaatacaa aaatcaaaaa gaggaaaata agttaaatta 540
agcccaagta acaaaaatac tggaattatt aaaacgtata gtatgctagc tatcctttta 600
aattatgcta attctcttct tctgaaatta tggtcacact atatactata gcatttcggt 660
12
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98I24808
tttatccttt gataaaactt ttcttttttc tttttttttt ttgagacagg gtctcacccc 720
gtcgcacagg ctggagtgca gtggcaaagt ctcgactcac tgcagccttg acctcccggg 780
cccaggtagt cctcccacct cagcctccca tgtagcagga accacaggca ggcaccacca 840
tacccagcta atttttgtat tttttgtaga gatggggttt cgccatgttg cccaggctag 900
tcttttcttt cttctttttt ttttttcccc acagtatata ttatacagca gtcataatat 960
ctataaatac atagagttta tgttgtgaag tttcccagtt cactgaaatg ttaggtttcc 1020
taaagggtac agtgccgtat aaaacaacct gcctcatata tttctcctca aaacgttgga 1080
ctatttggga aaaggaaaag agttgggaaa attggtttta aggtaagttt tagtcaaaag 1140
aattctttct tgaaactagc tggtttgtgg attcagatac tctgatcctt acagaatcca 1200
agaggaagct ttcataaaaa caattcagca aatatttcca atataatttg aatggctaat 1260
tttcagttgc taattaatta gcagctttgt aatacttgat ttgggagcat ttacttggaa 1320
atcctaagga ctataataaa agttttcaac atatttctaa attgtgtgag tttccagctg 1380
tagcttttgt ctttgtcaca ttttaaaaaa taataagcaa gacacattgg ggacactggc 1440
agcagttgcc aggttttagc tgccaccgct tcagtatgag atatagctgt cccatcttcc 1500
cccattcagg gtaggagata tagtgaccca gacttcatgc aaatggaaaa aaagttttaa 1560
ctgaaatatt tatttagatt tcagggtcta gatggatggg aaagtagaaa aacatatgca 1620
aatctcagtg ttctcactat gaccactctg agcagagatt tggttttgtt tccttttgta 1680
acaaagtgaa aacaggtgag acaatgtgcc caaaacaaag ggaagaagag aaccttctgt 1740
gactcctaaa atgttccatg ctgcattttt gtttcatttt ttatttttcc ttgctttgtt 1800
tttaaacatg aatataatgc ttacttcaaa ttgtttagta aaacaaaata actaaagaaa 1860
tgtgagcttc ccaaggtttc taaactatcg ctgttgtata ttctatagcg ttccttattc 1920
tttgagggaa actgtgcttg ctgtgatcca ttttgtctct agcttctagt tgtgattctt 1980
gtccataagc accaaatttg atgcccatga tttcaaaagg tcatttcttt tatctgaatg 2040
aaaatggtgg tactaagact gtgaaaatta tgtgaaacct aaagtagttg ccaaagtggc 2100
tcagggttgt aaaattcatt gacttaatta ttcatgtgcc agatcaaccc ctttattttc 2160
tctttagctg tgcatattta aaatattgga aagtatcaga tttacagatt ttctttgact 2220
aatttttttc acataacttt aggattttcg aaagttgtaa ccataactgg atatcttagc 2280
tgagcaaagg cggttataat ttgtcttttt aagatcactg gaaattgata aaattttgtg 2340
ataattatga ttattctgtg ccatttacag tttctaatac tatactgtat gaaatatgta 2400
taaatatatg atgctgagtc tgtggaatga tacttctgaa atcaaaattc ctcataaggc 2460
atgaagttgt aaaaacttga atgtgtatag ttagatattt aaatggttgc ttcttcatag 2520
aattgtctgc tttttaaaac tggaagtaca ggattttctt caggtaaaat ctgtgtgttc 2580
caattacagt tgtagctgaa ggaagtatgc tttggtgagt caattagtat gggaacttga 2640
ctaaagaccc ccagtgttgt aacgtacctt tgtacccaga caaaacaatt atgttacatt 2700
cctcaaagtg gcatgggctt tcttctctaa ttcttctgtt ttattagacc caagacaagt 2760
tctaaaaatt gaatgcaatg agagattgtc cagaaatgta atatatacta aaatatacca 2820
cttaagcatt gattgccttt tcttgtttgc ttcaagaata taaaacttgt tacttgagct 2880
tggaatcatg ggcttgattg aattaattac tcttggggaa aaaagacacc ttgtggcatt 2940
aagtcttgct ttggttaaag ccttatttca cataattgct aaaaactcat ttttgtttaa 3000
tatactacct atagtttaat tatcggcact tgtattttgt aacttgatat cttacctagg 3060
attgggaatt tgggacatga catgtactat aaaagtcagt ctatgtacat actgcttatt 3120
gatgtgctgt gatatgaggg aatctgaaat gtttcataaa aataaagctt aaaaattgtc 3180
aaaaaaaaaa aaaaaa 3196
<210> 15
<211> 101
<212> PRT
<213> Homo Sapiens
<400> 15
Met Gly Phe Leu Glu Glu Ala Leu Lys Leu Tyr Phe Pro Glu Leu His
1 5 10 15
Met Val Leu Leu Glu Ser Leu Val Glu Ile Ile Leu Val Ala Val Gln
20 25 30
His Val Asp Tyr Ser Leu Arg Cys Glu Gln Asp Pro Glu Lys Lys Ala
35 40 45
Phe Ile Arg Gln Asn Ala Ser Phe Leu Tyr Glu Thr Val Leu Pro Val
13
CA 02309624 2000-OS-18
WO 99/27079
PCT/US98/24808
50 55 60
Val Glu Lys Arg Phe Glu Glu Gly Val Gly Lys Pro Ala Lys Gln Leu
65 70 75 80
Gln Asp Leu Arg Asn Ala Ser Arg Leu Ile Arg Val Asn Pro Glu Ser
B5 90 95
Thr Thr Ser Val Val
100
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 16
angaagagat gcatttccat cagtctggg 29
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 17
anatctgaac tgaagccaag ttgtgcctc 29
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 18
gnctcatgca cttcagttgg ctgcccact 29
14
CA 02309624 2000-OS-18
WO 99/27079 PCT/US98/24808
<210> 19 _
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 19
angcctgtca caatactgta gtggtttgg 29
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 20
gngcaatacc ccaccagcaa ccaagattc 2g
<210> 21
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<220>
<221> misc_feature
<222> (2)
<223> biotinylated phosphoaramidite residue
<400> 21
tntgtcctga aagagctcag tactcagct 2g
<210> 22
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide
<400> 22
tgtggattat agtcttcgat gtgagcag 2g
1$
