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. 08/667,231, filed July
9, 1996.
FELD 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, interferons, CSFs and interleukins) has matured rapidly over the past decade.
The now routine hybridization cloning and expression cloning techniques clone novel
polynucleotides "directly" in the sense that ~hey 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
2 0 "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
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
2 5 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
3 0 In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:2;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:2 from nucleotide 2l0 to nucleotide 552;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2
from nucleotide 297 to nucleotide 552;
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(d) a polynucleotide comprising the nucleotide sequence of the ful~ length
protein coding sequence of clone BF245_1 deposited under accession number ATCC
98] 01;
(e) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone BF245_1 deposited under accession number ATCC 98101;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BF245_1 deposited under accession number ATCC
98101;
(g) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone BF245_1 deposited under accession number ATCC 98101;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:3;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:3 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:2from nucleotide 210 to nucleotide 552; the nucleotide sequence of SEQ ID NO:2 from
nucleotide 297 to nucleotide 552; the nucleotide sequence of the full length protein coding
sequence of clone BF245_ I deposited under accession number ATCC 98101; or the nucleotide
sequence of the mature protein coding sequence of clone BF245_1 deposited under accession
number ATCC 98101. In other preferred embodiments, the polynucleotide encodes the full
length or mature protein encoded by the cDNA insert of clone BF245_1 deposited under
accession number ATCC 98101.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:2, SEQ ID NO:I or SEQ ID NO:4 .
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:3;
(b) fragments of the amino acid sequence of SEQ ID NO:3; and
(c) the amino acid sequence encoded by the cDNA insert of clone
3 5 BF245_1 deposited under accession number ATCC 98101;
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the protein being substantially free from other m~mm7~ n proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO:3.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
5(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5;
(b) a polynucleotide co~ hlg the nucleotide sequence of SEQ ID NO:5
from nucleotide 477 to nucleotide 752;
(c) a polynucleotide comprising the nucleotide sequence of the full length
1 0protein coding sequence of clone AX56_8 deposited under accession number ATCC 98101;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AX56_8 deposited under accession number ATCC 98101;
(e) a polynucleotide comprising the nucleotide sequence of the mature
15protein coding sçquçnce of clone AX56_8 deposited under accession number ATCC
98101;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone AX56_8 deposited under accession number ATCC 98101;
(g) a polynucleotide encoding a protein comprising the amino acid
2 0sequence of SEQ ID NO:6;
(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 co~ hlg the nucleotide sequence of the full length
protein coding sequence of isolate AX56_28 deposited under accession number
25ATCC 98180;
(j) a polynucleotide encoding the full length protein encoded by the
cDNA insert of isolate AX56_28 deposited under accession number ATCC 98180;
(k) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of isolate AX56_28 deposited under accession number
3 0ATCC 98180;
(I) a polynucleotide encoding the mature protein encoded by the cDNA
insert of isolate AX56_28 deposited under accession number ATCC 98180;
(m) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(e) or (i)-(l) above;
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(n) 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:5from nucleotide 477 to nucleotide 752; the nucleotide sequence of the full length protein
coding sequence of clone AX56_8 deposited under accession number ATCC 98101; or the
nucleotide sequence of the mature protein coding ~equçnce of clone AX56_8 deposited under
accession number ATCC 98101. In other preferred embodiments, the polynucleotide encodes
the full length or mature protein encoded by the cDNA insert of clone AX56_8 deposited
under accession number ATCC 98101.
1 0 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:5.
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
1 5 (a) the amino acid sequence of SEQ ID NO:6;
(b) fragments of the amino acid sequence of SEQ ID NO:6;
(c) the amino acid sequence encoded by the cDNA insert of clone
AX56_8 deposited under accession number ATCC 98101; and
(d) the amino acid sequence encoded by the cDNA insert of isolate
2 0 AX56_2B deposited under accession number ATCC 98180;
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 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:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleotide 161 to nucleotide 553;
(c) a polynucleotide CUJ~ iSillg the nucleotide sequence of SEQ ID NO:7
3 0 from nucleotide 218 to nucleotide 553;
~d) a polynucleotide comprising the nucleotide sequence of the full length
protein coding sequ.-n~e of clone AC222_1 deposited under accession number ATCC
98101;
(e) a polynucleotide encoding the full length protein encoded by the
3 5 cDNA insert of clone AC222_1 deposited under accession number ATCC 98101;
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(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AC222_1 deposited under accession number ATCC
98101;
(g) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone AC222_1 deposited under accession number ATCC 9810];
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:8;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:8 having biological activity;
1 0 (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:7from nucleotide 161 to nucleotide 553; the nucleotide sequence of SEQ ID NO:7 from
nucleotide 218 to nucleotide 553; the nucleotide sequence of the full length protein coding
sequence of clone AC222_1 deposited under accession number ATCC 98101; or the
nucleotide sequence of the mature protein coding sequence of clone AC222_1 deposited under
accession number ATCC 98101. In other preferred embodiments, the polynucleotide encodes
2 0 the full length or mature protein encoded by the cDNA insert of clone AC222_1 deposited
under accession number ATCC 98101. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising the amino acid sequence
of SEQ ID NO:8 from amino acid 1 to amino acid 92.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 5 ID NO:7 or SEQ ID NO:9.
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:8;
3 0 (b) the amino acid sequence of SEQ ID NO:8 from amino acid 1 to amino
acid 92;
(c) fragments of the amino acid sequence of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AC222_1 deposited under accession number ATCC 98101;
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the protein being substantially free from other mammalian proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID N0:8 or the amino acid sequence of SEQ ID
N0:8 from amino acid I to amino acid 92.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0: 10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:10 from nucleotide 61 to nucleotide 693;
1 0 (c) a polynucleotide COlll~ illg the nucleotide sequence of the full length
protein coding sequence of clone 0289_1 deposited under accession number ATCC
98101;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone 0289_1 deposited under accession number ATCC 98101;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone 0289_1 deposited under accession number ATCC
98101;
(f) a polynucleotide encoding the mature protein encoded hy the cDNA
insert of clone 0289_1 deposited under accession number ATCC 98101;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:11;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0: 11 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
2 5 (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 N0: 10
from nucleotide 61 to nucleotide 693; the nucleotide sequence of the full length protein coding
3 0 sequence of clone 0289_1 deposited under accession number ATCC 98101; or the nucleotide
sequence of the mature protein coding sequence of clone 0289_1 deposited under accession
number ATCC 98101. In other preferred embodiments, the polynucleotide encodes the full
length or mature protein encoded by the cDNA insert of clone 0289_1 deposited under
accession number ATCC 98101. In yet other preferred embodiments, the present invention
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provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID
NO: l l from amino acid 9 l to amino acid 130.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO: l O or SEQ ID NO: 12.
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid se4uence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:I l;
(b) the amino acid sequence of SEQ ID NO:l l from amino acid 91 to
amino acid 130;
(c) fragments of the amino acid sequence of SEQ ID NO: 11; and
(d) the amino acid sequence encoded by the cDNA insert of clone
0289_1 deposited under accession number ATCC 98101;
the protein being substantially free from other m:-mm~ n proteins. Preferably such protein
comprises the amino acid se4uence of SEQ ID NO: l l or the amino acid sequence of SEQ ID
NO:l l from amino acid 9l to amino acid 130.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell, including bacterial, yeast,
insect and m~mm~ n cells, transformed with such polynucleotide compositions.
2 0 Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention
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 co-,-p~ise a pharmaceutically
acceptable carrier. Compositions comprising an antibody which specifically reacts with such
protein are also provided by the present invention.
3 0 Methods are also provided for preventing, treating or ameliorating a medical condition
which comprises ~flmini~toring to a m~mm~ n subject a therapeutically effective amount of
a composition comprising a protein of the present invention and a pharm~eutic~lly acceptable
carrier.
3 5 BRIE~F DESCRIPTION OF THE FIGURES
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Figure 1 is a schematic lepl~s~ lion of the pED6 and pNotS vectors used for deposit
of clones disclosed herein.
DETAILED DESCRIPTION
5 ISOLATEDPROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences are reported below for each clone and protein
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
10 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
encoded by a particular clone can also be determined by expression of the clone in a suitable
host cell, collecting the protein and d~le..--h~ g its sequence.
For each disclosed protein applicants have identified what they have determined to be
15 the reading frame best identifiable with sequence information available at the time of filing.
Because of the partial ambiguity in reported sequ~nre information, reported protein sequences
include "Xaa" dPcign~t~rs. These "Xaa" ~ ign~tc)rs indicate either (I) a residue which cannot
be identified 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
2 0 were dettonnined more accurately).
As used herein a "secreted" protein is one which, when expressed in a suitable host
cell, is l~ po.l~d across or through a membrane, including transport as a result of signal
sequPnres in its amino acid sequenre "Secreted" proteins include without limitation proteins
secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they
25 are expressed. "Secreted" proteins also include without limitation proteins which are
transported across the membrane of the endoplpasmic reticulum.
Clone"BF245 1"
A polynucleotide of the present invention has been identified as clone "BF245_1".
3 0 BF245_1 was isolated from a human fetal brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins. BF245_1 is a full-length clone, including the
entire coding sequence of a secreted protein (also referred to herein as "BF245_1 protein").
The nucleotide sequence of the 5' portion of BF245_1 as presently determined is
reported in SEQ ID NO:I. An additional internal nucleotide sequence from BF245_1 as
3 5 presently determined is reported in SEQ ID NO:2. What applicants believe is the proper
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reading frame and the predicted amino acid sequence encoded by such internal sequence is
reported in SEQ ID NO:3. Amino acids I to 29 of SEQ ID NO:3 are a predicted leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino acid 30.
Additional nucleotide sequence from the 3' portion of BF245_1, including the polyA tail, is
5 reported in SEQ ID NO:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing cloneBF245_1 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for BF245_1 was searched against the
GenBank ~l~t~hace using BLASTA/BLASTX and FASTA search protocols. BF245_1
10 demonstrated at least some homology with an EST identified as "yc91hO4.sl Homo sapiens
cDNA clone 23509 3"' (R39256, BlastN) and a human mRNA identified as "KIAA0052"
(D29641 (Fasta). Based upon homology, BF245_1 proteins and each homologous protein or
peptide may share at least some activity.
1 5 Clone "AX56 8"
A polynucleotide of the present invention has been identified as clone "AX56_8".AX56_8 was isolated from a human adult testes cDNA library using methods which are
selective for cDNAs encoding secreted proteins. AX56_8 is a full-length clone, including the
entire coding sequence of a secreted protein (also referred to herein as "AX56_8 protein").
2 0 The nucleotide sequence of AX56_8 as presently determined is reported in SEQ ID
NO:5. What applicants presently believe to be the proper reading frame and the predicted
amino acid sequence of the AX56_8 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:6. .
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone2 5 AX56_8 should be approximately 730 bp.
The nucleotide sequence disclosed herein for AX56_8 was searched against the
GenBank ~ h~ce using BLASTA/BLASTX and FASTA search protocols. No hits were
found in the ~:lt lh~ce The amino acid sequence of AX56_8 indicates that it may have some
homology with chicken cytotactin.
Clone"AC222 1"
A polynucleotide of the present invention has been identified as clone "AC222_1".
AC222_1 was isolated from a human adult placenta cDNA library using methods which are
selective for cDNAs encoding secreted proteins. AC2'~_1 is a full-length clone, including the
3 5 entire coding sequence of a secreted protein (also referred to herein as "AC222_1 protein").
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The nucleotide sequence of the 5' portion of AC222_1 as presently determined is
reported in SEQ ID N0:7. What applicants presently believe is the proper reading frame for
the coding region is indicated in SEQ ID N0:8. The predicted acid seqllenre of the AC222_1
protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
5 Amino acids 1 to 19 are the predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 20. Additional nucleotide sequence from the 3' portion
of AC222_1, including the polyA tail, is reported in SEQ ID N0:9.
The EcoRI/NotI restriction fragment obtainable from the deposit containing cloneAC222_1 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for AC222_1 was searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. AC222_1
demonstrated at least some homology with a chicken proteoglycan core protein, proteoglycan-
Lb (D10485, BlastX). Based upon homology, AC222_1 proteins and each homologous
protein or peptide may share at least some activity.
Clone"0289 1"
A polynucleotide of the present invention has been identified as clone "0289_1".0289_1 was isolated from a human dendritic cell cDNA library using methods which are
selective for cDNAs encoding secreted proteins. 0289_1 is a full-length clone, including the
2 0 entire coding sequence of a secreted protein (also referred to herein as "0289_1 protein").
The nucleotide sequence of the 5' portion of 0289_1 as presently determined is
reported in SEQ ID N0: 10. What applicants presently believe is the proper reading frame for
the coding region is int~ic~tr.d in SEQ ID N0:11. The predicted acid sequence of the 0289_1
protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0: 11.
2 5 Additional nucleotide sequence from the 3' portion of 0289_1, including the polyA tail, is
reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone0289_1 should be approximately 800 bp.
The nucleotide sequence disclosed herein for 0289_1 was searched against the
3 0 GenBank d~t~h~ce using BLASTA/BLASTX and FASTA search protocols. No hits were
found in the d~ h~e.
Deposit of Clones
Clones BF245_1, AX56_8, AC222_1 and 0289_1 were deposited on luly 9, 1996
3 5 with the American Type Culture Collection under accession number ATCC 98101, from which
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each clone comprising a particular polynucleotide is obtainable. Each clone has been
transfected into separate bacterial cells (E. coli) in this composite deposit. An additional
isolate of AX56_8 (identified as "AX56_28") was deposited on September 26, 1996 with the
American Type Culture Collection under accession number ATCC 98180, from which the
5 clone is also obtainable.
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 appropriately sized
fragment for such clone (approximate clone si~e fragment are identified below). Each clone
was deposited in either the pED6 or pNotS vector depicted in Fig. 1. In some instances, the
1 0 deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate.
In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI.
However, NotI will then produce the 5' cite and EcoRI will produce the 3' cite for placement
of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be
expressed from the vectors in which they were deposited. 0289_1 may be deposited in the
15 Bluescript vector. In such instance, the cDNA insert can be removed from the vector by
digestion with XhoI (5' cite) and NotI (3' cite).
Bacterial cells containing a particular clone can be obtained from the compositedeposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known
2 0 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.
2 5 Clone Probe Sequence
BF245_1 SEQ ID N0:13
AX56_~ SEQ I0 N0:14
AC222_1 SEQ ID N0:15
0289_1 SEQ ID N0: 16
In the sequences listed above which include an N at position 2, that position is occupied in
preferred probeslprimers by a biotinylated phosphoaramidite residue rather than a nucleotide
~ (such as, for example, that produced by use of biotin phosphoramidite (1 -dimethoxytrityloxy-
2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite)
(Glen Research, cat. no. 10-1953)).
11
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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
1 0 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 Ill of the stock used to inoculate a sterile culture flask containing 25 ml of
1 5 sterile L-broth COllL~ g ampicillin at 100 ~g/ml. The culture should preferably be grown to
saturation at 37~C, and the saturated culture should preferably be diluted in fresh L-broth.
Aliquots of these dilutions should preferably be plated to determine the dilution and volume
which will yield approximately 5000 distinct and well-separated colonies on solid
bacteriological media containing L-broth cont~ining ampicillin at 100 ~g/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-s~ 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 I hour with gentle agitation in 6X
2 5 SSC (20X stock is 175.3 g NaCUliter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH)
C~ g 0.5% SDS, 100 llg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per
150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration
greater than or equal to le+6 dpmlmL. The filter is then preferably inc~lb~t.qd 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 l~ ldLul~ without agitation, preferably followed by 500 mL of 2X SSC/0.1 %
SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X
SSC/0.5% SDS at 65~C for 30 minutes to 1 hour is optional. The filter is then preferably dried
and subjected to autoradiography for sufficient time to visualize the positives on the X-ray
film. Other known hybridization methods can also be empJoyed.
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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, hybridi~ation
analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting
biological activity are also encompassed by the present invention. Fragments of the protein
may be in linear form or they may be cyclized using known methods, for example, as described
in H.U. Saragovi, et a/., Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al., 3.
1 0 Amer. Chem. Soc. I 14,9245-9253 ( l 992), both of which are incorporated herein by reference.
Such fragments may be fused to carrier molecules such as immunoglobulins for many
purposes, including increasing the valency of protein binding sites. For example, fragments
of the protein may be fused through "linker" sequences to the Fc portion of an
immunoglobulin. 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
of the invention.
The present invention also provides both full-length and mature forms of the disclosed
proteins. The full-length form of the such proteins is identified in the sequence listing by
2 0 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
those deposited with ATCC) in a suitable m~mm~ n cell or other host cell. The sequence of
the mature form of the protein may also be determinable from the amino acid sequence of the
full-length form.
2 5 The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The corresponding genes can be isolated in accordance with known methods
using the sequence information disclosed herein. Such methods include the preparation of
probes or primers from the disclosed sequence information for identification and/or
amplification of genes in appropriate genomic libraries or other sources of genomic materials.
3 0 Where the protein of the present invention is ~ lbl~--e-bound (e.g., is a receptor), the
present invention also provides for soluble forms of such protein. In such forms part or all of
the intracellular and transmembrane domains of the protein are deleted such that the protein
is fully secreted from the cell in which it is expressed. The intracellular and transmembrane
domains of proteins of the invention can be identified in accordance with known techniques
3 5 for determination of such domains from sequence information.
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Species homologs of the disclosed polynucleotides and proteins are also provided by
the present invention. Species homologs may be isolated and identified by making suitable
probes or primers from the sequences provided herein and screening a suitable nucleic acid
source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or
proteins; that is, naturally-occurring altemative forms of the isolated polynucleotide which also
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 (1991), in order to produce the protein recombinantly.
Many suitable expression control sequences are known in the art. General methods of
expressing recombinant proteins are also known and are exemplified in R. K~llfm~n, Methods
in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the
isolated polynucleotide of the invention and an expression control se~uence are situated within
a vector or cell in such a way that the protein is expressed by a host cell which has been
transformed (transfected) with the ligated polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of the protein.
Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary
2 0 (CHO) cells, human kidney 293 cells, human epidermal A43 I cells, human Colo205 cells, 3T3
cells, CV-I cells, other transformed primate cell lines, normal diploid cells, cell strains derived
from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-
60, U937, HaK or Jurkat cells.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as
2 5 yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or
any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria,
3 0 it may be necessary to modify the protein produced therein, for example by phosphorylation
or glycosylation of the ap~ pliate sites, in order to obtain the functional protein. Such
covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of
the invention to suitable control se~uences in one or more insect expression vectors, and
3 5 employing an insect expression system. Materials and methods for baculovirus/insect cell
14
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expression systems are commercially available in kit form from, e.g., Invitrogen, San I~iego,
California, U.S.A. (the MaxBac~) kit), and such methods are well known in the art, as
described in Summers and Smith, Texas A~ricultural Experiment Station Bulletin No. 1555
(1987), incorporated herein by reference. As used herein, an insect cell capable of expressing
5 a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under
culture conditions suitable to express the recombinant protein. The resulting expressed protein
may then be purified from such culture (i.e., from culture medium or cell extracts) using known
purification processes, such as gel filtration and ion exchange chromatography. The
10 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-
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.
15Alternatively, the protein of the invention may also be expressed in a form which will
facilitate purification. For example, it may be expressed as a fusion protein, such as those of
- maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits
for expression and purification of such fusion proteins are commercially available from New
Lngland BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
2 0The protein can also be tagged with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("Flag") is commercially available from
Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl
2 5or other aliphatic groups, can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be employed to provide a
substantially homogeneous isolated recombinant protein. The protein thus purified is
substantially free of other m~mm~ n proteins and is defined in accordance with the present
invention as an "isolated protein."
3 0The protein of the invention may also be expressed as a product of transgenic animals,
e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are
characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are known
3 5to those skilled in the art. The synthetic~lly-constructed protein seqnenrpc~ by virtue of sharing
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primary, secondary or tertiary structural andlor conformational characteristics with proteins
may possess biological properties in common therewith, including protein activity. Thus, they
may be employed as biologically active or immunological substitutes for natural, purified
proteins in screening of therapeutic compounds and in immunological processes for the
5 development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification are naturally
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
10 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 exarnple, one or more
of the cysteine residues may be deleted or replaced with another amino acid to alter the
conformation of the molecule. Techniques for such alteration, substitution, replacement,
insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.
15 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains
the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be 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
20 disclosures herein. Such modifications are believed to be encompzl~ed by the present
invention.
USI~S AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to e~hibit one
2 5 or more of the uses or biological activities (including those associated with assays cited herein)
identified below. Uses or activities described for proteins of the present invention may be
provided by administration or use of such proteins or by administration or use of
polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors
suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research
community for various purposes. The polynucleotides can be used to express recombinant
protein for analysis, cl-a-d~;lelization or therapeutic use; as markers for tissues in which the
3 5 corresponding protein is preferentially expressed (either constitutively or at a particular stage
16
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WO 98/01~52 PCTIUS97111842
of tissue differentiation or development or in disease states); as molecular weight markers on
Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to
map related gene positions; to compare with endogenous DNA sequences in patients to identify
potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
5 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 exarnination of expression patterns; to raise anti-protein antibodies using
DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit
10 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
2 0 preferentially expressed (either constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
Where the protein binds or potentially binds to another protein (such as, for example, in a
receptor-ligand interaction), the protein can be used to identify the other protein with which
binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these
2 5 binding interactions can also be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade
or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the
3 0 art. References disclosing such methods include without limitation "Molecular Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch
and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning
Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
3 5 Nutritional Uses
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Polynucleotides and proteins of the present invention can also be used as nutritional
sources or supplements. Such uses include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a source of
carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the
5 feed of a particular organism or can be ~flmini~tered as a separate solid or li~uid preparation,
such as in the forln 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.
1 0 Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either
inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein factors
discovered to date, including all known c~tokines, have exhibited activity in one or more factor
15 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, DAIG, T10, B9, B9/11, BaF3, MC91G, M+ (preB M+), 2E8,
RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
2 0 The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter
2 5 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.
3 0 Assays for cytokine production andlor proliferation of spleen cells, Iymph node cells
or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A.M. and Shevach, E.M. In Current Protoco~s in Immlmology. J.E.e.a. Coligan
eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Mea-.u~ ut of
mouse and human Interferon y, Schreiber, R.D. In Cllrrent Protocols in Immunology. J.E.e.a.
3 5 Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
18
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Assays for proliferation and differentiation of hematopoietic and Iymphopoietic cells
include. without limitation, those described in: Measurement of Human and Murine Interleukin
2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Curre~lt Protocols i~l
Irnmllnology. J.E.e.a. Coligan eds. Vol I pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto.
1991; deVries et al., J. E~xp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692,
1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931 -2938, I 983; Measurement of
mouse and human interleukin 6 - Nordan, R. In C~rrent Protocols in Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.
Natl.Acad.Sci.U.S.A.83:1857-1861,1986;MeasurementofhumanInterleukin 11 -Bennett,1 0 F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocol~ in Immunology. J.E.e.a.
Coligan eds. Vol I pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse
and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current
Protocols in Immuno~ogy. 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
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,
2 0 In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular
receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.
Sci. USA 77:6091-6095, 1980; Weinberger et al., Lur. J. Immun. 11:405-411, 1981; Takai
et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
2 5 Immune Stimulatin~ or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immunesuppressing activity, in~lu~ing without limitation the activities for which assays are described
herein. A protein may be useful in the treatment of various immune deficiencies and disorders
~including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down)
3 0 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
caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from
autoill,l,l.llle 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, Leichm~ni~ spp., malaria
19
CA 022~99~7 1 999 - 01 - 08
WO 98/01552 PCT/US97/11842
spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of
the present invention may also be useful where a boost to the immune system generally may
be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention
5 include, for example, connective tissue disease, multiple sclerosis, systemic lupus
erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre
syndrome, autoimmune thyroiditis~ insulin dependent diabetes mellitis, myasthenia gravis,
graft-versus-host disease and ~uloinl~ l--e infl~mm~tory eye disease Such a protein of the
present invention may also to be useful in the treatment of allergic reactions and conditions,
10 such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions,
in which immune ~.u~pr~ssion 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
15 response already in progress or may involve preventing the induction of an immune response.
The functions of activated T cells may be inhibited by ~.u~ ssing T cell responses or by
inducing specific tolerance in T cells, or both. Immunosu~ ssion of T cell responses is
generally an active, non-antigen-specific, process which requires continuous exposure of the
T cells to the ~.u~ essive agent. Tolerance, which involves inducing non-responsiveness or
2 0 anergy in T cells, is distinguishable from immuno~.upplession in that it is generally antigen-
specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance
can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the
absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
2 5 limitation B Iymphocyte antigen functions (such as, for example, B7)), e.g., preventing high
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
3 0 by T cells, followed by an immune reaction that destroys the transplant. The Zlrlministration
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 forrn of a peptide having R7-2
activity alone or in conjunction with a monomeric form of a peptide having an activity of
another B Iymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation
3 5 can lead to the binding of the molecule to the natural ligand(s) on the immune cells without
CA 022~99~7 1999-01-08
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transmitting the corresponding costimulatory signal. Blocking B Iymphocyte antigen function
in this matter prevents cytokine synthesis by immune cells, such as T cells. and thus acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize
the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B
5 Iymphocyte antigen-blocking reagents may avoid the necessity of repeated ~rlministration of
these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject,
it may also be necessary to block the function of a combination of B 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.
1 0 Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats
and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine
the immunosuppressive effects of CTLA4Ig fusion proteins in vi~Jo as described in Lenschow
et a~., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89: 11 I02-
11105 (1992). ~n addition, murine models of GVHD (see Paul ed., Fundamental Immunology,
Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking
B Iymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune
diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that
are reactive against self tissue and which promote the production of cytokines and
20 autoantibodies involved in the pathology of the diseases. Preventing the activation of
autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents
which block costim~ ion 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.
2 5 Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells
which could lead to long-term relief from the disease. The efficacy of blocking reagents in
preventing or alleviating autoimmune disorders can be determined using a number of well-
characterized animal models of human autoimmune diseases. Examples include murine
experimental autoimmune encephalitis, systemic lupus erythmatosis in MRLllpr/lpr mice or
3 0 NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and
BB rats, and murine ~ hl~ tal myasthenia gravis (see Paul ed., Fnn~ m~nt~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
3 5 immune responses may be in the form of enhancing an existing immune response or eliciting
CA 022~99~7 1999-01-08
WO 98/01552 PCT/US97/11842
an initial immune response. For example, enhancing an immune response through stim~ ting
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
~lminictration of stimulatory forms of B Iymphocyte antigens systemically.
Altematively, anti-viral immune responses may be enhanced in an infected patient by
removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed
APCs either expressing a peptide of the present invention or together with a stimulatory form
of a soluble peptide of the present invention and reintroducing the in vi~ro activated T cells into
the patient. Another method of enhancing anti-viral immune responses would be to isolate
infected cells from a patient, transfect them with a nucleic acid encoding a protein of the
present invention as described herein such that the cells express all or a portion of the protein
on their surface, and reintroduce the l~ r~led cells into the patient. The infected cells would
now be capable of delivering a costimulatory signa] to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably
B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor
cells (e.g., sarcoma, melanoma, 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 c~lllbhldLion of peptides . For example, tumor cells obtained from
2 0 a patient can be transfected ex vivo with an expression vector directing the expression of a
peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like
activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to
result in expression of the peptides on the surface of the transfected cell. Alternatively, gene
therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B
Iymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation
signal to T cells to induce a T cell mP.rliz~tP.d 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
3 0 transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated
portion) of an MHC class I a chain protein and ~2 microglobulin protein or an MHC class II
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 IIMHC in
conjunction with a peptide having the activity of a B Iymphocyte antigen (e.g., B7-1, B7-2, B7-
3 5 3) induces a T cell mPfii~tPd immune response against the transfected tumor cell. Optionally,
CA 022~99~7 1999-01-08
WO 98/015S2 PCTIUS97J11842
a gene encoding an Anli~en~e construct which blocks expression of an MHC class II associated
protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide
having the activity of a B Iymphocyte antigen to promote presentation of tumor associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated
5 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,
10 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); 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. 137:3494-3~00, 1986; Takai et al., J.
Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3~00, 1986; Bowmanet al., J.
Virology 61 1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,
20 CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which
will identify, among others, proteins that modulate T-cell dependent antibody responses and
that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J.
Immunol. 144:3028-3033,1990; and Assays for B cell function: In vitro antibody production,
2 5 Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed 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.
30 Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7,
Immunologic studies in Humans); Takai et al., l. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol 149:3778-3783, 199'~.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed
3 5 by dendritic cells that activate naive T-cells) include, without limitation, those described in:
23
CA 022 ~ 99 ~ 7 1999 - 01 - 08
WO 98/01552 PCT/US97/11842
Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine
~73 549 ssg, Igg~ r~tonj~ et al., Journal of Immunology 154:5071-~079, 1995; Porgador
et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology
67:4062-4069, 1993; Huang et al., Science 264:96]-965, 1994; Macatonia et al., Journal of
Experimental Medicine 169:1255-1264, 19Bg; 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 survivaUapoptosis (which will identify, among others, proteins
that prevent apoptosis after superantigen induction and proteins that regulate Iymphocyte
1 0 hom~.os~cis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry
13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer
Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of
Immunology 145:4037-404~, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczycaet
al., International Journal of Oncology I :639-648, 1992.
1 5 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 15~: 111-122, 1994; Galy et al., Blood 85 :2770-2778, 1995; Toki
et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
2 0 Hematopoiesis Regulatin~ ActivitY
A protein of the present invention may be useful in regulation of hematopoiesis and,
consequently, in the l~ca~ ent of myeloid or Iymphoid cell deficiencies. ~ven marginal
biological activity in support of colony forming cells or of factor-dependent cell lines indicates
involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of
erythroid progenitor cells alone or in combination with other cytokines, thereby indicating
utility, for example, in treating various anemias or for use in conjunction withirradiation/chemotherapy to s~im~ e the production of erythroid precursors and/or erythroid
cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and
monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with
3 0 chemotherapy to prevent or treat conce~ .nt myelo-~u~l,-e~sion; in supporting the growth and
proliferation of ~l,e~ak~yocytes and conse~uently of platelets thereby allowing prevention or
ll~d~lllell~ of various platelet disorders such as thrombocytopenia, and generally for use in place
of or complh,,c;lll~y to platelet transfusions; andlor in sl~ppo~ g 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
24
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disorders (such as those usually treated with transplantation, including, without limitation,
aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem
cell co~ lent post irradiation/chemotherapy, either in-vivo or e~--vivo (i.e., in conjunction
with bone marrow transplantation or with peripheral progenitor cell transplantation
5 (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines are
cited above.
Assays for embryonic stem cell differentiation (which will identify, among others,
proteins that influence embryonic differentiation hematopoiesis) include, without limitation,
those described in: Johansson et al. Cellular Biology 15: 141 - 151,1995; Keller et al., 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,
15 proteins that regulate Iympho-hematopoiesis) include, without limitation, those described in:
Methylcellulose colony forming assays, Freshney, M.G. In Cultllre 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 Clllture of
2 0 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York,
NY. 1994; Neben et al., Expe,il,.ellL~I Hematology 22:353-359, 1994; Cobblestone area
forming cell assay, Ploemacher, R.E. In Clllture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures
in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Cl~lt~lre of
2 5 Hematopoietic Cells. R.l. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York,
NY.1994; Long term culture ini~iAling cell assay, Sutherland, H.l. In Culture of Hematopoietic
Cells. R.I. Freshney, etal. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
CA 022~99~7 1 999 - 01 - 08
WO 98/01552 PCTIUS97/11842
Tissue Growth Activit~
A protein of the present invention also may have utility in compositions used for bone,
cartilage, tendon, ligament andlor nerve tissue growth or regeneration, as well as for wound
healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in
circumstances where bone is not normally formed, has application in the healing of bone
fractures and cartilage damage or defects in humans and other animals. Such a pr~pa.~lLion
employing a protein of the invention may have prophylactic use in closed as well as open
fracture reduction and also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair of congenital, trauma
induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic
plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease,
and in other tooth repair processes. Such agents may provide an environment to attract bone-
forming cells, stimulate growth of bone-forming cells or induce 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.) me~ ed by infl ITnm Itory processes.
2 0 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 l;h~;ul~ ces where
such tissue is not normally formed, has application in the healing of tendon or ligament tears,
deformities and other tendon or ligament defects in humans and other animals. Such a
2 5 ,~ ion employing a tendon/ligament-like tissue inducing protein may have prophylactic
use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation
of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament
tissue. De novo tendonAigament-like tissue formation induced by a composition of the present
invention contributes to the repair of congenital, trauma induced, or other tendon or ligament
3 0 defects of other origin, and is also useful in cosmetic plastic surgery for ~ chment or repair
of tendons or ligaments. The compositions of the present invention may provide an
environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming
cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return i~1 vivo to
3 5 effect tissue repair. The compositions of the invention may also be useful in the treatment of
26
CA 022~99~7 1999-01-08
W O 98/01552 PCTrUS97/11842
tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions
may also include an appropriate matrix and/or sequestering agent as a carrier as is well known
in the art.
The protein of the present invention may also be useful for proliferation of neural cells
and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and traumatic disorders,
which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically,
a protein may be used in the treatment of diseases of the peripheral nervous system, such as
peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central
nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be
treated in accordance with the present 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
insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for
2 0 generation or regeneration of other tissues, such as organs (including, for example, pancreas,
liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells comprising such
tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to
allow norrnal tissue to regenerate. A protein of the invention may also exhibit angiogenic
2 5 activity.
A protein of the present invention may also be useful for gut protection or regeneration
and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions
resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting
3 0 differentiation of tissues described above from precursor tissues or cells; or for inhibiting the
growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assays for tissue generation activity include, without limitation, those described in:
International Patent Publication No. W095/16035 (bone. cartilage, tendon); International
CA 022 ~ 99 ~ 7 1999 - 01 - 08
WO 98/01552 PCT/US97111842
Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No.
WO91 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healin~ pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest.
Dermatol 71:382-84 (19783.
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related
1 0 activities. Inhibins are char~t~.ri7~d by their ability to inhibit the release of follicle ctimnl~ting
horinone (FSH), while activins and are characterized by their ability to stimulate the release
of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in
heterodimers with a member of the inhibin oc family, may be useful as a contraceptive based
on the ability of inhibins to decrease fertility in female m~mm~lc and deerease spermatogenesis
1 5 in male m~mm~l~. Adminictration of sufficient amounts of other inhibins can induce infertility
in these m~mm:~lc. 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
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
2 0 protein of the invention may also be useful for advancement of the onset of fertility in sexual1y
imm~tllre m lmm~lc, so as to increase the lifetime reproductive performance of domestic
animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
2 5 Assays for activin/inhibin activity include, without limitation, those described in: Vale
et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al.,
Nature 321 :776-77g, 1986; Mason et al., Nature 318:659-663,1985; Forage et al., Proc. Natl.
Acad. Sci. USA 83:3091-3095, 1986.
3 0 Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g.,
act as a chemokine) for m~mm~ n cells, including, for example, monocytes, fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic
and chemokinetic proteins can be used to mobilize or attract a desired cell population to a
3 5 desired site of aetion. Chemotaetic or chemo'Kinetic proteins provide particular advantages in
28
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WO 98/01552 PCT/US97/11842
treatment 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
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
5 slim~ n directly or indirectly, the directed orientation or movement of such cell population.
Preferably, the protein or peptide has the ability to directly stimulate directed movement of
cells. Whether a particular protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any known assay for cell
chemotaxis.
1 0 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,
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines
6.12.1-6.12.28; Taub et al. J. Clin. Invest. g5:1370-1376, 1995; Lind et al. APMIS
103:140-146, 1995;MulleretalEur.J.lmmunol.25: 1744-1748;Gruberetal.J.ofImmunol.
152:5860-5867, 1994; Johnston et al. l. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As
2 5 a result, such a protein is expected to be useful in treatment of various coagulation disorders
(including hereditary disorders, such as hemophilias) or to enhance coagulation and other
hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein
of the invention may also be useful for dissolving or inhibiting formation of thromboses and
for llcallllGllt and prevention of conditions resulting therefrom (such as, for example, infarction
3 0 of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis
29
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WO 98/01552 PCTIUS97111842
Res. 45:413-4]9, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins
35:467-474, 1988.
Receptor/Li~and Activity
A protein of the present invention may also demonstrate activity as l~C~ OI~, receptor
ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors
and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases
and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell
interactions and their ligands (including without limitation, cellular adhesion molecules (such
as selectins, integrins and their ligands) and receptorlligand pairs involved in antigen
presentation, antigen recognition and development of cellular and humoral immune responses).
Receptors and ligands are also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein of the present invention
(in~lu-ling, without limitation, fragments of receptors and ligands) may themselves be useful
1 5 as inhibitors of receptorlligand interactions.
The activity of a protein of the invention may, among other means, be measured by the
- following methods:
Suitable assays for receptor-ligand activity include without limitation those described
in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and 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 84:6864-6X68, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et
al., J. lmmunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1g95.
Anti-lnfl~mm~tnry Activity
Proteins of the present invention may also exhibit anti-infl~mm It~ ry activity. The anti-
infl~mm~tory activity may be achieved by providing a stimulus to cells involved in the
infl~mm~t~ry response, by inhibiting or promoting cell-cell interactions (such as, for example,
3 0 cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the infl~mm~t~ ry
process, inhibiting or promoting cell extravasation, or by stimulating or ~u~ hlg production
of other factors which more directly inhibit or promote an inflammatory response. Proteins
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 inflal.l"latu-y response syndrome (SIRS)), ischemia-
CA 022~99~7 1999 - 01 - 08
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reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis, cytokine or chemokine-induced lung injury, infl~rnm~tnry bowel disease, Crohn's
disease or resulting from over production of cytokines such as TNF or IL-I . Proteins of the
invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
5 or material.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention
of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may
10 inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may
exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by
inhibiting formation of tissues necessary to support tumor growth (such as, for example, by
inhibiting angiogenesis), by causing production of other factors, agents or cell types which
inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types
15 which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional
2 0 activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents,
inclu-ling, without limitation, bacteria, viruses, fungi and other parasites; effecting (supL,lc~s~ g
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);
2 5 effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female
subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or
elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other
nutritional factors or component(s); effecting behavioral characteristics, including, without
limitation, appetite, libido, stress, cognition (including cognitive disorders), depression
3 0 (including depressive disorders) and violent behaviors; providing analgesic effects or other
pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages
other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes,
correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity
3 5 (such as, for example, the ability to bind antigens or complement); and the ability to act as an
CA 022~99~7 1 999 - 01 - 08
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antigen in a vaccine composition to raise an immune response against such protein or another
material or entity which is cross-reactive with such protein.
5 ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including without
limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical
composition when combined with a pha,.,lac~ulically acceptable carrier. Such a composition
may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers,
10 solubili~ers, and other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The characteristics of the carrier will depend on
the route of ~l~f~ alion. The ph~.~ fculic~l composition of the invention may also contain
cytokines, Iymphokines, or other he".alol)oietic factors such as M-CSF, GM-CSF, TNF, IL-I,
IL-2, IL-3, IL~, IL-5, IL-6, L-7, IL-8, L-9, IL-10, IL-11, IL-12, L-13, IL-14, IL-IS, IFN,
TNF0, TNFI, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
The pharmaceutical composition may further contain other agents which either enhance the
activity of the protein or compliment its activity or use in treatment. Such additional factors
and/or agents may be inclu(ied in the pharmaceutical composition to produce a synergistic
2 0 effect with protein of the invention, or to minimi7e side effects. Conversely, protein of the
present invention may be included in formulations of the particular cytokine, Iymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to
minimi7e side effects of the cytokine, Iymphokine, other hematopoietic factor, thrombolytic
or anti-thrombotic factor, or anti-inflal----.aloly agent.
2 5 A protein of the present invention may be active in multimers (e.g., heterodimers or
homodimers) or complexes with itself or other proteins. As a result, pharmaceutical
compositions of the invention may comprise a protein of the invention in such multimeric or
complexed form.
The pharrn~e--~ic~l composition of the invention may be in the form of a complex of
3 0 the protein(s) of present invention along with protein or peptide antigens. The protein and/or
peptide antigen will deliver a stimulatory signal to both B and T Iymphocytes. B Iymphocytes
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
3 5 class II MHC genes on host cells will serve to present the peptide antigen(s) to T Iymphocytes.
CA 022 ~ 99 ~ 7 1999 - 01 - 08
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The antigen components could also be supplied as purified MHC-peptide complexes alone or
with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able
to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to
bind the TCR and other molecules on T cells can be combined with the pharmaceutical
5 composition of the invention.
The pharm~ceutic~l composition of the invention may be in the form of a liposome in
which protein of the present invention is combined, in addition to other pharmaceutically
acceptable carriers, with a~ l)ipdlhic agents such as lipids which exist in aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable
10 lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides,
sulfatides, Iysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such
liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S.
Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent
No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "therlpeutic~lly effective amount" means the total amount of
each active component of the pharm~.euti~l composition or method that is sufficient to show
a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant
medical condition, or an increase in rate of treatment, healing, prevention or amelioration of
such conditions. When applied to an individual active ingredient, administered alone, the term
2 0 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 ~-lminic~red in
combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically
effective amount of protein of the present invention is administered to a m:~mm~l having a
2 5 condition to be treated. Protein of the present invention may be ~-lminictered in accordance
with the method of the invention either alone or in combination with other therapies such as
tre~-mf~ntc employing cytokines, Iymphokines or other hematopoietic factors. When co-
:lrlminict~.red with one or more cytokines, Iymphokines or other hematopoietic factors, protein
of the present invention may be administered either simultaneously with the cytokine(s),
3 0 Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If administered sequentially, the attending physician will decide on the
appropriate sequence of administering protein of the present invention in combination with
cytokine(s), Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
CA 0 2 2 ~ 9 9 ~ 7 1 9 9 9 - 0 l - 0 8
WO 98101552 PCT/US97/11842
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried out in a variety
of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, p~ e,dl or intravenous injection. Intravenous administration
to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a tablet, capsule,
powder, solution or elixir. When administered in tablet form, the pharmaceutical composition
of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and
preferably from about 25 to gO% 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,
mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline solution~ dextrose or
other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene
glycol. When administered in liquid form, the pharmaceutical composition contains from
about 0.5 to 90% by weight of protein of the present invention, and preferably from about l
to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is
~mini~t~red by intravenous, cutaneous or subcutaneous injection, protein of the present
invention will be in the form of a pyrogen-free, pa~ lly acceptable aqueous solution. The
pl~ar~l~ion of such parenterally acceptable protein solutions, having due regard to pH,
isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical
composition for intravenous, cutaneous, or ~ubcu~ Pous injection should contain, in addition
2 5 to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, T.~rt~.o.d
Ringer's lnjection, or other vehicle as known in the art. The pharmaceutical composition of
the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art.
3 0 The amount of protein of the present invention in the pharmaceutical composition of
the present invention will depend upon the nature and severity of the condition being treated,
and on the nature of prior treatments which the patient has undergone. Ultimately, the
attending physician will decide the amount of protein of the present invention with which to
treat each individual patient. Initially, the attending physician will administer low doses of
3 5 protein of the present invention and observe the patient's response. Larger doses of protein of
34
CA 022~99~7 1999-01-08
W O 98/01552 PCTAJS97111842
the present invention may be ~fiminist~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
various pharrnaceutical compositions used to practice the method of the present invention
should contain about 0.01 llg to about 100 mg (preferably about 0.1 ~g to about 10 mg, more
5 preferably about 0.1 ~lg to about I mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the
present invention will vary, depending on the severity of the disease being treated and the
condition and potential idiosyncratic response of each individual patient. lt is contemplated
that the duration of each application of the protein of the present invention will be in the range
10 of 12 to 24 hours of continuous intravenous ~lministration. 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
15 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
20 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
2 5 protein may be useful in detecting and preventing the met~ct~tic spread of the cancerous cells,
which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon
or ligament regeneration, the therapeutic method includes administering the composition
topically, systematically, or locally as an implant or device. When :l-lminis~ered, the
3 0 therapeutic composition for use in this invention is, of course, in a pyrogen-free,
physiologically acceptable form. Further, the composition may desirably be encapsulated or
injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical
administration may be suitable for wound healing and tissue repair. Therapeutically useful
agents other than a protein of the invention which may also optionally be included in the
3 5 composition as described above, may alternatively or additionally, be administered
CA 022~99~7 l999-0l-08
WO 98/01552 PCT/US97/11842
simultaneously or sequentially with the composition in the methods of the invention.
Preferably for bone andlor cartilage formation, the composition would include a matrix capable
of delivering the protein-containing composition to the site of bone and/or cartilage damage,
providing a structure for the developing bone and cartilage and optimally capable of being
5 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,
mechanical properties, cosmetic appearance and interface properties. The particular
application of the compositions will define the appropriate formulation. Potential matrices for
10 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
dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix
culllpo~ ts~ Other potential matrices are nonbiodegradable and chemically defined, such as
15 sintered hydroxapatite, bioglass, alumin~s, or other ceramics. Matrices may be comprised
of col..bh,dlions of any of the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in
composition, such as in calcium-~lllmin:ltP-phosphate and processing to alter pore size, particle
size, particle shape, and biodegradability.
2 0 Presently preferred is a 50:50 ~mole weight) copolymer of lactic acid and glycolic acid
in the form of porous particles having diameters ranging from 150 to 800 microns. In some
applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose
or autologous blood clot, to prevent the protein compositions from disassociating from the
matrix.
2 5 A preferred family of sequestpring agents is cellulosic m~eri~c such as alkylcelluloses
~including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and
carboxymethylcellulose, the most l"~r~"~d being cationic salts of carboxymethylcellulose
~CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate,
3 0 poly~ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-}0 wt% based on
total formulation weight, which represents the amount necessary to prevent desorbtion of the
protein from the polymer matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from infiltrating the matrix, thereby
3 5 providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
36
CA 022~99~7 1 999 - 01 - 08
WO 98/01552 PCT/US97/11842
In further compositions, proteins of the invention may be combined with other agents
beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
These agents include various growth factors such as epidermal growth factor (EGF), platelet
derived growth factor (PDGF), transforming growth factors (TGF-cc and TGF-~), and insulin-
like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications.
Particularly domestic animals and thoroughbred horses, in addition to humans, are desired
patients for such treatment with proteins of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be usedin 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
tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of
administration and other clinical factors. The dosage may vary with the type of matrix used
in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
For example, the addition of other known growth factors, such as IGF I (insulin like growth
factor I), to the final composition, may also effect the dosage. Progress can be monitored by
periodic assessment of tissue/bone growth and/or repair, for example, X-rays,
histomorphometric determinations and tetracycline labeling.
2 0 Polynucleotides of the present invention can also be used for gene therapy. Such
polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a
m Imm~ n subject. Polynucleotides of the invention may also be :~flmini~rto.red by other
known methods for introduction of nucleic acid into a cell or organism (including, without
limitation, in the form of viral vectors or naked DNA).
2 5 Cells may also be cultured ex vivo in the presence of proteins of the present invention
in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells
can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully
3 0 set forth.
CA 022~99~7 l999-0l-08
WO 98tO1552 PCT/US97/11842
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John
LaVallie, Edward
Racie, Lisa
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Bowman, Michael
~ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CarnbridgePark Drive
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: U.S.A.
(F) ZIP: 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 #1.0, 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:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 339 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
38
CA 022~99~7 1999-01-08
W O 98/01552 PCTAUS97/11842
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GCGGCCGCAG GTCTAGAATT CAATCGGGAG AGAGATACTG CCTGGTTCTT ACAGACACAG 60
ATTATGTCAT CCTTGCAGCC TTCACCCAAA GTTGCTCCCT CCTTCTAGGG CATTTTGTTT 120
TCCTACTTAA TACCAAGTGT CAGCATGTTA GTAATAAACA GGTGTCTCTA CCATTAGTCA 180
AAGGTGGGAG TTAAGCCTTT CATCTTTGTA GCTTTCTCCA GTACCTAACC ATGATTTACT 240
TCATGGGAAG TCCCTCAAAG TACTATTAAT TATCCTGTGT TCTCCTGCCT TGCCTCTTAA 300
CAAAAATTCT GCTGTTCCTG ATTATTTCCA TTTTACCAG 339
(2~ INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 552 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AAATANAAAT ANAACAAATT NTAGGGAAGG ACTAAACTGT CTAAAGAAAT GTAAAATCCA 60
AAGACTTGGA TTTTCAACCT ATATCAGAAG ACA~ TTCAGTTCCC ATGTGAAATT 120
CTTTNTAGGC CAAGGAAGGA CAAATACAAA TTTTGATTAC AAATTATTTT TAGAACTTTG 180
ACACCTACAC TTAAATTCTG AGTCATTAAA CAGGCCTACA TTTATCAACT GTGGAAATAT 240
CAGCCAGTTT TTGCAAACCT CTTCTTAGGA CACTAAGTTG TTTGCAGAAA TCACTAGCAT 300
TGACTGACTC AGCAACAATG TGGTTATATT CTTTGATTAA CTTAGTCCTT TTTCTTGGTC 360
AAGAGTCAGT AGACAGGACT GAAGCTTATG CCCCTTGCCC CCCCACCACC ACTCCATTAC 420
TACCACCTTG GTTTAGCCAT CCTTTTCTTG ATCTGTTCTC CCCACTTCTA CTGTGCTACT 480
CTACAGACTT GCCCTGAATG TAAGAGCAAC AATTACCTTG TAAAGTCCAA GTTGGGGCAG 540
GTCACTCCCA AA 5S2
(2) INFORMATION FOR SEQ ID NO:3:
39
CA 022~99~7 1999-01-08
WO 98/01552 PCTNS97/11842
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B~ TYPE: amino acid
(C) STRANDEDNESS:
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Thr Gly Leu His Leu Ser Thr Val Glu Ile Ser Ala Ser Phe Cys Lys
l 5 l0 15
Pro Leu Leu Arg Thr Leu Ser Cys Leu Gln Lys Ser Leu Ala Leu Thr
Asp Ser Ala Thr Met Trp Leu Tyr Ser Leu Ile Asn Leu Val Leu
35 40 45
(2) INFORMATION FOR SEQ ID No:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 308 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
~ llNGGA ATCACCAAAA TCAAGNGNGA TATTGTGTTT GCTGCCAGCC TNNANTTGTA 60
GAGTCAGCTA AAGGAATGTG NGATTTTAAA TTATTGACCA CCTGTTTGAT TACAGTTGAN l20
NACAAATGCC TGCAAGTGTG GATTTGGTTT TCCCANACAT TTTAATATGT ATTATATTTA 180
AATCAAACAT CATTCATAGA AAGCATATNA CANANATGTT TANACATAAG CATNACATTT 240
TTTTAATAAA AATGTANACA GGTGGGGCAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 300
AAAAAAAA 308
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1077 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
CA 022~99~7 1999-01-08
W O98/01552 PCTAUS97/11842
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CATTTGATCT TTCCACNACT ATCCAACTCA CAACGTGGCA NTGGGGTTGT GCCGCCTTTG 60
CAGGTGTATA TNANACACGT GGTTTTGGCC GCAGAGGCAC CNGTCGNCAG GTGGGGGGTT l20
CCGCTGCCTG CAAAGGGTCG CTACAGACGT lG~ l~lNTT CAAGAAGCTT CCAGAGGAAN l80
TGCTTCCTTC ACGACATTCA ACAGACCTTG CATTCCTTTG GCGAGAGGGG AAAGACCCCT 240
AGGNATGNTT GTCAAGAAGA CAGGGNCAGG TTTTCCGGCC CTTNACATTG CCAAAAGACG 300
GCAATATGGT GGNAAATAAC ATATAGACAA ACGCACACCG GCCTTATTCC AAGCGGNTTC 360
GGCCAGTAAC GTTAGAATTG CGGCCGCAGG TCTAGAATTC AGTCTTTTCA AGAGAACAAT 420
AAAATAGGCA GTCTCCTACC TCTTGTCTTA CTCTAATATA AACTCCATGA AGATAAGTAT 480
TGTATCCATA CTGTTCATGC TGCACAGCAG TTGCCCTTAT CTGCAGGGCG ACGCATCCCA 540
AGACCCCCAG TGGATGCTTG AAACTGCAGA ANANTNACAC ACGTGATTGC CACCATCGGA 600
ACACATTTCT GTTCACGTCT TCCACCCACA GATTTAATGC CTTTTCCATC TTAACTAAGC 660
ACTCATCATG GACTGTGGCC ATAACTTTTG CAGTTTTAGA TGCAACAGCA AAACTAACAT 720
TAATTTTTTC TTCTTCTTCA CAATTTCATG GGTAGATTTG TTCTTACCGT AGATCTTAKC 780
AACCTCAGCA TATGATGTTT lllCll~ GA GAACTTTCAC CTTTTNTCTT AAAGAAAGCA 840
CTTTACAGCT TNTCTTTGGC ATATCTCAAC TGCCAGCATC ACTGNTCTTG AACTTTGGGG 900
CCATTATTAA GTCAGNAAAG GGTTANTTCA AACNAGCTTT TGTANTCCCA CNTACTTGGG 960
AGGNTAAGGC AGGAGAATGG NATGAACCCG AGAGGCAGAG GTTGCAGTGA GCTGAGATCG 1020
TGCCACTGCA CTCCAGCGTG GGTGACAGAG AGATTNTGTC TAAAAAAAAA AAAAAAA l077
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
41
CA 022~99~7 l999-0l-08
WO 98/01552 PCT/US97111842
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Leu Tyr Pro Tyr Cys Ser Cys Cys Thr Ala Val Ala Leu Ile Cys
1 5 10 15
Arg Ala Thr His Pro Lys Thr Pro Ser Gly Cys Leu Lys Leu Gln Xaa
Xaa Xaa Thr Arg Asp Cys His His Arg Asn Thr Phe Leu Phe Thr Ser
Ser Thr His Arg Phe Asn Ala Phe Ser Ile Leu Thr Lys His Ser Ser
Trp Thr Val Ala Ile Thr Phe Ala Val Leu Asp Ala Thr Ala Lys Leu
65 70 75 80
Thr Leu Ile Phe Ser Ser Ser Ser Gln Phe His Gly
t2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 553 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AGGGCCAGGT TTTCCGGGCC NTCACATTGC CAAAAGACGG CAATATGGTG GGAAATAACA 60
TATAGACAAA CGCACACCGG CCTTATTCCA AGCGGNTTCG GCCAGTAACG TTAGAATTGC 120
GGCCGCAGGT YTAGGTCAGA GCCAAAGGAA AGCTTGAAAA ATGAAGACAT TAGCAGGACT 180
TGTTCTGGGA CTTGTCATCT TTGATGCTGC TGTGACTNCC CCAACTCTAG AGTCCATCAA 240
CTATGACTCA GAAACCTATG ATGCCACCTT AGAAGACCTG GATAATTTGT ACAACTATGA 300
AAACATACCT GTTGATAAAG TTGAGATTGA AATAGCCACA GTGATGCCTT CAGGGAACAG 360
AGAGCTCCTC ACTCCACCCC CACAGCCTGA GAAGGCCCAG GAAGAGGAAG AGGAGGAGGA 420
ATCTACTCCC AGGCTGATTG ATGGCTCTTC TCCCCAGGAG CCTGAATTCA CAGGGGTTCT 480
GGGGCCACAC ACAAATGAAG ACTTTCCAAC CTGTCTTTTG TGTACTTGTA TAAGTACCAC 540
42
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W O 98/01552 PCTtUS97tllX42
CGTGTACTGT GAT 553
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 131 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Lys Thr Leu Ala Gly Leu Val Leu Gly Leu Val Ile Phe Asp Ala
1 5 10 15
Ala Val Thr Xaa Pro Thr Leu Glu Ser Ile Asn Tyr Asp Ser Glu Thr
Tyr Asp Ala Thr Leu Glu Asp Leu Asp Asn Leu Tyr Asn Tyr Glu Asn
Ile Pro Val Asp Lys Val Glu Ile Glu Ile Ala Thr Val Met Pro Ser
Gly Asn Arg Glu Leu Leu Thr Pro Pro Pro Gln Pro Glu Lys Ala Gln
Glu Glu Glu Glu Glu Glu Glu Ser Thr Pro Arg Leu Ile Asp Gly Ser
Ser Pro Gln Glu Pro Glu Phe Thr Gly Val Leu Gly Pro His Thr Asn
100 105 110
Glu Asp Phe Pro Thr Cys Leu Leu Cys Thr Cys Ile Ser Thr Thr Val
115 120 125
Tyr Cys Asp
130
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
43
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TTATAATATA ATGAAAATGT AGTAATTTAA GAAAACACCA GATGAGTTAG GAATAAACCT 60
ATAACATTTA CAAAAAGAGC AAAATTAAGT GATAGAAAAT ATTTCACACA TGTTCTTATA l20
GATCATGTAT CACTTGCAAG TTTTNGGAGT TCATATCCTA TATCATTTCA AATTAAGNAC l80
ATAATAAAGT AAAATTTTGA AATGAACACT TTAGGTATTT TTGCCAAGAT TTAGATGTTT 240
TTAATTAAAC ~ Cll~C ~"l"l"l"l"l"l"l"l"l' CACTAAAGCA TGTTTATTCC CCTAATCCAT 300
TAAAGAGCAT GAAAAAAAGA ATAAATGTAT TTGAAAATTA AAAAAAAAAA AAAAAAAA 358
(2) INFORMATION FOR SEQ ID NO:lO:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 693 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
GCGGCCGCTC TANAACTAGT GGATCCCCCG GGCTGCAGGA ATTCGGCCAA AGAGGCCTAG 60
AATGAAAATC CAGGTGTTTG TCATTCATCA GCAACAGGTG ATCCCCATTG CAGGCAGCCG l20
GAACCGACGT CTCCTGGACC ACTGAGCTGG CTGTTCTCAT TACTGCCCTT TCCGCCCAGG l80
CTGGCGGTGA CTCACCGTGA GACAAGTCAG CTAGGTGTTC AGGACAGGGA TTTCAGAGTA 240
~lL~ GlCCA AAGAGGAAAG GGATGATTTC TACGGATCAC TACCAGTTGG TTTACTGTTA 300
GCTCATCGTG TTGATCACAC CAAGTCTTGC CAATTTGGTT TTCTAAGTAT TTTCACGCCT 360
TCTCCTCGTG TCCGCGTCAC TGCTCTGATT CAGGCCCTTG TCATTTCTCA TCTTTGCCAT 420
TTTAGTAGTT TTTGGATTGG GCTCCCGGCT GCTAATTTTG TCCCCTTTTC CACTATCTTC 480
CACATTGTCA CCGCAGTCAT GTTTCTAAGG CAGAATCTCA CTGTGCCCCW CATCGTGTTG 540
CTGGGCCCTT GCATGCCGTA CCCTGGCCTT TGTGAAATGC CCTTCATCTG TGCTCTTCCC 600
TCCACCTGGA ATGTCCGTCT CT~ GCCAACCCAC NCGACCCCTC CCTCCTNCAA 660
GCCCGTGAGT GTCCCCNCCC TCCATGTCCT GTG 693
(2) INFORMATION FOR SEQ ID NO:ll:
44
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WO 98/01552 PCT/US97/11842
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Asn Glu Asn Pro Gly Val Cys His Ser Ser Ala Thr Gly Asp Pro His
1 5 10 15
Cys Arg Gln Pro Glu Pro Thr Ser Pro Gly Pro Leu Ser Trp Leu Phe
Ser Leu Leu Pro Phe Pro Pro Arg Leu Ala Val Thr His Arg Glu Thr
Ser Gln Leu Gly Val Gln Asp Ar~ Asp Phe Arg Val Phe Leu Ser Lys
Glu Glu Arg Asp Asp Phe Tyr Gly Ser Leu Pro Val Gly Leu Leu Leu
Ala His Arg Val Asp His Thr Lys Ser Cys Gln Phe Gly Phe Leu Ser
Ile Phe Thr Pro Ser Pro Arg Val Arg Val Thr Ala Leu Ile Gln Ala
100 105 110
Leu Val Ile Ser His Leu Cys His Phe Ser Ser Phe Trp Ile Gly Leu
115 120 125
Pro Ala
130
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 327 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
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TTCGTGGGGC CTGCCCGTTC NGATNTNGNT GCNGCCTCGC TGTCCCCCTC CTCCCTCCNG 60
TGGTTNATAT TCCAGGAATT CTGAATTAGT TGCACCGTGN TCTNATATTT ACTGCAAGAA l20
TAGACCAGTG GTTCTCCAGC TTTTCTGCAC TCTGGAATCA CCTGGGGGTC TTTAAAAAAC 180
ACTGCCTGGC TCCTAGTCCT AAATTTGGAG ATTTAACTGG ACTTACAGTT TTTCAAAGCA 240
CCCCAAAAGA TTNTAATGTG CAGCAAAGTT TGGGAACCAC TGGTATAGAC TGTCTTCTGC 300
TTGTTTTCTT GAAAAACACA AACACAA 327
(2) INFORMATION FOR SEQ ID NO:l3:
(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:13:
GNTAAACCAA GGTGGTAGTA ATGGAGTGG 29
(2) INFORMATION FOR SEQ ID NO:14:
(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:14:
GNTCTTGGGA TGCGTCGCCC TGCAGATAA 29
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTTCS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
46
.... , . . .. , ~ . ...
CA 02259957 1999-01-08
W O 98/01552 PCTrUS97/11842
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GNAGGCATCA CTGTGGCTAT TTCAATCTC 29
(2) INFORMATION FOR SEQ ID NO:16:
(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:16:
TNCCTTAGAA ACATGACTGC GGTGACAAT 29
47
