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 application Ser. No. 08/702,420, filed
August 14, 1996, entitled "SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM", filed in the name of some or all of the inventors of the presentapplication.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such
polynucleotides, along with therapeutic, diagnostic and research utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g., cytokines, such
as Iymphokines, intc.re,ons, CSF;s and interleukins) has matured rapidly over the past decade.
The now routine hybridization cloning and expression cloning techniques clone novel
polynucleotides "directly" in the sense that they rely on information directly related to the
discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of
hybridization cloning; activity of the protein in the case of expression cloning). More recent
"indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences
based on the presence of a now well-recognized secretory leader sequence motif, as well as
various PCR-based or low stringency hybridization cloning techniques, have advanced the state
2 5 of the art by making available large numbers of DNA/amino acid sequences for proteins that
are known to have biological activity by virtue of their secreted nature in the case of leader
sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques.
It is to these proteins and the polynucleotides encoding them that the present invention is
directed.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:I;
(b) a polynucleotide collllJIisillg the nucleotide sequence of SEQ ID
NO:I from nucleotide 27 to nucleotide 413;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1
from nucleotide 129 to nucleotide 413;
(d) a polynucleotide comprising the nucleotide sequence of the full length
protein coding sequence of clone AM349_2 deposited under accession number ATCC
98155;
(e) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AM349_2 deposited under accession number ATCC 98155;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AM349_2 deposited under accession number ATCC
98]55;
(g) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone AM349_2 deposited under accession number ATCC 98155;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:2 having biological activity;
(1) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
2 0 of (h) or (i) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: I
from nucleotide 27 to nucleotide 413; the nucleotide sequence of SEQ ID NO:I from
nucleotide 129 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
2 5 nucleotide sequence of the mature protein coding sequence of clone AM349_2 deposited under
accession number ATCC 98155. In other preferred embodiments, the polynucl~.c tide encodes
the full length or mature protein encoded by the cDNA insert of clone AM349_2 deposited
under accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
3 0 ID NO: I or SEQ ID NO:.
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid sequ. nce selected from the group
consisting of:
(a) the amino acid sequence of SE~Q ID NO:2;
3 5 (b) fragments of the amino acid sequence of SEQ ID NO:2; and
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(c) the amino acid sequence encoded by the cDNA insert of clone
AM349_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:2.
In alternate embodiments, isolate AM349_1 deposited under accession number ATCC
98140 may be substituted for AM349_2 in any of the foregoing.
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
1 0 NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5
from nucleotide 189 to nucleotide 389;
(c) a polynucleotide COlllpli~illg the nucleotide sequence of the full length
protein coding sequence of clone AR310_3 deposited under accession number ATCC
98155;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AR310_3 deposited under acces~ion number ATCC 98155;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AR310_3 deposited under accession number ATCC
2 0 98155;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone AR310 3 deposited under accession number ATCC 98155;
(g) a polynucleotide enco-ling a protein comprising the amino acid
sequence of SEQ ID NO:6;
2 5 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:6 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein
3 0 of (g) or (h) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:5from nucleotide 189 to nucleotide 389; 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
3 5 accession number ATCC 98155. In other preferred embodiments, the polynucleotide encodes
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the full length or mature protein encoded by the cDNA insert of clone AR310_3 deposited
under accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:5, SEQ ID NO:4 or SEQ ID NO:7 .
In other cmbodiments, the present invention provides a composition comprising a
protein, whcrein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) fragments of the amino acid sequence of SEQ ID NO:6; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AR310_3 deposited underaccession numberATCC 98155;
the protein being substantially free from other m~mm~ n proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO:6.
In alternate embodiments, isolate AR3 10_2 deposited under accession number ATCC98 ] 40 may be substituted for AR3 10_3 in any of the foregoing.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide cu~ ishlg the nucleotide sequence of SEQ ID
NO:8;
2 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 448 to nucleotide 603;
(c) a polynncleoti~lç CC ~ hlg the nucleotide sequence of SEQ ID NO:8
from nucleotide 547 to nucleotide 603;
(d) a polynucleotide COIl~ ing the nucleotide sequence of the full length
2 5 protein coding sequence of clone AS 186_3 deposited under accession number ATCC
98155;
(e) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AS186_3 deposited under accession number ATCC 98155;
(f) a polynucleotide comprising the nucleotide sequence of the mature
3 0 protein coding sequence of clone AS 186_3 deposited under accession number ATCC
98155;
(g) a polynucleotide enco-~ing the mature protein encoded by the cDNA
insert of clone AS186_3 deposited under accession number ATCC 98155;
(h) a polynucleotide encoding a protein comprising the amino acid
3 5 sçquenre of SEQ ID NO:9;
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(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:9 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 .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:8from nucleotide 448 to nucleotide 603; the nucleotide sequence of SEQ ID NO:8 from
nucleotide 547 to nucleotide 603; the nucleotide sequence of the full length protein coding
sequenceofcloneAS186_3depositedunderaccessionnumberATCC98155;orthenucleotide
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 insen of clone AS 186_3 deposited under
accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:8 or SEQ ID NO:.
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein co,.,p~ise~ an amino acid sequence selected from the group
consisting of:
2 0 (a) the amino acid sequence of SEQ ID NO:9;
(b) fragments of the amino acid sequence of SEQ ID NO:9; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AS 186_3 deposited under accession number ATCC 98 l 55;
the protein being substantially free from other m~mm~ n proteins. Preferably such protein
2 5 comprises the amino acid sequence of SEQ lD NO:9.
In alternate embo~lim~n~c, isolate AS186_2 deposited under accession number ATCC98 l 40 may be substituted for AS 186_3 in any of the foregoing.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
3 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 11 ;
(b) a polynucleQtirle comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 207 to nucleotide 533;
(c) a polynucleotide co."~lish-g the nucleotide sequence of SEQ ID
3 5 NO: 11 from nucleotide 252 to nucleotide 533;
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(d) a polynucleotide comprising the nucleotide sequence of the full length
protein coding sequence of clone AY 160_2 deposited under accession number ATCC
98 l S5;
(e) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AY160_2 deposited under accession number ATCC 98155;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AY 160_2 deposited under accession number ATCC
98155;
(g) a polynucleotide encoding the mature protein encoded by the cDNA
1 0 insert of clone AY 160_2 deposited under accession number ATCC 98155;(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO: 12;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO: 12 having biological activity;
1 5 (i) 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 .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: I I
from nucleotide 207 to nucleotide 533; the nucleotide sequence of SEQ ID NO:lI from
nucleotide 252 to nucleotide 533; the nucleotide sequence of the full length protein coding
sequence of clone AY160_2 deposited under accession number ATCC 98155; or the
nucleotide .sequPnre of the mature protein coding sequence of clone AY 160_2 deposited under
accession number ATCC 98155. In other preferred embodiments, the polynucleotide encodes
2 5 the full length or mature protein encoded by the cDNA insert of clone AY160_2 deposited
under accession number ATCC 98155.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO: 11 or SEQ ID NO:.
In other embodiments, the present invention provides a composition comprising a
3 0 protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO: 12;
(b) fragments of the amino acid sequence of SEQ ID NO: 12; and
(c) the amino acid sequence encoded by the cDNA insert of clone
3 5 AY 160_2 deposited under accession number ATCC 98155;
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the protein being substantially free from other m~mmaliAn proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO~
In alternate embodiments, isolate AY160_1 deposited under accession number ATCC
98140 may be substituted for AY160_2 in any of the foregoing.
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:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:14 from nucleotide 84 to nucleotide 548;
(c) a polynucleotide comprising the nucleotide sequence of the full 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
1 5 cDNA insert of clone BD 127_ 16 deposited under accession number ATCC 98155;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BD127_16 deposited under accession number
ATCC 98155;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
2 0 insert of clone BD127_16 deposited under accession number ATCC 98155;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:15;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO: 15 having biological activity;
2 5 (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 14
3 0 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
3 5 deposited under accession number ATCC 98155.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO: ] 4 or SEQ ID NO:.
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: 15;
(b) fragments of the amino acid sequence of SEQ ID NO: 15; and
(c) the amino acid sequence encoded by the cDNA insert of clone
BD 127_l 6 deposited under accession number ATCC 98155;
1 0 the protein being substantially free from other m~nnm~li;ln proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO: 15.
In alternate embodiments, isolate BD127_11 deposited under accession number ATCC98 l 40 may be substituted for BD127_16 in any of the foregoing.
In one embodiment, the present invention provides a composition comprising an
1 5 isolated polynucleotide selected from the group consisting of:
(a) a po}ynucleotide comprising the nucleotide sequence of SEQ ID
NO: 18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:18 from nucleotide 51 to nucleotide 598;
2 0 (c) a polynuc!~o~ide co~ hlg the nucleotide sequence of the full length
protein coding sequence of clone BL205_ 14 deposited under accession number ATCC98155;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone BL205_14 deposited under accession number ATCC 98155;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BL205_ 14 deposited under accession number ATCC98155;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone BL205_14 deposited under accession number ATCC 98155;
(g) a polynucleotide encoding a protein con.~,isi.,g the amino acid
sequ~n~e of SEQ ID NO: 19;
(h) a polynucleotide encoding a protein COlll~lisillg a fragment of the
amino acid sequence of SEQ ID NO:19 having biological activity;
(i) a polynucl~ootide which is an allelic variant of a polynucleotide of (a)-
3 5 ~d) above;
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(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18
from nucleotide ~ l to nucleotide 598; 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 c~one 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 NO: l 9 from amino acid 84 to amino acid 182.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NOtl8, SEQ ID NO: l 7 or SEQ ID NO:20 .
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: l 9;
(b) the amino acid sequence of SEQ ID NO: l 9 from amino acid 84 to
amino acid 182;
2 0 (c) fragments of the amino acid sequence of SEQ ID NO: l 9; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BL205_14 deposited under accession number ATCC 98155;
the protein being substzlnti~lly free from other m:lmm ~ n proteins. Preferably such protein
comprises the amino acid s~quçn~e of SEQ ID NO:I9 or the amino acid sequence of SEQ ID
2 5 NO:I9 from amino acid 84 to amino acid 182.
In alternate embodiments, isolate BL205_7 deposited under accession number ATCC
98140 may be substituted for BL205_14 in any of the foregoing.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell, in-~lurling bacterial, yeast,
3 0 insect and m~mm~ n 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
5 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 ~lmini$~f~rjng to a m~mm~ n subject a therapeutically effective amount of
a composition co,~ ing a protein of the present invention and a pharmaceutically acceptable
1 0 carrier.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences are reported below for each clone and protein
15 disclosed in the present application. In some instances the sequences are preliminary and may
include some incorrect or ambiguous bases or amino acids. The actual 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 sequence of the protein
2 0 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 avai]able at the time of filing.
Because of the partial ambiguity in reported sequence information, reported protein sequences
2 5 include "Xaa" designators. These "Xaa" designators indicate either ( I ) a residue which cannot
be i<l,o.n~ifi~d because of nucleotide sequence ambiguity or (2) a stop codon in the determined
nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence
were determined more accurately).
As used herein a "secreted" protein is one which, when expressed in a suitable host
3 0 cell, is transported across or through a membrane, including ll~"~,uu,L as a result of signal
sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins
secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they
are expressed. "Secreted" proteins also include without limitation proteins which are
transported across the membrane of the endoplpasmic reticulum.
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Clone"AM349 2"
A polynucJeotide 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. AM349_2 is a full-length clone, including
the entire coding sequencc of a secreted protein (also referred to herein as "AM349_2
protein").
The nucleotide sequence of the 5' portion of AM349_2 as presently determined is
reported in SEQ ID NO: I . What applicants presently believe is the proper reading frame for
the coding region is indicated in SEQ Ir) NO:2. The predicted acid sequence of the AM349_2
protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
Amino acids I to 34 are the predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 35. Additional nucleotide sequence from the 3' portion
of AM349_2, including the polyA tail, is reported in SEQ ID NO:3.
The EcoRI/NotI restriction fragment obtainable from the deposit containing cloneAM349_2 should be approximately 3450 bp.
The nucleotide sequence disclosed herein for AM349_2 was searched against the
GenBank ll~t~b~ce using BLASTA/BLASTX and FASTA search protocols. AM349_2
demonstrated at least some identity with a ESTs identified as "zd84dO9.rl Soares fetal hean
NbHH19W Homo sapiens cDNA" (W81648, BlastN) and "yl72elO.sl Homo sapiens cDNA
43276 3"' (H06061, BlastN). Based upon identity, AM349_2 proteins and each identical
protein or peptide may share at least some activity.
Clone "AR3 10 3"
A polynucleotide of the present invention has been identified as clone "AR3 10_3".
AR310_3 was isolated from a human adult retina cDNA library using methods which are
selective for cDNAs Pnco-ling secreted proteins. AR3 10_3 is a full-length clone, inclu~ling the
entire coding sequence of a secreted protein (also referred to herein as "AR3 ] 0_3 protein").
The nucleotide sçqu~n~e of the 5' portion of AR310_3 as ,~"eser.lly determined is
reported in SEQ ID NO:4. An additional internal nucleotide sequence from AR310_3 as
3 0 presently determined is reported in SEQ ID NO:5. What applicants believe is the proper
reading frame and the predicted amino acid sequence encoded by such internal sequence is
reported in SEQ ID NO:6. Additional nucleotide sequence from the 3' portion of AR3 10_3,
including the polyA tail, is reported in SEQ ID NO:7.
The nucleotide sequence disclosed herein for AR310_3 was searched against the
GenBank d~t~bace using BLASTA/BLASTX and FASTA search protocols. AR310_3
11
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demonstrated at least some identity with ESTs identified as "yy20bOl.sl Homo sapiens cDNA
clone 271753 3"' (N35123, BlastN) and "yy33fO3.sl Homo sapiens cDNA clone 273053 3"'
(N36408, BlastN). Based upon identity, AR310_3 proteins and each identical protein or
peptide may share at least some activity.
Clone "AS 186 3 "
A polynucleotide of the present invention has been identified as clone "AS 186_3".
AS186_3 was isolated from a human fetal brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins. AS 186_3 is a full-length clone, including the
1 0 entire coding sequence of a secreted protein (also referred to herein as "AS 186_3 protein").
The nucleotide sequence of the 5' portion of AS186_3 as presently detennined is
reported in SEQ ID NO:8. What applicants presently believe is the proper reading frame for
the coding region is in-lir:~t~d in SEQ ID NO:9. The predicted acid sequence of the AS 186_3
protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:9.
1 5 Amino acids I to 33 are the predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 34. Additional nucleotide sequence from the 3' portion
of AS 186_3, including the polyA tail, is reported in SEQ ID NO: 10.
The nucleotide sequence disclosed herein for AS186_3 was searched against the
GenBank fl~t~h~e using BLASTA/BLASTX and FASTA search protocols. No hits were
2 0 found in the ~3~t~h~cç
Clone "AY 160 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
2 5 selective for cDNAs encoding secreted proteins. AY160_2 is a full-length clone, including
the entire coding seqn~nce of a secreted protein (also referred to herein as "AY 160_2 protein ").
The nucleotide sequence of the 5' portion of AY160_2 as pl~se~ltly determined isreported in SEQ ID NO: 11. What applicants presently believe is the proper reading frame for
the coding region is indicated in SEQ ID NO:12. The predicted acid sequence of the
3 0 AY160_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
NO:12. Amino acids I to 15 are the predicted leader/signal sequence, with the predicted
mature amino acid sequence bçginning at amino acid 16. Additional nucleotide sequence from
the 3' portion of AY160_2, including the polyA tail, is reported in SEQ ID NO:13.
The nucleotide sequence disclosed herein for AY160_2 was searched against the
35 GenBank r~ h~ce using BLASTA/BLASTX and FASTA search protocols. AY160_2
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demonstrated at least some identity with an EST identified as "yz84hl2.rl Homo sapiens
cDNA clone 289799 5"' (N77069, BlastN). Based upon identity, AY]60_2 proteins and each
identical protein or peptide may share at least some activity.
Clone "BD 127 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
selective for cDNAs encoding secreted proteins. BD127_16 is a full-length clone, including
the entire coding sequence of a secreted protein (also referred to herein as "BD127_16
1 0 protein").
The nucleotide sequence of the 5' portion of BD127_16 as presently deterrnined is
reported in SEQ ID NO: 14. What applicants presently believe is the proper reading frame for
the coding region is indicated in SEQ ID NO:IS. The predicted acid sequence of the
BD127_16 protein corresponding to the foregoing nucleotide sequence is repor~ed in SEQ ID
NO:15. Additional nucleotide sequence from the 3' portion of BD127_16, including the
polyA tail, is reported in SEQ ID NO: 16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing cloneBD127_16 should be approximately 1080 bp.
The nucleotide sequence disclosed herein for BD127_16 was searched against the
2 0 GenBank database using BLASTAJBLASTX and FASTA search protocols. No hits were
found in the database.
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
selective for cDNAs encoding secreted proteins. BL205_14 is a full-length clone, including
the entire coding sequence of a secreted protein (also referred to herein as "BL205_14
protein").
The nucleotide sequence of the 5' portion of BL205_14 as presently deterrnined is
reported in SEQ ID NO:17. An additional internal nucleotide seque.nce from BL205_14 as
presently determined is reported in SEQ ID NO: 18. What applicants believe is the proper
reading frame and the predicted amino acid sequence encoded by such internal sequence is
reported in SEQ ID NO: 19. Additional nucleotide sequence from the 3' portion of BL205_14,
including the polyA tail, is reported in SEQ ID NO:20.
. .
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The nucleotide sequence disclosed herein for BL205_14 was searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were
found in the database.
Deposit of Clones
Clones AM349_2, AR310_3, ASIB6_3, AY160_2, BD127_16 and BL205_14 were
deposited with the American Type Culture Collection on August 23, 1996 under accession
number 98155, from which each clone comprising a particular polynucleotide is obtainable.
An additional isolate of each clone (AM349_1, AR310_2, AS 186_2, AY 160_1, BD127_11
1 0 and BL205_7) was deposited on August 14,1996 with the American Type Culture Collection
under accession number ATCC 98140.
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' cite, EcoRI; 3' cite, NotI) to produce the appropriate fragment for
such clone (approximate clone size fragment are identified above). Bacterial cells containing
a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be d~igned 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.
Clone Probe Sequence
AM349_2 SEQ ID NO:21
2 5 AR310_3 SEQ ID NO:22
AS 186_3 SEQ ID NO:23
AY160_2 SEQ ID NO:24
BD127_16 SEQ ID NO:25
BL205_14 SEQ ID NO:26
In the sequ~ n~çs 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 exarnple, that produced by use of biotin phosphoramidite ( I -dimethoxytrityloxy-
2-(N-biotinyl-4-arninobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite)
3 5 (Glen Research, cat. no. 10- 1953)).
14
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The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ~ C (assuming 2~ for each A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-3~P ATP (specific activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling
oligonucleotides. Other labeling techniques can also be used. Unincorporated label should
preferably be removed by gel filtration chromatography or other established methods. The
10 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 ~l of the stock used to inoculate a sterile culture flask containing 25 ml of
15 sterile L-broth containing ampicillin at 100 llg/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 cont~ining L-broth c~ g ampicillin at 100 llg/ml and agar at 1.5%
2 0 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 Iyse, denature and bake them.
The filter is then preferably incubated at 65~C for l hour with gentle agitation in 6X
2 5 SSC (20X stock is l 75.3 g NaCVliter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH)
containing 0.5 % SDS, l 00 llg/ml of yeast RNA, and l 0 mM EDTA (approximately 10 mL per
150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration
greater than or equaJ to le+6 dpm/mL. The filter is then preferably inf~ub~ff~d at 65~C with
gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5%
3 0 SDS at room twlll.eldlu~ without dgitation, preferably followed by 500 mL of 2X SSC/0. 1%
SDS at room t~.l,peldtu~ with gentle shaking for 15 minutes. A third wash with 0.1X
SSC/0.5% SDS at 65''C for 30 minutes to I 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 Icnown hybridization methods can also be employed.
, . , , .. .. _ _
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The positive colonies are picked, grown in culture, and plasmid DNA isolated using
standard procedures. The clones can then be verified by restriction analysis, hybridization
analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting
5 biological aclivity 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 10, 773-778(1992) and in R.S. McDowell, et al., J.
Amer. Chem. Soc. 1 14,9245-9253(1992), both of which are incorporated herein by reference.
Such fragments may be fused to carrier molecules such as immunoglobulins for many
10 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 be used to generate such
fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein
15 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 lisling by
translation of the nucleotide sequence of each disclosed clone. The mature form of such
protein may be obtained by expression of the disclosed full-length polynucleotide (preferably
2 0 those deposited with ATCC) in a suitable m~lnm~ n cell or other host cell. The sequence of
the mature form of the protein may also be deterrninable from the amino acid sequence of the
full-length form.
The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The cu-lc~ullding genes can be isolated in accordance with known methods
2 5 using the sequence information disclosed herein. Such methods include the pl~l)d~ ion 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 ll-e,nbldne-bound (e.g., is a receptor), the
present invention also provides for soluble forms of such protein. In such forms part or all of
3 0 the intracellular and tran~ ..,hldne domains of the protein are deleted such that the protein
is fully secreted from the cell in which it is expressed. The intracellular and transmembrane
domains of proteins of the invention can be identified in accordance with known techniques
for determination of such domains from sequence information.
Species homologs of the ~ < lo~ed polynucleotides and proteins are also provided by
3 5 the present invention. Species homologs may be isolated and identified by making suitable
16
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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 fonns of the isolated polynucleotide which also
5 encode proteins which are identical, homologous or related to that encoded by the
polynucleotides .
The isolated polynucleotide of the invention may be operably linked to an expression
control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al.,
Nucleic Acids Res. 19, 4485-4490 (l99l), in order to produce the protein recombinantly.
10 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 18~, 537-566 (l990). As defmed herein "operably 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
1 5 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 protein.
M~mm~ n host cells include, for example, monkey COS cells, Chinese Hamster Ovary(CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells,3T3
cells, CV-I cells, other transformed primate cell lines, normal diploid cells, cell strains derived
2 0 from m vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-
60, U937, HaK or Jurkat cells.
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
2 5 any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subfilis~ Salmonella typhimurium, or any bacterial
strain capable of e~ s~hlg 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
3 0 covalent a~ mpnts 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,
3 5 California, U.S.A. (the MaxBac(~ kit), and such methods are well known in the art, as
17
. , . , . _
CA 02263192 1999-02-18
WO ~ "0~5 PCT/US97/14641
described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. ] 555
~1987), incorporated herein by reference. As used herein, an insect cell capable of expressing
a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under
5 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 containing agents which will
bind to the protein; one or more column steps over such affinity resins as concanavalin A-
10 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.
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
15 maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits
for expression and purification of such fusion proteins are commercially available from New
England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
The protein can also be tagged with an epitope and subsequent1y purified by using a specific
antibody directed to such epitope. One such epitope ("Flag") is commercially available from
2 0 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 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
25 substantially homogeneous isolated recombinant protein. The protein thus purified is
s.lbsl~n~ lly free of other m~rnm~ n proteins and is defined in accordance with the present
invention as an "isolated protein."
The protein of the invention may also be e~ ed as a product of transgenic animals,
e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are
3 0 ch ~Ir71~t ri7f d by somatic or germ cells Co~ lg a nucleotide sequence encoding the protein.
The protein may a3so 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 synlh.otic:~lly-constructed protein sequences, by virtue of sharing
primary, secondary or tertiary structural andlor conformational characteristics with proteins
3 5 may possess biological properties in common therewith, including protein activity. Thus, they
18
CA 02263192 1999-02-18
W 098/07855 PCTAUS97/14641
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 characteri~ed by amino acid
5 sequences similar to those of purified proteins but into which modification are naturally
provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known techniques. Modifications of
interest in the protein sequences may include the alteration, substitution, replacement, insertion
or deletion of a selected amino acid residue in the coding sequence. For example, one or more
10 of the cysteine residues may be deleted or replaced with another amino acid to alter the
conformation of the molecule. Techniques for such alteration, substitution, replacement,
insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.
4,51~,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains
the desired activity of the protein.
Other fragments and derivatives of the se~ n~ec of proteins which would be expected
to retain protein activity in whole or in part and may thus be useful for screening or other
immunological methodologies may also be easily made by those skilled in the art given the
disclosures herein. Such modifications are believed to be encompassed by the present
invention .
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one
or more of the uses or biological activities (including those associated with assays cited herein)
identified below. Uses or activities described for proteins of the present invention may be
2 5 provided by ~imini~tration 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
3 0 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 particuJar stage
of tissue differentiation or development or in disease states); as molecular weight markers on
3 5 Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to
19
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CA 02263192 1999-02-18
W 098/07855 PCTAUS97/14641
map related gene positions; to compare with endogenous DNA sequences in patients to identify
potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a
probe to "subtract-out" known sequences in the process of discovering other novel
polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other
support, including for examination of expression patterns; to raise anti-protein antibodies using
DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit
another immune response. Where the polynucleotide encodes a protein which binds or
potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the
polynucleotide can also be used in interaction trap assays (such as, for 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.
The proteins provided by the present invention can similarly be used in assay todetermine biological activity, including in a panel of multiple proteins for high-throughput
screening; to raise antibodies or to elicit another immune response; as a reagent (including the
labeled reagent) in assays designed to quantitatively determine levels of the protein (or its
receptor) in biological fluids; as markers for tissues in which the corresponding protein is
preferentially expressed (either constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
2 0 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.
2 5 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
3 0 and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning
Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional
3 5 sources or ~upplcll~cnl~. Such uses include without limitation use as a protein or amino acid
CA 02263192 1999-02-18
W 098/07855 PCT~US97/14641
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 forrn of powder, pills, solutions, suspensions or capsules. In the case of
microorganisms, the protein or polynucleotide of the invention can be added to the medium
in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either
1 0 inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein factors
discovered to date, including all known cytokines, have exhibited activity in one or more factor
dependent cell proliferation assays, and hence the assays serve as a convenient confirmation
of cytokine activity. The activity of a protein of the present invention is evidenced by any one
of a number of routine factor dependent cell proliferation assays for cell lines including,
without limitation, 32D, DA2, DAlG, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8,
RB5, DAI, 123, T1165, HT2, CTLL2, TF-I, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
2 0 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-lnterscience (Chapter
3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol.
145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991;
Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-
1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, Iymph node cells
or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation,
3 0 Kruisbeek, A.M. and Shevach, E.M. In Curren~ Protocols in Immunology. J.E.e.a. Coligan
eds. Vol I pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of
mouse and human Interferon y, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol I pp. 6.8.1 -6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and Iymphopoietic cells
3 5 include, without limitation, those described in: Measurement of Human and Murine Interleukin
21
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2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Currenl Protocols in
Immunolo~. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto.
1991;deVriesetal.,J.Exp.Med. 173:1205-121]~ 1991;Moreauetal.,Nature336:690-692,
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 Immunolo~y. J.E.e.a.
Coligan eds. Vol I 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 Currettt Protocols in Immunology. J.E.e.a.
Coligan eds. Vol I pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse
1 0 and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current
Protoco~s in Immunology. J.E.e.a. Coligan eds. Vol I pp. 6.13.1, John Wiley and Sons,
Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others,
proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring
1 5 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. USA77:6091-6095, 1980;Weinbergeretal.,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 S~imnl~tin~ or Suy~ sin~ Activity
A protein of the present invention may also exhibit immune stimulating or immune2 5 ~U~yplc~ lg activity, inr!u~iing without limitation the activities for which assays are described
herein. A protein may be useful in the ll~,d~lllCIII of various immune ~ieficierlcie~ and disorders
(including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down)
growth and proliferation of T and/or B Iymphocytes, as well as effecting the cytolytic activity
of NK cells and other cell populations. These immune deficiencies may be genetic or be
3 0 caused by viral (e.g., HlV) as well as bacterial or fungal infections, or may result from
ohl~ e 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, T ricl,n~.,ia spp., malaria
spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of
CA 02263192 1999-02-18
W O 98/07855 PCTAUS97/14641
the present invention may also be useful where a boost to the immune system generally may
be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention
include, for example, connective tissue disease, multiple sclerosis, systemic lupus
5 erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre
syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,
graft-versus-host disease and autoimmune inflammatory eye disease. 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,
10 in which immune suppression is desired (including, for example, organ transplantation), may
also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a
number of ways. Down regulation may be in the form of inhibiting or blocking an immune
response already in progress or may involve preventing the induction of an immune response.
15 The functions of activated T cells may be inhibited by ~ul~pleSSillg T cell responses or by
inducing specific tolerance in T cells, or both. Immuno~ul,p.~s~ion of T cell responses is
generally an active, non-antigen-specific, process which requires continuous exposure of the
T cells to the su~ cssive agent. Tolerance, which involves inducing non-responsiveness or
anergy in T cells. is distinguishable from immuno~u~Jplession in that it is generally antigen-
2 0 specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerancecan be ~lemonctrated 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 Iymphocyte antigen functions (such as, for example, B7)), e.g., preventing high
2 5 level Iymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and
organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T
cell function should result in reduced tissue destruction in tissue transplantation. Typically,
in tissue transplants, rejection of the transplant is initiated through its recognition as foreign
by T cells, followed by an immune reaction that destroys the transplant. The administration
3 0 of a molecule which inhibits or blocks interaction of a B7 Iymphocyte antigen with its natural
ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2
activity alone or in conjunction with a monomeric form of a peptide having an activity of
another B Iymphocyte antigen (e.g., B7-I, B7-3) or blocking antibody), prior to ~ lantation
can lead to the binding of the molecule to the natural ligand(s) on the immune cells without
35 ~ hlgtheco~ olIdingcostim~ orysignal. BlockingBIymphocyteantigenfunction
23
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W O9B/078S5 PCT~US97/14641
in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an
immuno~up~,essant. 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
Iymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of
5 these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject,
it may also be necessary to block the function of a combination of B Iymphocyte 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
1 0 and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine
the immunosuppressive effects of CTLA41g fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-
11105 ~1992). In addition, murine models of GVHD (see Paul ed.~ Fun(l~m.~llt:-l Immunology,
Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking
1 5 B Iymphocyte antigen function irl vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune
e~. Many ~-ltoimm~lne disorders are the result of hlayp.ul"iate 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
2 0 autoreactive T cells may reduce or elimin~t~ disease symptoms. Administration of reagents
which block costim~ tion of T cells by disrupting receptor:ligand interactions of B
Iymphocyte antigens can be used to inhibit T cell activation and prevent production of
autoantibodies or T cell-derived cytokines which may be involved in the disease process.
Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells
2 5 which could lead to long-term relief from the disease. The efficacy of blocking reagents in
preventing or alleviating autoimmune disorders can be determined using a number of well-
characterized animal models of human autoimmune diseases. Examples include murine
experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or
NZB hybrid mice, murine .r..~uil..~ collagen arthritis, diabetes mellitus in NOD mice and
3 0 BB rats, and murine exp~lhll~..tal myasthenia gravis (see Paul ed., Funri~lnent~l Immunology,
Raven Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B Iymphocyte antigen function), as
a means of up regulating immune responses, may also be useful in therapy. Upregulation of
immune l~yonses may be in the form of enhancing an existing immune response or eliciting
3 5 an initial immune response. For example, ~-.hal-~ g an immune response through stim~ ting
24
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B Iymphocyte antigen function may be useful in cases of viral infection. In addition, systemic
viral diseases such as influenza, the common cold~ and encephalitis might be alleviated by the
administration of stimulatory forms of B Iymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by
5 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
10 present invention as described herein such that the cells express all or a portion of the protein
on their surface, and reintroduce the transfected cells into the patient. The infected cells would
now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably
B Iymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor
15 cells (e.g., sarcoma, melanoma, Iymphoma, 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 . For example, tumor cells obtained from
a patient can be transfected ex vivo with an expression vector directing the expression of a
2 0 peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like
activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to
result in expression of the peptides on the surface of the transfected cell. Alternatively, gene
therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B
2 5 Iymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation
signal to T cells to induce a T cell melliA~ed immune response against the transfected tumor
cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which
fail to reexpress sufficient amounts of MHC class I or MHC class Il molecules, can be
transfected with nucleic acid encoding all or a portion of (e.g., a cytolula~l,~ic-domain truncated
3 0 portion) of an MHC class I a chain protein and ,B~ microglobulin protein or an MHC class 11
a chain protein and an MHC class II ,~ chain protein to thereby express MHC class I or MHC
class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in
conjunction with a peptide having the activity of a B Iymphocyte antigen (e.g., B7-1, B7-2, B7-
3) induces a T cell m,oriiA~d immune response against the transfected tumor cell. Optionally,
3 5 a gene çncodirlg an au~ ce construct which blocks expression of an MHC class Il associated
CA 02263192 1999-02-18
W O 9~Uro~S PCTAJS97/14641
protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide
having the activity of a B Iymphocyte antigen to promote presentation of tumor associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell mefti~ted
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,
1 0 D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7,
Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., 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,
lg81; 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.
2 0 Assays for T-cell-dependent immunoglobulin responses and isotype switching (which
will identify, among others, proteins that modulate T-cell dependent antibody responses and
that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J.
Immunol.144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production,
Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
I pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed Iymphocyte reaction (MLR) assays (which will identify, among others, proteins
that generate predominantly Thl and CTL responses) include, without limitation, those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-3 0 Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7,
Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-rl~.penden~ assays (which will identify, among others, proteins expressed
by dendritic cells that activate naive T-cells) include, without limitation, those described in:
3 5 Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine
26
CA 02263192 1999-02-18
W 098/0785~ PCT~US97/14641
173 :549-559,1991; Macatonia et al., Joumal 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; Mac~tonia et al., Journal of
Experimental Medicine ]69:1255-1264, 19~9; 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 Iymphocyte survival/apoptosis (which will identify, among others, proteins
that prevent apoptosis after superantigen induction and proteins that regulate Iymphocyte
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 53:1945-19~1, 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 I :639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development
include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine
et al., Cellular Immunology 155: 111 - 122, 1994; Galy et al., Blood 85 :2770-2778, 1995; Toki
et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re~ulatin~ Activity
2 0 A protein of the present invention may be useful in regulation of hematopoiesis and,
consequently, in the treatment of myeloid or Iymphoid 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, thereby indicating
2 5 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/~"a~.uphages (i.e., traditional CSF activity) useful, for example, in conjunction with
chem~nhPrapy to prevent or treat concequen~ myelo-~u~plc;~sion; in ~up,uu.~ g the growth and
3 0 proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or
Ll~alllient of various platelet disorders such as thrombocytopenia, and generally for use in place
of or c~ plh~elll~i y to platelet transfusions; andlor 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
3 5 disorders (such as those usually treated with transplantation, including, without limitation,
27
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W O 98/07855 PCTrUS97/146~
aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem
cell Coll~p~uLlll~''l 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 dif.ferentiation (which will identify, among others,
1 0 proteins that influence embryonic differentiation hematopoiesis) include, without limitation,
thosedescribedin:Johanssonetal.CellularBiology 15:141-l51,1995;Kelleretal.,Molecular
and CellularBiology 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 Iympho-hematopoiesis) include, without limitation, those described in:
1 5 Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I.
Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss. Inc., New York, NY. 1994; Hirayama et
al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming
cells with high proliferative potential, McNiece, I.K. 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, 1994; Cobblestone area
forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.l. 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, etal. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York,
2 5 NY. 1994; Long term culture initi~ting cell assay, Sutherland, H.J. In Culture of Hematopoietic
Cells. R.I. Freshney, etal. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone,
3 0 cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound
healing and tissue repair and repl~emPnt and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in
circllmct:mc~c 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
3 5 employing a protein of the invention may have prophylactic use in closed as well as open
28
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W O 9~ o~5 PCTrUS97/14641
fracture reduction and also in the improved fixation of artificia1 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 differentiation of progenitors
of bone-forming cells. A protein of the invention may also be useful in the treatment of
osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by
blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast
activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein
of the present invention is tendon/ligament formation. A protein of the present invention,
which induces tendon/ligament-like tissue or other tissue formation in circumstances where
such tissue is not norrnally formed, has application in the healing of tendon or ligament tears,
deforrnities and other tendon or ligament defects in humans and other animals. Such a
pl~J~alion 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
2 0 tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present
invention contributes to the repair of congenital, trauma induced, or other tendon or ligament
defects of other origin, and is also useful in cosmetic plastic surgery for ~t~ m~nt or repair
of tendons or ligaments. The compositions of the present invention may provide an
environment to attract tendon- or ligament-forming cells, ctim~ e growth of tendon- or
2 5 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
intheart.
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 ~l~at~ lt 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,
3 5 a protein may be used in the t~allllellt of diseases of the pe.i~he.~l nervous system, such as
29
CA 02263192 1999-02-18
W O 9X~ o~5 PCTrUS97/14641
peripheral nerve injuries, peripheral neuropathy and localized n~u,ol)alllie~, and central
nervous system diseases, such as ~17heimer'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 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 non-
healing wounds, including without limitation pressure ulcers, ulcers associated with vascular
1 0 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) and vascular
(in-~lu-lin~ vascular endothelium) tissue, or for ~u,u-noli--g the growth of cells comprising such
1 5 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 angiogenic
activity.
A protein of the present invention may also be useful for gut protection or regeneration
and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions
2 0 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
2 5 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 ).
3 0 Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healin~e, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by F~gl~tein and Mertz, J. Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
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WO 98/07855 PCTrUS97/14641
A protein of the present invention may also exhibit activin- or inhibin-re1ated
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
5 heterodimers with a member of the inhibin a family, may be useful as a contraceptive based
on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis
in male m~-nm~ lminictration of sufficient amounts of other inhibins can induce infertility
in these m~mm~l~ Alternatively, the protein of the invention, as a homodimer or as a
heterodimer with other protein subunits of the inhibin-,B group, may be useful as a fertility
1 0 inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release
from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A
protein of the invention may also be useful for advancement of the onset of fertility in sexually
immature m~mm~l~, so as to increase the lifetime reproductive performance of domestic
animals such as cows, sheep and pigs.
1 5 The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assays for activinlinhibin 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.
20 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 m~rnm~lism cells, including, for example, monocytes, fibroblasts,
2 5 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. Ch~mot~rtic or chemokinetic proteins provide particular advantages in
cnl of wounds and other trauma to tissues, as well as in treatment of localized infections.
For example, attraction of Iymphocytes, monocytes or neutrophils to tumors or sites of
3 0 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
stim~l Itf', 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
ceJls. Whether a particular protein has chemotactic activity for a population of cells can be
CA 02263192 1999-02-18
W O ~ d55 PCTrUS97/14641
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
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,
1 0 A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
Associates and Wiley-In~ ,lce (Chapter 6.12, Measurement of alpha and beta Chemokines
6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS
103:140-146, 1995;MulleretalEur.J.Immunol.25: 1744-1748;Gruberetal.J.ofImmunol.
]52:5860-5867, 1994; Johnston etal. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic 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
2 0 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 lled~ lt 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
2 5 following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis
Res. 45:413419, 1987; Humphrey et al., Fibrinolysis 5:71 -79 (1991); Schaub, Prostaglandins
35:467-474, 1988.
Receptor/Li~and Activity
A protein of the present invention may also d~,~llon~ activity as receptors, receptor
ligands or inhibitors or agonists of receptorlligand interactions. Examples of such receptors
and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases
3 5 and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell
32
CA 02263192 1999-02-18
W O 9~ o~5 PCT~US97/14641
interactions and their ligands (including without limitation, cellular adhesion molecules (such
as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen
presentation, antigen recognition and development of cellular and humoral immune responses).
Receptors and ligands are also useful for screening of potential peptide or small molecule
5 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/ligand interactions.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
1 0Suitable assays for receptor-ligand activity include without limitation those described
in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H Margulies,
E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22),
Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
15168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborget
al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 199~.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-infl~-nm~ory activity. The anti-
2 0 infl:-mm~tory 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 stim~ ting or ~u~ ssing productio
of other factors which more directly inhibit or promote an inflammatory response. Proteins
2 5 exhibiting such activities can be used to treat infl~mm~tory conditions including chronic or
acute conditions), including without limitation inflammation associated with infection (such
as septic shock, sepsis or systemic infl~nm~tory response syndrome (SIRS)), ischemia-
reperfusion injury, endotoxin lethality, arthritis, complement-me~ tf d hyperacute rejection,
nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's
3 0 disease or resulting from over production of cytokines such as TNF or IL-l . Proteins of the
invention may also be useful to treat anaphylaxis and hy~Jel~cnsiLivity to an antigenic substance
or material.
Tumor Inhibition Activity
_,
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W O 98/07855 PCTrUS97/14641
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
5 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 ~,u~ c~,~,ing, elimin~ting or inhibiting factors, agents or cell types
which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional
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
15 or enhancing) bodily characteristics, including, without limitation, height, weight, hair color,
eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape
(such as, for example, breast augmentation or diminution, 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
2 0 elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other
nutritional factors or component(s); effecting behavioral characteristics, including. without
limitation, appetite, 2ibido, stress, cognition (including cognitive disorders), depression
(including depressive disorders) and violent behaviors; providing analgesic effects or other
pain reducing effects; promoting dirre~ iation and growth of embryonic stem cells in lineages
2 5 other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes,
correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for example, the ability to bind antigens or complement); and the ability to act as an
antigen in a vaccine cu~ o~,ilion to raise an immune response against such protein or another
3 0 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
3 5 limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical
34
CA 02263192 1999-02-18
W O~ lo~5 PCTAUS97/14641
composition when combined with a pharmS~eutic~lly acceptable carrier. Such a composition
may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials well known in the art. The term "pharrnaceutically
acceptable" means a non-toxic material that does not interfere with the effectiveness of the
5 biological activity of the active ingredient(s). The characteristics of the carrier will depend on
the route of ~lmini~tratjon. The pharm:l~elltic~l composition of the invention may also contain
cytokines, Iymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-I,
IL-2, IL-3, IL-4, IL-5, IL-6, L-7, IL-8, L-9, IL-IO, L-11, IL-12, IL-13, IL-14, IL-15, IFN,
TNFO, TNFI, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
10 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 pha----ac~uLical composition to produce a synergistic
effect with protein of the invention, or to minimi7f side effects. Conversely, protein of the
present invention may be included in formulations of the particular cytokine, Iymphokine, other
15 hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to
minimize side effects of the cytokine, Iymphokine, other hematopoietic factor, thrombolytic
or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers or
homodimers) or complexes with itself or other proteins. As a result, pharmaceutical
2 0 compositions of the invention may comprise a protein of the invention in such multimeric or
complexed form.
The l ha..n~ ic~l composition of the invention may be in the form of a complex of
the protein(s) of present invention along with protein or peptide antigens. The protein and/or
peptide antigen will deliver a 5tim~ ry signal to both B and T Iymphocytes. B Iymphocytes
2 5 will respond to antigen through their surface immunoglobulin receptor. T Iymphocytes will
respond to antigen through the T cell receptor (TCR) following presentation of the antigen by
MHC proteins. MHC and structurally related proteins including those encoded by class I and
class II MHC genes on host cells will serve to present the peptide antigen(s) to T Iymphocytes.
The antigen components could also be supplied as purified MHC-peptide complexes alone or
3 0 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 pharn a~ellti~l
composition of the invention.
The phann~reutic~l composition of the invention may be in the form of a liposome in
3 5 which protein of the present invention is combined, in addition to other pl.a--lJace~ltically
CA 02263192 1999-02-18
W O ~ SS PCTAUS97/14641
acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable
lipids for liposomal formulation include, without limitatiom monoglycerides, diglycerides,
sulfatides, Iysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such
5 liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S.
Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent
No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of
each active component of the pl~ llic~l composition or method that is sufficient to show
1 0 a m~.~ningful patient benefit, i.e., ll~aLlnent~ healing, prevention or amelioration of the relevant
medical condition, or an increase in rate of treatment, healing, prevention or amelioration of
such conditions. When applied to an individual active ingredient, zlrl ninictf red 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 :lflminict~red in
15 combination, serially or simultaneously.
In practicing the method of tlt;allllent or use of the present invention, a therapeutically
effective amount of protein of the present invention is ;~drrinistt~red to a mammal having a
condition to be treated. Protein of the present invention may be :~lminictçred in accordance
with the method of the invention either alone or in combination with other therapies such as
20 tre~tmf ntc employing cytokines, Iymphokines or other hematopoietic factors. When co-
~minic~red with one or more cytokines, Iymphokines or other hematopoietic factors, protein
of the present invention may be z~-lminictered either simultaneously with the cytokine(s),
Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If ~Aminictçred sequentially, the attending physician will decide on the
25 app,up,-ate sequçnce of ~ ini~lr.ling protein of the present invention in combination with
cytokine(s), Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
A~minictration of protein of the present invention used in the pharml~eu~ic~l
composition or to practice the method of the present invention can be carried out in a variety
3 0 of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous,
SUt~C~ OUS, i~ Iilulleal, pa~t;nl~l~l orintravenous injection. Intravenous administration
to the patient is l"~l~lled.
When a therapeutically effective amount of protein of the present invention is
~h..il~ ~l~d orally, protein of the present invention will be in the form of a tablet, capsule,
3 5 powder, solution or elixir. When a-lminict~red in tablet form, the phal~ac~ulical composition
CA 02263192 1999-02-18
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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. When administered in liquid
form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil,
5 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 ~flminicl~red in liquid forrn, the pharmaceutical composition contains from
about 0.5 to 90% by weight of protein of the present invention, and preferably from about l
10 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
invention will be in the form of a pyrogen-free, p~.,t~ lly acceptable aqueous solution. The
pl~paldtion of such parenterally acceptable protein solutions, having due regard to pH,
15 isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition
to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, I ~ct~ted
Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composi~ion of
2 0 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 pl.d....aceutical 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
2 5 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 ~(lminict~red 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
3 0 various pharmaceutical compositions used to practice the method of the present invention
should contain about 0.01 llg to about l OO mg (preferably about O. l ~lg to about 10 mg, more
preferably about 0. l ~g to about l 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"lepen-ling on the severity of the disease being treated and the
3 5 condition and potential idiosyncratic response of each individual patient. It is contemplated
37
CA 02263192 1999-02-18
W O g~ o~5 PCTflUS97/14641
that the duration of each application of the protein of the present invention will be in the range
of ] 2 to 24 hours of continuous intravenous administMtion. 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 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 R.P. Merrifield, J. Amer.Chem.Soc. 85,
2149-2154 (1963); J.L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonal
antibodies binding to the protein of the invention may be useful diagnostic agents for the
immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein
may also be useful therapeutics for both conditions associated with the protein and also in the
treatment of some forms of cancer where abnormal expression of the protein is involved. In
the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the
protein may be useful in detecting and preventing the metastatic spread of the cancerous cells,
which may be me(li~d by the protein.
For cu~ o~ilions 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 A~lminictPred, 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
2 5 a-lministration may be suitable for wound healing and tissue repair. Therapeutically useful
agents other than a protein of the invention which may also optionally be included in the
composition as described above, may alternatively or additionally, be administered
simultaneously or sequentially with the composition in the methods of the invention.
Preferably for bone and/or cartilage formation, the col"~uo~ilion would include a matrix capable
3 0 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 applications.
The choice of matrix material is based on biocompatibility, biodegradability,
3 5 mech~nir~l properties, cosmetic a~ e~nce and interface properties. The particular
38
CA 02263192 1999-02-18
W O 98/07855 PCT~US97/14641
application of the co~ )osilions will define the d~ lid~t~ formulation. Potential matrices for
the compositions may be biodegradable and chemically defined calcium sulfate,
tricalciumphosphate, hydroxyapatite, polylactic acid. polyglycolic acid and polyanhydrides.
Other potential materials are biodegradable and biologically well-defined, such as bone or
5 dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix
components. Olh~r potential matrices are nonbiodegradable and chemically defined, such as
sintered hydroxapatite, bioglass, alumin~ , or other 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
10 composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle
size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid
in the form of porous particles having diameters ranging from 150 to 800 microns. In some
applications, it will be useful to utili~e a sequestering agent, such as carboxymethyl cellulose
15 or autologous blood clot, to prevent the protein compositions from disassociating from the
matrix.
A p.~r~..ed family of seq~l~Cl~ring agents is cellulosic materials such as alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and
2 0 carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose
(CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate,
poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
The amount of se~lue~ lg agent useful herein is 0.5-20 wt%, preferably l-l0 wt% based on
total formulation weight, which represents the amount necessary to prevent desorbtion of the
2 5 protein from the polymer matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cel}s 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 Lr~dllnent of the bone and/or cartilage defect, wound, or tissue in question.
3 0 These agents include various growth factors such as epidermal growth factor (EGF), platelet
derived growth factor (PDGF), "~.~rO- ...hlg growth factors (TGF-~ and TGF-,~), and insulin-
like growth factor (IGF).
The th~ld~,~. lic compositions are also presently valuable for veterinary applications.
Particularly domestic animals and thoroughbred horses, in addition to humans, are desired
3 5 patients for such l~alm~.-t with proteins of the present invention.
39
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The dosage regimen of a protein-containing phal.l.aceulical ~o~ )osilion 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 a wound, type of damaged
5 tissue (e.g., bone3, 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 matnx used
in the reconstitution and with inclusion of other proteins in the pharrnaceutical composition.
For example, the addition of other known growth factors, such as IGF I (insulin like growth
factor 1), to the final composition, may also effeet the dosage. Progress can be monitored by
10 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. Suchpolynucleotides can be introduced either in vivo or ex vivo into cells for expression in a
m~mm~lian subject. Polynucleotides of the invention may also be administered by other
15 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 eultured ex vivo in the plesence of proteins of the present invention
in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells
ean then be introdueed in vivo for therapeutie purposes.
Patent and literature ~ nces cited herein are ineorporated by reference as if fully
set forth.
CA 02263192 1999-02-18
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SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John
LaVallie, Edward
Racie, Lisa
Merberg, David
Treacy, Maurice
Spaulding, Vikki
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
( C ) C I TY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: U.S.A.
(F) ~IP: 02140
(v) COMPUTER READABLE FORM:
(A~ MEDIUM TYPE: Floppy disk
(B~ COMPUTER: IBM PC compatible
(C~ OPERATING SYSTEM: PC-DOS/MS-DOS
(D~ SOFTWARE: PatentIn Release #l.0, Version #1.30
(vi~ CURRENT APPLICATION DATA:
(A~ APPLICATION NUMBER:
(B~ FILING DATE:
(C~ CLASSIFICATION:
(viii~ ATTORNEY/AGENT INFORMATION:
(A~ NAME: Brown, Scott A.
(B~ REGISTRATION NUMBER: 32,724
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8224
(B) TELEFAX: (617~ 876-5851
(2~ INFORMATION FOR SEQ ID NO:l:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 413 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: double
(D~ TOPOLOGY: linear
(ii~ MOLECULE TYPE: cDNA
41
~ .. .. . . . .
CA 02263192 1999-02-18
W O 98/07855 PCTAJS97/14641
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: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 GAC 413
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 a~ino acids
(B) TYPE: a~ino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
Glu Cys Ala Pro Gln Thr Asp Gly Asn Ala Ser Leu Pro Gly Val Val
Gly Glu Asn Tyr Gly Lys Glu Tyr Tyr Gln Ala Leu Leu Gln Glu Gln
Glu Glu His Tyr Gln Thr Arg Ala Thr Ser Leu Lys Arg Gln Ile Ala
Gln Leu Lys Gln Glu Leu Gln Glu Met Ser Glu Lys Met Arg Ser Leu
Gln Glu Arg Arg Asn Val Gly Ala Asn Gly Ile Gly Tyr Gln Ser Asn
100 105 110
42
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Lys Glu Gln Ala Pro Ser Asp Leu Leu Glu Phe Leu His Ser Gln Ile
115 120 125
Asp
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TAGTTTAGTG A~ AA AAGATGTGTG AAATGTTCTC TGCAAAATAA TTCAGGCCAC 60
TGTCTCCTTT TATATATTAT TATAATTATT TATTANGAAG ACCAGTGAAT TACGATATTT 120
AAAGTGAGAG AACTTAATTA TTTGCAAAGG TAAGTTACAG CTT~l~ ll GAGAGAATCA 180
AATGAGTTTA CTTTTGTTCT T~ll~ll~ lN AACTAGCTTT AAGTTTAAAG ATGGAAGCTA 240
AGCAATGGAA ATGCTATACG TTTTTGACAT TTATTAAATG GTACCAATAA AGTATTTTAT 300
TACCAAAAAA ~APAAAAAAA AA 322
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 409 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAGAACTCGG TTTGGTAGAC TTGGACATCT CTCTGGCTTC TGAANANCCT GAAGCTGGCC 60
TGGACCATTC CTGTCCCTTT GTTACCATAC TGTCTCTGGA GTGATGGTGT CCTTCCCTGC 120
CCCACCACGC ATGCTCAGTG C~llllG~ll TCACCTTCCC TCNACTTGAC CCTTCCCTCC 180
CCCAGCGTCA GTTTCACTCC CTCTTGGTTT TTATCAAATT TGCCATGACA TTTCATCTGG 240
43
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GTGGTCTGAA TATTAAAGCT CTTCATTTCT GGANATGGGG CAGCAGGTGG ~l~Tl~ CT 300
GGGGCTGACT TGTCCAGAAG GGGACAAAGT GCAATACANA ACCTTCCCTA CCCTGACGCC 360
TCCCANTCAT CATCTCCAAA ACTCCCACGG GGCTCCCTGA ACTCTCAAG 409
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 389 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TTCTAAGAAG ATTGGGCAGT TGGGTTTCTG GCTTGAGATG AATCCAAGCA GCAGAATGAG 60
CCAGGAGTAG CAGGAGATGG GCAAAGAAAA CTGGGGTGCA NTCAGCTCTC ACAGGGGTAA 120
TNATCTCAAG TGGTATTTGT AGCCAAGTGG GAGCTATTTT Cll~ G CATATAGATA 180
TTTCTTAAAT GAAGCTGCTT TCTTGTCTTT TATTTCTAAA AGCCCCCTTA TACCCCACTT 240
TGTGCAGCAA AGATCCCCGT GCAGGTCACA GCCTGATTTG TGGCCAGGCT GGACAAATTC 300
CTGAGGCACA ACTTGGCTTC AGTTCAGATT TCAAGCTGTG TTGGTGTTGG GACCAGCAGA 360
AGGCAAACGT CCAGCCAACA CACAGGACT 389
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Lys Leu Leu Ser Cys Leu Leu Phe Leu Lys Ala Pro Leu Tyr Pro
1 5 10 15
Thr Leu Cys Ser Lys Asp Pro Arg Ala Gly His Ser Leu Ile Cys Gly
44
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Gln Ala Gly Gln Ile Pro Glu Ala Gln Leu Gly Phe Ser Ser Asp Phe
Lys Leu Cys Trp Cys Trp Asp Gln Gln Lys Ala Asn Val Gln Pro Thr
50 55 60
His Arg Thr
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 292 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AATCTGGCAT CTTCTTANGC TTCTAGTGTT TGGGCCAAAC ATCAACCAAG GGGTTTAATT 60
TATCCAATGC TNGACGANAT GTTCAGGAGG GGCTGGATCA AATTTTGAGA GGGTTATGGG l20
AAAGGGNGGG GGAGAAGAAA TTGACATTTA TTTTATTATT TATTTTAAAT GTTAACATTT l80
TCTTTATGTT GTATCAANCC TGAATAGAAA CTGATAGCAT TAAAANACTC CGTTCCTNTT 240
TNTAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 292
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 603 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGGCCAAAGA NGCCTACTTA GGTAGATGGT GCAACCAGTG GTTCCAGATG GAGAAAAGGT 60
GAAAACAAGT TGGCATTTTT TTGTGCCCTT CAANATCTGA CTTGCTTTAT TTTTTAATTT 120
TTA~ C TANCACATTT GAAAGTGTGA ACATTTAAAC TCTTATTCTG TTTCAGTTTG l80
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CATATGAAGA TGTTTTAAGT AAGTTCTGGA ATTATATAAA AAAAAAATAG AGAGAGTGAG 240
GATGCCCAXA TGACAACAAG CAGAAAAATT CATCCTTTAA ATAAAAGCCT CTATTCTCAT 300
TNGGAAAGCA AAATGTTNTT TNTTAAAAGT 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
CGA 603
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Lys Val Phe Lys Thr Met Ala Gln Gly Ser Leu Ile Tyr Gly Ala
l 5 lO 15
Gly Gly Lys Pro Gly Cys Phe Gln Ser Ala Ala Leu Thr Gly Ala Cys
Ala Ser Pro Val Leu Cys Cys Ser Gly Gln Pro Thr Glu Val His Glu
35 40 45
Ser Asn Ala Arg
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
46
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
AGAAACTTNA TNTATATTAA AAATACAAAA ATTAGCTGGG TGCAGTGGTN CACGCCTNTA 60
TTCCCAATTG CAATTGNTAG GGAGGCTGAG GTATAAGAAA TGAGAATTGC TTNAGATGTT l20
GTAANTTTTA AGCTTGGCCC CAGGAGGCAG AGANTNTANT GACCCGAGAT TACCCCANTN l80
TANTCCAGCC TGGGCNACAG AGTGAGANTC CATNTCAAAA AAAAAAAAAA AAAAAAA 237
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 533 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
TTTGCCTTTN TTTTCCAACA GGTGTCCATT CCCAGGTCNA ANTGCAGGCG AGCCTGAATT 60
CGGCCAAAGA GGCTACATCG CCCYTCTTCT YTCCAGGTCC CCCTTCCCCG CAAYTTCCCA 120
CGAGTGCCAG GTGCCGCGAG CGCCGAGTTC CGCGCATTGG AAAGAAGCGA CCGCGGCGGC l80
TGGAACCCTG ATTGCTGTCC TTCAACATGT TCATTATGAA GTTATTAGTA ATA~ 'l' 240
TTTCTGGACT TATAACTGGT TTTAGAAGTG ACTCTTCCTC TAGTTTGCCA CCTAAGTTAC 300
TACTAGTATC CTTTGATGGC TTCAGAGCTG ATTATCTGAA GAACTATGAA TTTCCTCATC 360
TCCAGAATTT TATCAAAGAA G~~ llGG TAGAGCATGT TAAAAATGTT TTTATCACAA 420
AAACATTTCC AAACCACTAC AGTATTGTGA CAGGCTTGTA TGAAGAAAGC CATGGCATTG 480
TGGCTAATTC CATGTATGAT GCAGTCACAA AGAAACACTT TTCTGACTCT AAT 533
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l09 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
47
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Phe Ile Met Lys Leu Leu Val Ile Leu Leu Phe Ser Gly Leu Ile
1 5 10 15
Thr Gly Phe Arg Ser Asp Ser Ser Ser Ser Leu Pro Pro Lys Leu Leu
Leu Val Ser Phe Asp Gly Phe Arg Ala Asp Tyr Leu Lys Asn Tyr Glu
Phe Pro His Leu Gln Asn Phe Ile Lys Glu Gly Val Leu Val Glu His
Val Lys Asn Val Phe Ile Thr Lys Thr Phe Pro Asn His Tyr Ser Ile
Val Thr Gly Leu Tyr Glu Glu Ser His Gly Ile Val Ala Asn Ser Met
85 90 95
Tyr Asp Ala Val Thr Lys Lys His Phe Ser Asp Ser Asn
100 105
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 395 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ACAAAGGTCT TGGATTANTC ACAAAACTAA GAATACATCC AAGAATAGTG TGTAACTATG 60
AAAAAGAATA CTTTTGAAAG ACAAAGAACT TAGACTAAGC ATGTTAAAAT TATTACTTTG 120
TTTTCCTTGT GTTTTGTTTC GGTGCATTTG CTAATAAGAT AACGCTGACC ATAGTAAAAT 180
TGTTAGTAAA TCATTAGGTA ACATCTTGTG GTAGGAAATC ATTAGGTAAT ATCAATCCTA 240
ACTAGAAATA CTAAAAATGG CTTTTGAGAA AAATACTTCC TCTGCTTGTA TTTTGCGATG 300
AAGATGTGAT ACATCTTTAA ATGAAAATAT ACCAAAATTT AGTAGGCATG ~ AAT 360
AAATTTATAT ATTTGTAAAG ~AAAAAAAAA AAAAA 395
t2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
48
CA 02263l92 1999-02-18
W O 98/07855 PCTAUS97/14641
(A) LENGTH: 548 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECUBE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCGAGATCTC GCCACTGCAC TCCAGCCTGG GTGAAAAGGG AAAGAAACCA ACAAGCCAGG 60
CTGATTTTCT AGAGGGATCA GTGATGTGGG GTACAATGAC ACCTTCCCTG TGGCTTGTTA 120
TGCCTCCGGT TTTGTTTTTG AATCTTGGTT GCTGGTGGGG TATTGCCCCC TCGGCTCCTC 180
TATGCTTTCG C~l~lGlGAA 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
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
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
Leu Cys Phe Arg Val Cys Glu Asn Ala Gly Val Asp His Cys Ala Gln
49
. .
CA 02263l92 l999-02-l8
W O 9X~'~/o55 PCTrUS97/1464
Gln Asp His Gly Cys Glu Gln Leu Cys Leu Asn Thr Glu Asp Ser Phe
Val Cys Gln Cys Ser Glu Gly Phe Leu Ile Asn Glu Asp Leu Lys Thr
Cys Ser Arg Val Asp Tyr Cys Leu Leu Ser Asp His Gly Cys Glu Tyr
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
Ser Phe Val Cys Gln Cys Phe Glu Gly Tyr Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
TTGCATTCAG GGGACAGATG AGGAAAAACT GTAGCTCAGA TTAAGTAAAG AGCCTAAGGT 60
CACCAGNTAG NGAGTGGTGG GTTCAAANTC TGCTGGCTGT GATTCCAAAG TCAGTCCTGG 120
GAGAAGAGGA TGCTCCTGAG AAACGCTTTG CCNTGNTTTC TGACCCCGGT GATCTCACAG 180
CACATGGTGA GGCTGGCAGT GATGTGTCCT GGGNACATAT TTCCTTGCAG CTGGTCCCAA 240
ATNTTGTCTC AATTAAAAAA AA~AAAAAA 269
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 365 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02263192 1999-02-18
W O 9~/~7OSS PCTrUS97/146~1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
AGCGGCCGCA GGGGCGAGCT GGCTGGACTC GGAGCGCGGT CGANGCTTTC TGCGTTCGCG 60
GCGGCGGAAT GGCCCGTGCG CGGCTCGCCG CGTCGCGGCT CTGTGGTCCC TANACGTCGG 120
CTCCCGCCCT CGGCGCTGAT CTCCGGCGCG GGCACTGCTT TCCACTCGGC TCCTGTCGTC l80
CGTTCTCTCA GGCTCCCGTT CAGGATTTTT AGACTCTGAG GAGCAGTTGG AGCTAATCCA 240
CATTATGGAA ATGGAAACCA CCGAACCTGA GCCAGACTGT GTAGTGCAGC CTCCCTCTCC 3 0 0
TCCTGATGAC TTTTCATGCC AAATGAGACT CTCTGAGAAG ATCACTCCAT TGAAGANTTG 3 60
TTTTA 3 65
(2) INFORMATION FOR SEQ ID NO:l8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 598 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTTTTAGATN TAGGAACCAG AGTACAGATG AGAACAGCTT ACATGAACCT ATGATGAAGA 60
AAGCCATGGA AATCAATTCA TCATGCCCAC CAGCAGAAAA TAATATGTCT GTTNTGATTC l20
CTGATAGGAC AAATGTTGGG GACCAGATAC CGGAAGCCCA TCCTTCCACT GAAGCTCCAG l80
AACGAGTGGT TCCAATCCAA GATCACAGCT TTCCATCAGA AACCCTCAGT GGGACGGTGG 240
CAGATTCCAC ACCAGCTCAC TTCCAGACTG A~lCllllGCC AGTTTCAAGT GATGTTCCTA 3 00
CTAGTCCTGA CTGCTTAGAC AAAGTCATAG ATTATGTTCC AGGCATTTTC CCAGAAAACA 3 60
GTTTTACAAT CCAATACATT CTGGACACCA GTGATAAGCT GAGTACTGAG CTCTTTCAGG 420
ACAAAAGTGA AGAGGCTTCC CTTGACCTCG TGTTTGAGCT GGTGAACCAG TTGCAGTACC 480
ACACTCACCA AGAGAACGGA ATTGAAATTT GCATGGACTT TCTGCAAGGC ACTTGTATTT 540
ATGGCAGGGA TTGTTTGAAG CACCACACTG TCTTGCCATA TCATTGGCAG ATCAAAAG 598
(2) INFORMATION FOR SEQ ID NO:l9:
CA 02263l92 l999-02-l8
W O 98/07855 PCT~US97/14641
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 182 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Met Met Lys Lys Ala Met Glu Ile Asn Ser Ser Cys Pro Pro Ala Glu
1 5 10 15
Asn Asn Met Ser Val Xaa Ile Pro Asp Arg Thr Asn Val Gly Asp Gln
Ile Pro Glu Ala His Pro Ser Thr Glu Ala Pro Glu Arg Val Val Pro
Ile Gln Asp His Ser Phe Pro Ser Glu Thr Leu Ser Gly Thr Val Ala
Asp Ser Thr Pro Ala His Phe Gln Thr Asp Leu Leu Pro Val Ser Ser
Asp Val Pro Thr Ser Pro Asp Cys Leu Asp Lys Val Ile Asp Tyr Val
Pro Gly Ile Phe Pro Glu Asn Ser Phe Thr Ile Gln Tyr Ile Leu Asp
100 105 110
Thr Ser Asp Lys Leu Ser Thr Glu Leu Phe Gln Asp Lys Ser Glu Glu
115 120 125
Ala Ser Leu Asp Leu Val Phe Glu Leu Val Asn Gln Leu Gln Tyr His
130 135 140
Thr His Gln Glu Asn Gly Ile Glu Ile Cys Met Asp Phe Leu Gln Gly
145 150 155 160
Thr Cys Ile Tyr Gly Arg Asp Cys Leu Lys His His Thr Val Leu Pro
165 170 175
Tyr His Trp Gln Ile Lys
180
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
52
CA 02263l92 l999-02-l8
W O9~ 55 PCT~US97/14641
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CTGTAGGGAC CACTTTGGAT GGAGAGAGTA TCCCGAGTNT GTCATTNGAT TGATTGAAGA 60
AGCCAACTCT CGGGGTCTGA AAGAGGTTCG ATTTATGATG TGGAATAACC ACTACATCCT 120
CCACAATTCA TTTTTCAGGA GAGAGATAAA AAGGAGACCC CTCTTCCGCT CCTGTTTTAT 180
ACTGCTTCCA TATTTACAGT AAGTGTCGAG TATGAAGTTG CAATATTTAC TCTCATTTTA 240
TGTAAATGCA TTCCTGAATA CTAGAGATAA AAAATAAATA AGAGTCTACC TTGGTTAGTA 300
CCCCTAAAAA AAAAAAAAAA AAAA 324
~2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ANGAAGAGAT GCATTTCCAT CAGTCTGGG 29
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CA 02263192 1999-02-18
WO 98/0785~ PCT~US97/14641
ANATCTGAAC TGAAGCCAAG TTGTGCCTC 29
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GNCTCATGCA CTTCAGTTGG CTGCCCACT 29
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ANGCCTGTCA CAATACTGTA GTGGTTTGG 29
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GNGCAATACC CCACCAGCAA CCAAGATTC 29
CA 02263192 1999-02-18
W O9~ 55 PCT~US97/14641
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TNTGTCCTGA AAGAGCTCAG TACTCAGCT 29
_ .. . .. _ .
