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 c~ Lillualion-in-part of application Ser. No. 08/686,878, filed July
26, 1996, entitled "SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING
- THEM", filed in the name of some or all of the inventors of the present application.
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 lymphokines, 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 they rely on information directly related to the
discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of
2 0 hybridization cloning; activity of the protein in the case of expression cloning). More recent
"indirect" cloning techniques such as signal sequt~nce 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
2 5 are known to have biological activity by virtue of their secreted nature in the case of leader
sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based ~Pc'~niques
It is to these proteins and the polynucleotides encoding them that the present invention is
directed.
3 o SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide col~p,i~,h~g the nucleotide sequence of SEQ ID
NO:2;
3 5 (b) a polynucleotirl.o coTnrricing the nucleotide sequen~e of SEQ ID NO:2
from nucleotide l to nucleotide 591;
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(c) a polynucleotide cu.,.p.i~i"g the nucleotide sequence of the full length
protein coding sequen~e of clone BL89_13 deposited under accession number ATCC
98153;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone BL89_13 deposited under accession number ATCC 98153;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BL89_13 deposited under accession number ATCC
98153;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone BL89_13 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:3;
(h) 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)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:22 0 from nucleotide ] to nucleotide 591; the nucleotide sequence of the full length protein coding
sequence of clone BL89_13 deposited under accession number ATCC 98153; or the nucleotide
sequence of the mature protein coding sequence of clone BL89_13 deposited under accession
number ATCC 98153. In other preferred embodiments, the polynucleotide encodes the full
length or mature protein encoded by the cDNA insert of elone BL89_13 deposited under
2 5 accession number ATCC 98153. In yet other preferred embodiments, the present invention
provides a polynucleotide ~n~o(ling a protein comprising the amino acid sequence of SEQ ID
NO:3 from amino aeid 80 to amino aeid 105.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1, SEQ ID NO:2 or SEQ ID NO:4.
3 0 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:3;
(b) the amino acid sequence of SEQ ID NO:3 from amino acid 80 to
3 5 amino acid 105;
... . . .
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(c) fragments of the amino acid sequence of SEQ ID NO:3; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BL89_13 deposited under accession number ATCC 98153;
the protein being ~ub~lantially free from other m~mm~ n proteins. Preferably such protein
co.ll~lises the amino acid sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID
NO:3 from amino acid 80 to amino acid 105.
In alternate emborlim~.ntc, isolate BL89_10 deposited under accession number ATCC
98115 may be substituted for BL89_13 in any of the foregoing.
In one embodiment, the present invention provides a composition colll~ hlg an
1 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:S;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:S
from nucleotide I to nucleotide 390;
1 5 ~c) a polynucleotide comprising the nucleotide sequence of the full length
protein coding seqnrnre of clone BV239_3 deposited under accession number ATCC
98153;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone BV239_3 deposited under accession number ATCC 98153;
2 0 (e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BV239_3 deposited under accession number ATCC
98153;
(f) a polynnrleoti(le encoding the mature protein encoded by the cDNA
insert of clone BV239_3 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding a protein comprising t'ne amino acid
sequence of SEQ ID NO:6;
(h) a polynucleotide encoding a protein cu~ hlg a fragment of the
amino acid sequence of SEQ ID NO:6 having biological activity;
(i) a polynl~r!P-otide which is an allelic variant of a polynucleotide of (a)-
3 0 (d) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
Preferably, such polynucleotide colllylises the nucleotide sequence of SEQ ID NO:5
from nllrleotillto I to mlr!~otide 390; the nucleotide sequence of the full length protein coding
35 sequence of clone BV239_3 deposited under accession number ATCC 98153; or the
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nucleotide sequence of the mature protein coding sequence of clone BV239_3 deposited under
accession number ATCC 98153. In other preferred embodiments, the polynucleotide encodes
the full length or mature protein encoded by the cDNA insert of clone BV239_3 deposited
under accession number ATCC 98153. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising the amino acid sequence
of SEQ ID NO:6 from amino acid 50 to amino acid 130.
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:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 50 to
amino acid 130;
(c) fragments of the amino acid sequence of SEQ ID NO:6: and
(d) the amino acid sequence encoded by the cDNA insert of clone
BV239_3 deposited under accession number ATCC 98153;
the protein being substantially free from other m~rnm~ n proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID
NO:6 from amino acid 50 to amino acid 130.
In alternate embo-lim~n~c isolate BV239_2 deposited under accession number ATCC
98115 may be substituted for BV239_3 in any of the foregoing.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide ColllpliSil-g the nucleotide sequence of SEQ ID
NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7
from nucleotide 2 to nucleotide 415;
(c) a polynll~leotide COlllplisillg the nucleotide sequence of the full length
3 0 protein coding sequence of clone CC25_17 deposited under accession number ATCC
98153;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone CC25_17 deposited under accession number ATCC 98153;
,~. . . .
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(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CC25_17 deposited under accession number ATCC
98153;
(f) a polynucleotide encoding the mature protein encoded by the cDNA
insert of clone CC25_17 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:8;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:8 having biological activity;
1 0 (i) a polynucleotide which is an allelic variant of a polynl~clçoti~ of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7from nucleotide 2 to nucleotide 415; the nucleotide sequence of the full length protein coding
sequence of clone CC25_17 deposited under accession number ATCC 98153; or the
mlcleotide sequence of the mature protein coding seq~llonce of clone CC25_17 deposited under
accession number ATCC 98153. In other preferred embo-lim.-ntc, the polynucleotide encodes
the full length or mature protein encoded by the cDNA insert of clone CC25_17 deposited
under accession number ATCC 98153. In yet other preferred embo-iiment~, the present
invention provides a polynucleotide encoding a protein comprising the amino acid sequence
of SEQ ID NO:8 from amino acid 56 to amino acid 138.
Other embodiments provide the gene corresponding to the cDNA sequ~nce of SEQ
ID NO:7.
2 5 In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino acid 56 to
3 0 amino acid 138;
(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
CC25_17 deposited under accession number ATCC 98153;
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the protein being substantially free from other m~mm~ n proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO:8 or the amino acid sèquence of SEQ ID
NO:8 from amino acid 56 to amino acid 138.
In alternate embo-iimf~tc, isolate CC25_16 deposited under accession number ATCC98115 may be substituted for CC25_17 in any of the foregoing.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide seleeted from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:lO;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 10 from nucleotide 242 to nucleotide 322;
~c) a polynucleotide c(~ isillg the nucleotide sequence of the full length
protein coding sequence of clone CC397_19 deposited under accession number ATCC
98153;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone CC397_19 deposited under accession number ATCC 98153;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of elone CC397_19 deposited under accession number ATCC
98153;
2 0 (f) a polynucleotide encoding the mature protein eneoded by the cDNA
insert of clone CC397_19 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO: 11;
(h) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID NO: 11 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynueleotide of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above .
3 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:I0
from nucleotide 242 to nucleotide 322; the nucleotide sequence of the full length protein
coding sçquence of clone CC397_19 deposited under accession number ATCC 98153; or the
nucleotide sequence of the mature protein coding sequence of clone CC397_19 deposited
under aceession number ATCC 98153. In other preferred embo-limP,n~c, the polynucleotide
... . . .
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encodes the full length or mature protein encoded by the cDNA insert of clone CC397_19
deposited under accession number ATCC 98153.
Other emborlim~rltc provide the gene corresponding to the cDNA sequence of SEQ
ID NO:10, SEQ ID NO:9 or SEQ ID NO:12 .
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a, the amino acid sequence of SEQ ID NO: 11;
(b) fragments of the amino acid sçqu~.nce of SEQ ID NO: 11; and
(c) the amino acid sequence encoded by the cDNA insert of clone
CC397_19 deposited under accession number ATCC 98153;
the protein being substantially free from other m~mm~ n proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO: 11.
In alternate embodiments, isolate CC397_11 deposited under accession number ATCC1 5 98115 may be substituted for CC397_19 in any of the foregoing.
In certain plcr~lled emborlimen~c, the polynucleotide is operably linked to an
expression control sçquen~e The invention also provides a host cell, including t!~ r~ yeast,
insect and m~mm~ n cells, transformed with such polynucleotide compositions.
Processes are also provided for producing a protein, which comprise:
2 0 (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture me-lium; 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
2 5 mature form of the protein.
Protein c~,."po~ilions of the present invention may further cc""~.. ise a pha" . ,:~e~l ~ic~'ly
acc~l~le carrier. Compositions comprising an antibody which specif1cally reacts with such
protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition
3 0 which comprises ~rlmini~t~ring to a m~mm~ m subject a therapeutically effective amount of
a cu",~o~ilion compri~ing a protein of the present invention and a pl.~ rlllic~lly acc~pl~lc
carrier.
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DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences are reported below for each clone and protein
disclosed in the present application. In some inct~nc~.~ the sequences are preliminary and may
5 include some incorrect or ambiguous bases or amino acids. The actual nucleotide sequence
of each clone can readily be determined by sequencing of the deposited clone in accordance
with known methods. The predicted amino acid sequence (both full length and mature) can
then be determined from such nucleotide sequence. The amino acid sequence of the protein
encoded by a particular clone can also be determined by expression of the clone in a suitable
1 0 host cell, collecting the protein and determining its sequence.
For each disclosed protein applicants have irientified what they have determined to be
the reading frame best identifiable with sequence information available at the time of filing.
Because of the partial ambiguity in reported sequence information, reported protein sequences
include "Xaa" designators. These "Xaa" design~ors indicate either (I ) a residue which cannot
1 5 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
were determined more accurately).
As used herein a "secreted" protein is one which, when expressed in a suitable host
cell, is transported across or through a membrane, including transport as a result of signal
2 0 sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins
secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they
are expressed. "Secreted" proteins also include without limitation proteins which are
transported across the membrane of the endoplpasmic reticulum.
2 5 Clone "BL89 13"
A polynucleotide of the present invention has been identified as clone "BL89_13".
BL89_13 was isolated from a human adult testes cDNA library using methods which are
selective for cDNAs encoding secreted proteins. BL89_13 is a full-length clone, including the
entire coding sequence of a secreted protein (also referred to herein as "BL89_13 protein").
The nucleotide sequence of the 5' portion of BL89_13 as presently determined is
reported in SEQ ID NO:I. An additional internal nucleotide sequence from BL89_13 as
presently determined is reported in SEQ ID NO:2. What applicants believe is the proper
reading frame and the predicted amino acid sequence encoded by such internal sequence is
reported in SEQ ID NO:3. Additional nucleotide sequence from the 3' portion of BL89_13,
3 5 including the polyA tail, is reported in SEQ ID NO:4.
~. , I
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The EcoRVNotI restriction fragment obtainable from the deposit containing clone
BL89_13 should be approximately 3290 bp.
The nucleotide sequence disclosed herein for BL89_13 was searched against the
GenBank ~t~bztce using BLASTA/BLASTX and FASTA search protocols. No hits were
5 found in the d~t:lh~ce
Clone"BV239 3"
A polynucleotide of the present invention has been identified as clone "BV239_3".
BV239_3 was isolated from a human adult brain cDNA library using methods which are
1 0 selective for cDNAs encoding secreted proteins. BV239_3 is a full-length clone, including the
entire coding sequence of a secreted protein (also referred to herein as "BV239_3 protein").
The n~ .oti~f, sequence of BV239_3 as presently determined is reported in SEQ IDNO:5. What applicants plese~ y believe to be the proper reading frame and the predicted
amino acid sequence of the BV239_3 protein corresponding to the foregoing nucleotide
15 sequence is reported in SEQ ID NO:6.
The EcoRVNotI restriction fragment obtainable from the deposit containing clone
BV239_3 should be approximately 300 bp.
The nucleotide sequ~nce, disclosed herein for BV239_3 was searched against the
GenBank d~t~h~e using BLASTA/BLASTX and FASTA search protocols. No hits were
2 0 found in the tl~ h:3~e,
Clone "CC25 17"
A polynucleotide of the present invention has been identified as clone "CC25_17".
CC25_17 was isolated from a human adult brain cDNA library using methods which are
2 5 selective for cDNAs encoding secreted proteins. CC25_17 is a full-length clone, including the
entire coding sequen~e of a secreted protein (also referred to herein as "CC25_17 protein").
The nucleotide sequenre of CC25_17 as presently determined is reported in SEQ IDNO:7. What applicants presently believe to be the proper reading frame and the predicted
amino acid sequence of the CC25_17 protein corresponding to the foregoing nucleotide
3 0 sequence is reported in SEQ ID NO:8.
The EcoRVNotI restriction fragment obtainable from the deposit co,.~ g clone
CC25_17 should be approximately 280 bp.
The nucleotide sequence disclosed herein for CC25_17 was searched against the
GenBank d~h~ce using BLASTA/BLASTX and FASTA search protocols. No hits were
3 5 found in the rl~t:~h~e,
~ . . .. .. . , . ~ . .
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Clone "CC397 19"
A polynucleotide of the present invention has been identified as clone "CC397_19".
CC397_19 was isolated from a human adult brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins. CC397_19 is a full-length clone, including
the entire coding sequence of a secreted protein (also referred to herein as "CC397_19
protein").
The nucleotide sequence of the 5' portion of CC397_19 as presently determined isreported in SEQ ID NO:9. An additional internal nucleotide sequence from CC397_19 as
presently determined is reported in SEQ ID NO:I0. What applicants believe is the proper
1 0 reading frame and the predicted amino acid sequence encoded by such internal sequence is
reported in SEQ ID NO: I l . Additional nucleotide sequence from the 3' portion of CC397_19,
including the polyA tail, is reported in SEQ ID NO:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing cloneCC397_19 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for CC397_19 was searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were
found in the d~s~h:~ce.
Deposit of Clones
Clones BL89_13, BV239_3, CC25_17 and CC397_19 were deposited with the
American Type Culture Collection on August 23, 1996 under accession number 98153, from
which each clone comprising a particular polynucleotide is obtainable. An additional isolate
of each clone (BL89_10, BV239_2, CC25_16 and CC397_11) was deposited on July 25,1996
with the American Type Culture Collection under accession number ATCC 98115.
2 5 Each clone has been transfected into separate bacterial cells (E. coli) in this composite
deposit. Each clone can be removed from the vector in which it was deposited by performing
an EcoRItNotI digestion (5' cite, EcoRI; 3' cite, NotI) to produce the appropriate fragment for
such clone (approximate clone size fragment are identified above). Bacterial cells Cf".~;.i"i"g
a particular clone can be obtained from the composite deposit as follows:
3 0 An oligonucleotide probe or probes should be designed to the sequence that is known
for that particular clone. This sequence can be derived from the sequences provided herein,
or from a ~o...bi,.alion of those sequences. The seq~l~nce 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.
r
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Clone Probe Sequence
BL89_13 SEQ ID NO:13
BV239_3 SEQ ID NO: 14
CC25_17 SEQ ID NO:I5
CC397_19 SEQ ID NO:16
In the sequences listed above which include an N at position 2, that position is occupied in
preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide
(such as, for example, that produced by use of biotin phosphul ~it~ dimethoxytrityloxy-
1 0 2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite)
(Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest
1 5 ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. ~0 ~ C (~csllming 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
Cilmmole) and T4 polynucleotide kinase using commonly employed techniques for labeling
2 0 oligonucleotides. Other labeling techniques can also be used. Unincorporated label should
preferably be removed by gel filtration chromatography or other established methods. The
amount of radioactivity incollJolaled 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.
2 5 The bacterial culture containing the pool of full-length clones should preferably be
thawed and 100 ~1 of the stock used to inoculate a sterile culture flask cont~uning 25 ml of
sterile L-broth COl,~ g ampicillin at 100,ug/ml. The culture should preferably be grown to
saturation at 37~C, and the saturated culture should preferably be diluted in fresh L-broth.
Aliquots of these dilutions should preferably be plated to deterrnine the dilution and volume
3 0 which will yield approximately 5000 distinct and well-separated colonies on solid
bacteriological media cv.~ g L-broth cont~ining ampicillin at 100 llg/ml and agar at 1.5%
in a 150 mm petri dish when grown overnight at 37DC. Other known methods of obtaining
distinct, well-separated colonies can also be employed.
Standard colony hybridization l)~ucedu~es should then be used to transfer the colonies
3 5 to nitrocellulose filters and Iyse, denature and bake them.
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The filter is then preferably incubated at 65~C for 1 hour with gentle agitation in 6X
SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH)
containing 0.5% SDS,10011g/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 dpm/mL. The filter is then preferably incubated at 65~C with
gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5%
SDS at room ~ ure without agitation, preferably followed by 500 mL of 2X SSC/0.1 %
SDS at room t~lllpe'dlule 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
1 0 and subjected to autoradiography for sufficient time to visualize the positives on the X-ray
film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using
standard procedures. The clones can then be verified by restriction analysis, hybridization
analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting
biological activity are also encompassed by the present invention. Fragments of the protein
may be in linear form or they may be cyclized using known methods, for example, as described
2 0 in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al., J.
Amer. Chem. Soc.114, 9245-9253 (1992), both of which are incorporated herein by reference.
Such fragments 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
2 5 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
3 0 proteins. The full-length form of the such proteins is identified in the sequence listing by
translation of the nucleotide seq~-enre 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~rnm~ n cell or other host cell. The sequence of
the mature form of the protein may also be d~lel",i"able from the amino acid sequence of the
3 5 full-length form.
12
,
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The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The cu~ ponding genes can be isolated in accordance with known methods
using the sequence information disclosed herein. Such methods include the p~c?al~lion of
probes or primers from the disclosed sequence information for identification and/or
amplification of genes in appropriate genomic libraries or other sources of genomic materials.
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the
present invention also provides for soluble forms of such protein. In such forms part or all of
the intracellular and transmembrane domains of the protein are deleted such that the protein
is fully secreted from the cell in which it is expressed. The intracellular and transmembrane
1 0 domains of proteins of the invention can be identified in accordance with known techniques
for determination of such domains from sequence information.
Species homologs of the 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
1 5 source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or
p~vlehls, 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 .
2 0 The isolated polynucleotide of the invention may be operably linked to an expression
control ~equence such as the pMT2 or pED expression vectors disclosed in K~llfm~n 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 l~col-~l)inant proteins are also known and are exemplified in R. K~--fm:ln, Methods
in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the
isolated polynucleotide of the invention and an expression control seqll~nre 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 seqnence
A number of types of cells may act as suitable host cells for expression of the protein.
3 0 1~ rnm~ n host cells include, for example, monkey COS cells, Chinese Hamster Ovary
(CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells,3T3
cells, CV-I cells, other Ll~~ .ed 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.
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WO 9~ 1C91 PCT/US97/12884
Alternatively, it may be possible to produce the protein in lower eukaryotes such as
yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or
any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial
5 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,
it may be necessary to modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the functional protein. Such
covalent ~ hments may be accomplished using known chemical or enzymatic methods.The protein may also be produced by operably linking the isolated polynucleotide of
the invention to suitable control sequences in one or more insect expression vectors, and
employing an insect expression system. Materials and methods for baculovirus/insect cell
expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego,
California, U.S.A. (the MaxBac~) kit), and such methods are well known in the art, as
15 described in Summers and Smith, Texas A~ricultural Experiment Station Bulletin No. 1555
(] 987), incorporated herein by reference. As used herein, an insect cell capable of expressing
a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under
culture conditions suitable to express the recombinant protein. The resulting expressed protein
2 0 may then be purified from such culture (i.e., from culture medium or cell extracts) using known
purification processes, such as gel filtration and ion exchange chromatography. The
purification of the protein may also include an affinity column containing agents which will
bind to the protein; one or more column steps over such affinity resins as concanavalin A-
agarose, heparin-toyopearl~) or Cibacrom blue 3GA Sepharose~); one or more steps involving
2 5 hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will
facilitate purification. For example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits
3 0 for expression and purification of such fusion proteins are commercially available from New
England BioLab (Beverly, MA), Pharmacia (Pisc~w~ly, NJ) and InVitrogen, respectively.
The protein can also be tagged with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("Flag") is commercially available from
Kodak (New Haven, CT).
14
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Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl
or other aliphatic groups, can be employed to further purify the protein. Some or all of the
foregoing purification steps. in various combinations, can also be employed to provide a
substantially homogeneous isolated recombinant protein. The protein thus purified is
~ub~ ially free of other m~mm~ n proteins and is defined in accordance with the present
invention as an "isolated protein."
The protein of the invention may also be expressed as a product of l.~llsge~lic animals,
e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are
characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are known
to those skilled in the art. The synthetic:~lly-constructed protein sequences, by virtue of sharing
primary, secondary or tertiary structural and/or conformational characteristics with proteins
may possess biological properties in common therewith, in~ ing protein activity. Thus, they
may be employed as biologically active or immunological substitutes for natural, purified
proteins in screening of therapeutic compounds and in immunological processes for the
development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification are naturally
provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known techniques. Modifications of
interest in the protein seqnen~pc may include the alteration, ~ ion, repl:l~emP.nt insertion
or deletion of a selected amino acid residue in the coding sequ.~nre For example, one or more
2 5 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, replzlrpm~nt~
insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.
4,518,S84). Preferably, such alteration, ~ub~ ulion, repl~ ."r.~l insertion or deletion retains
the desired activity of the protein.
3 0 Other fr~gm~ntc and derivatives of the selluences of proteins which would be expected
to retain protein activity in whole or in part and may thus be useful for screening or other
immunological methodologies may also be easily made by those skilled in the art given the
disclosures herein. Such modifications are believed to be t;"cu.,.p~csed by the present
invention.
~ .. . ......
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USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one
or more of the uses or biological activities (including those associated with assays cited herein)
identified below. Uses or activities described for proteins of the present invention may be
5 provided by ~ mini~tration or use of such proteins or by administration or use of
polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors
suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research
community for various purposes. The polynucleotides can be used to express recombinant
protein for analysis, characterization or therapeutic use; as markers for tissues in which the
corresponding protein is preferentially expressed (either constitutively or at a particular stage
of tissue differentiation or development or in disease states); as molecular weight markers on
15 Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to
map related gene positions; to compare with endogenous DNA seqllences in patients to identify
potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a
probe to "subtract-out" known sequences in the process of discovering other novel
2 0 polynucleotides; for selecting and making oligomers for ~n~hmt~nt to a "gene chip" or other
support, in~ln.1ing for ~x~min:-tion of expression patterns; to raise anti-protein antibodies using
DNA immnni7~tion techniques; and as an antigen to raise anti-DNA antibodies or elicit
another immune response. Where the polynucleotide encodes a protein which binds or
potentially binds to another protein (such as, for example~ in a receptor-ligand interaction), the
2 5 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 enco~iing 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
3 0 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
~.~rel~ ially 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.
3 5 Where the protein binds or potentially binds to another protein (such as, for example, in a
16
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CA 02261~81 1999-01-2~
WO g~ PCT/US97/12884
receptor-ligand interaction), the protein can be used to identify the other protein with which
binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these
binding interactions can also be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade
or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the
art. References disclosing such methods include without limitation "Molecular Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch
and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning
Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional
sources or supplements. Such uses include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a source of
carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the
feed of a particular c ,~ is... or can be a-lmini~t~.red as a separate solid or liquid ~,~t;p~ion,
such as in the form of powder, pills, solutions, suspensions or capsules. In the case of
2 0 microorg~ni~mc, the protein or polynucleotide of the invention can be added to the medium
in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either
2 5 inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein factors
discovered to date, including all known cytokines, have exhibited activity in one or more factor
dependent cell proliferation assays, and hence the assays serve as a convenient confirmation
of cytokine activity. The activity of a protein of the present invention is evidenced by any one
3 0 of a number of routine factor dependent cell proliferation assays for cell lines including,
without limitation, 32D, DA2, DAlG, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8,
RBS, DA1, 123, Tl 165, HT2, CTLL2, TF-l, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be ~lle~ul~d by the
following methods:
CA 02261 ~ 81 1999 - 01 - 2 ~
WO 98~'~ 1C~4 PCT/US97/12884
Assays for T-cell or thymocyte proliferation include without limitation those described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter
3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. 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.t J. Immunol. 152: 1756-
1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, Iymph node cells
1 0 or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan
eds. Vol I pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of
mouse and human Interferon y, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
1 5 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 Current Protocols in
Immunology. J.E.e.a. Coligan eds. Vol I pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto.
l991;deVriesetal.,J.Exp.Med. 173:1205-1211, 1991;Moreauetal.,Nature336:690-692,
1988;Greenl)~ letal.,Proc.Natl.Acad.Sci.U.S.A.80:2931-2938,1983;Measulel--~ntof
mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.
Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measu,~ "t of human Interleukin 11 - Bennett,
F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse
and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol I pp. 6.13.1, John Wiley and Sons,
Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others,
3 0 proteins that affect APC-T cell interactions as well as direct T-cell effects by m~cming
proliferation and cytokine production) include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M.
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3,
In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular
3 5 receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.
18
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CA 02261~81 1999-01-2~
WO 981'~ 1~4 PCT/US97112884
Sci. USA 77:6091 -6095, 1980; Weinberger et al., Eur. J. Immun. I ~ :405-411, 1981; Takai
et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-51~, 1988.
Immune Stimulatin~ or Su~ p-cssill~ Activity
A protein of the present invention may also exhibit immune stim~ ting or immune
~.U~)~,lcs~ lg activity, inrh~-ling without limitation the activities for which assays are described
herein. A protein may be useful in the ~lcalll,c.lt of various immune deficiencies and diso.dt;.-.
(including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down)
growth and proliferation of T and/or B Iymphocytes, as well as effecting the cytolytic activity
of NK cells and other cell populations. These immune deficiencies may be genetic or be
caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from
a.lloi---...ulle 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, Leichmzlni~ spp., malaria
15 spp. and various fungal infections such as c~n~i.1i~cic 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.
Aulohn~l~u~e disorders which may be treated using a protein of the present invention
include, for example, connective tissue disease, multiple sclerosis, systemic lupus
2 0 erythenn~tos--c rhe--m~toid arthritis, autoi,.. u"e pulmonary inflammation, Guillain-Barre
syndrome, autoimmune thyroiditis, insulin depen-l~nt diabetes mellitis, myasthenia gravis,
graft-versus-host disease and autoi--,l,-u~.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,
such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions,
2 5 in which immune ~;.u~ cssion is desired (including, for example, organ transplantation), may
also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a
number of ways. Down regulation may be in the form of inhibiting or blocking an immune
response already in progress or may involve preventing the induction of an immune response.
3 0 The functions of activated T cells may be inhibited by ~u~ cssillg T cell responses or by
inducing specific tolerance in T cells, or both. Immunosupl)rcssion of T cell responses is
generally an active, non-antigen-specific, process which requires continuous exposure of the
T cells to the ~.u~lcs~7ive agent. Tolerance, which involves inducing non-responsiveness or
anergy in T cells, is distinguishable from immuno.u~ .c.~ion in that it is generally antigen-
3 5 specific and persists after cxposu.c to the tolerizing agent has ceased. Operationally, tolerance
19
CA 02261~81 1999-01-2~
WO 98/OlC91 PCT/US97/12884
can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the
absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B Iymphocyte antigen functions (such as, for example, B7)), e.g., preventing high
5 level Iymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and
organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T
cell function should result in reduced tissue destruction in tissue transplantation. Typically,
in tissue transplants, rejection of the transplant is initiated through its recognition as foreign
by T cells, followed by an immune reaction that destroys the transplant. The a-lminic~ration
1 0 of a molecule which inhibits or blocks interaction of a B7 Iymphocyte antigen with its natural
ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2
activity alone or in conjunction with a monomeric form of a peptide having an activity of
another B Iymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation
can lead to the binding of the molecule to the natural ligand(s) on the immune cells without
1 5 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
Iymphocyte antigen-blocking reagents may avoid the necessity of repeated A~lrninictration of
2 0 these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject,
it may also be necessary to block the function of a combination of B Iymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection
or GVHD can be assessed using animal models that are predictive of efficacy in humans.
Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats
2 5 and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine
the immunosuyy,~ ive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et aL, Proc. Natl. Acad. Sci USA, 89: 11102-
1110~ (1992). In addition, murine models of GVHD (see Paul ed., F--n~lArnenf~l Immunology,
Raven Press, New York, 1989, pp. 846-847~ can be used to determine the effect of blocking
3 0 B Iymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be thc~AI~u~icAlly useful for treating autoimmune
diseases. Many A~ .. e disorders are the result of inayylu~: ,iate activation of T cells that
are reactive against self tissue and which promote the production of cytokines and
autoantibodies involved in the pathology of the ~1ic~Ac~c Preventing the activation of
3 5 autoreactive T cells may reduce or eliminate disease symptoms. .A~ninic~ration of reagents
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CA 02261~81 1999-01-2~
WO 98/04694 PCTIUS97/12884
which block costimulation of T cells by disrupting receptor:ligand interactions of B
Iymphocyte antigens can be used to inhibit T cell activation and prevent production of
autoantibodies or T cell-derived cytokines which may be involved in the disease process.
Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells
5 which could lead to long-term relief from the disease. The efficacy of blocking reagents in
preventing or alleviating autoimmune disorders can be determined using a number of well-
characterized animal models of human autoimmune diseases. Examples include murine
experimental autoimmune çnceph~litis, systemic lupus erythmatosis in MRL/lpr/lpr mice or
NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and
10 BB rats, and murine experimental myasthenia gravis (see Paul ed., Funrl~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
immune responses may be in the form of enhancing an existing immune response or eliciting
15 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 ~nceph:~litic might be alleviated by the
administration of stim~ tory forms of B Iymphocyte antigens syst.-mi~lly.
Alternatively, anti-viral immune responses may be ~nh~n~ed in an infected patient by
2 0 removing T cells from the patient, costimnl~ting the T cells in vitro with viral antigen-pulsed
APCs either ~ cs~ing a peptide of the present invention or together with a stimulatory form
of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into
the patient. Another method of enhancing anti-viral immune responses would be to isolate
infected cells from a patient, transfect them with a nucleic acid encoding a protein of the
2 5 present invention as described herein such that the cells express all or a portion of the protein
on their surface, and n;inL~uduce the transfected cells into the patient. The infected cells would
now be capable of delivering a costimnl~t( ry signal to, and thereby activate, T cells in vivo.
In another application, up regulation or ~nh~nc~m.ont of antigen function (preferably
B Iymphocyte antigen function) may be useful in the induction of tumûr immunity. Tumor
3 0 cells (e.g., sarcoma, melanoma, Iymphoma, lenk~mi~ neuroblastoma, carcinoma) ~ r~;led
with a nucleic acid encoding at least one peptide of the present invention can be zl.l,..i..i~l~.~d
to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can
be ~ reu~d to express a combination of peptides . For example, tumor cells obtained from
a patient can be ll~nsre.;led ex vivo with an expression vector directing the expression of a
3 5 peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like
.. . . . . . . . . .... .. .. .. .. .... . . ... ..
CA 02261 ~ 81 1999 - 01 - 2 ~
WO 98/04694 PCT/US97112884
activity andlor 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 mefli It--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 II molecules, can be
transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated
1 0 portion) of an MHC class I a chain protein and ~12 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 II MHC in
conjunction with a peptide having the activity of a B Iymphocyte antigen (e.g., B7-1, B7-2, B7-
3) induces a T cell me~ Pd immune response against the transfected tumor cell. Optionally,
1 5 a gene ~nco-ling an ~nticence construct which blocks expression of an MHC class II ~ccoci rted
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 hlllllunily. Thus, the induction of a T cell mP~ ted
immune response in a human subject may be sufficient to overcome tumor-specific tolerance
2 0 in the subject.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek,
2 5 D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7,
Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J.
3 0 Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
r- ~ l
CA 02261 ~ 81 1999 - 01 - 2 ~
WO 98/04694 PCT/US97/12884
Assays for T-cell-dependent immunoglobulin le~l,ol)ses and isotype switching (which
will identify, among others, proteins that modulate T-cell dependent antibody responses and
that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J.
Immunol. 144:3028-3033,1990; and Assays for B cell function: In vitro antibody production,
Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
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 l. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-el~. ience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7,
Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed
1 5 by dendritic cells that activate naive T-cells) include, without limitation, those described in:
Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine
173:549-559, 1991; Macatonia et al., Joumal of Immunology 154:5071-5079, 1995; Porgador
et al., Journal of E~l,c:.ill-t;--L~I Medicine 182:255-260, 1995; Nair et al., Journal of Virology
67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; M~c:~tc-nia et al., Journal of
~ hllell~l Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., lournal of Exr~rimen~l Medicine 172:631 -
640, l99o.
Assays for Iymphocyte survival/apoptosis (which will identify, among others, proteins
that prevent apoptosis after ~u~e~ igen induction and proteins that regulate Iymphocyte
2 5 hom~-ost~cic) include, without limitation, those described in: Darzynkiewicz et al., Cytometry
13:795-808, 1992; Gorczyca et al., l.rllk~mi~ 7:659-670, 1993; Gorczyca et al., Cancer
Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of
Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et
al., International Journal of Oncology I :639-648, 1992.
3 0 Assays for proteins that inflnrnre early steps of T-cell co~u"ilnlent and development
include, without limitation, those described in: Antica et al., Blood 84: 111 -117, 1994; Fine
et al., Cellular I~lullul~ology 155: 111 -122,1994; Galy et al., Blood 85:2770-2778, 1995; Toki
et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
3 5 Hematopoiesis Re~ulatin~ Activity
.. .. . ... . ... .
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A protein of the present invention may be useful in regulation of hematopoiesis and,
consequently, in the treatment of myeloid or Iymphoid cell deficiencies. Even marginal
biological activity in support of colony forming cells or of factor-dependent cell lines indicates
involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of
erythroid progenitor cells alone or in combination with other cytokines, thereby indicating
utility, for example, in treating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid
cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and
monocytes/ma~;lopl-ages (i.e., traditional CSF activity) useful, for exalrnple, in conjunction with
1 0 chemotherapy to prevent or treat consequent myelo-~.uppl~s~,ion; in supporting the growth and
proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or
treatment of various platelet disorders such as thrombocytopenia, and generally for use in place
of or complimentary to platelet transfusions; and/or in ~.u~,~u-ling the growth and proliferation
of hematopoietic stem cells which are capable of maturing to any and all of the above-
mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell
disorders (such as those usually treated with transplantation, including, without limitation,
aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem
cell conlp~ Ll.,ent post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in con)unction
with bone marrow transplantation or with pe;ipheral progenitor cell transplantation
2 0 (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 dirre,~ iation of various hematopoietic lines are
cited above.
2 5 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 Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others,
3 0 proteins that regulate Iympho-hematopoiesis) include, without limitation, those described in:
Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I.
Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et
al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming
cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of
3 5 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York,
24
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WO 98/04694 PCTIUS97/12884
NY. 1994; Neben et al., E~pt;~ cntal Hematology 22:353-359, 1994; Cobblestone area
forming cell assay, Plo~m~r~ler, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures
in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
5 Hematopoietic Cells. R.I. Freshney, etal. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York,
NY. 1994; Long term culture initi~ting cell assay, Sutherland, H.J. In Culture of Hem~topoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone,
cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound
healing and tissue repair and repl~r-ç~ t, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in
circllmct~nrçc where bone is not normal}y formed, has application in the healing of bone
15 fractures and cartilage damage or defects in humans and other animals. Such a plep~cllion
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
2 0 plastic surgery.
A protein of this invention may also be used in the ~ elll of periodontal disease,
and in other tooth repair processes. Such agents may provide an envh~mllenl to attract bone-
forming cells, stim~ tr 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
2 5 osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by
blocking inll~mm~tion or processes of tissue destruction (collagenase activity, ostroc!~ct
activity, etc.) mf r~ trd by infl~.. ~lc.,y processes.
Another category of tissue legen~ldlion activity that may be attributable to the protein
of the present invention is tendon/ligament formation. A protein of the present invention,
3 0 which induces tendon/ligament-like tissue or other tissue formation in ~ lcec 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
pl~ lion employing a tendon/ligament-like tissue inducing protein may have prophylactic
use in preventing damage to tendon or lig~m~nt tissue, as well as use in the improved fixation
3 5 of tendon or ligament to bone or other tissues, and in repairing defects to tendon or lig;~m~nt
CA 0226 1 ~81 1 999 - 01 - 2~
WO ~8/01 Cg ~ PCT/US97/12884
tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present
invention contributes to the repair of congenital. trauma in~ cerl or other tendon or ligament
defects of other origin, and is also useful in cosmetic plastic surgery for ~tt~hm~.nt or repair
of tendons or lig~mPnts The compositions of the present invention may provide an5 environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming
cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to
effect tissue repair. The compositions of the invention may also be useful in the treatment of
tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions
10 may also include an ap~.~,p~id~e matrix and/or se4u. ~ hlg 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 neu.op~l,ies, as well as mechanical and traumatic disorders,
15 which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically,
a protein may be used in the lled~lllellt of diseases of the pefi~Jhel~l nervous system, such as
peripheral nerve injuries, peli~heldl neu.opdllly and localized neuropathies, and central
nervous system diseases, such as Al7h.-.im~r's, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be
2 0 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.
Pelipheldl neul~dlhies 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-
2 5 healing wounds, including without limitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for
generation or legellcldliOn of other tissues, such as organs (including, for example, pancreas,
liver, int~.stin,o kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
3 0 (including vascular endothelium) tissue, or for promoting the growth of cells comprising such
tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to
allow normal tissue to l~,~,eneld~e. A protein of the invention may also exhibit angiogenic
activity.
26
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CA 02261~81 1999-01-2~
wos~,la1~s1 rCT/US97/12884
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
5 dirr~lcnliation 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. WO95/16035 (bone, cartilage, tendon); International
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 Healing, pps. 71-112 (Maibach, HI and Rovee, DT. eds.), Year
15 Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related
2 0 activities. Inhibins are ch~ud~;le,i~ed by their ability to inhibit the release of follicle stim~ ing
hormone (FSH), while activins and are characterized by their ability to stimulate the release
of follicle s~imul:~ing hormone (FSH). Thus, a protein of the present invention, alone or in
heterodimers with a member of the mhibin a family, may be useful as a contldc~ulive based
on the ability of inhibins to decrease fertility in female m~mm~lc and decrease ~ ,."a~ugenesis
25 inmalem~mm~lc A.l.,.;ll,~l~dlionofsufficientamountsofotherinhibinscaninduceinfertility
in these mzlmm~lc Alternatively, the protein of the invention, as a homodimer or as a
heterodimer with other protein subunits of the inhibin-~ group, may be useful as a fertility
inducing th~dpeulic, based upon the ability of activin molecules in stim~ ting FSH release
from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A
3 0 protein of the invention may also be useful for ad~ e. . .~t of the onset of fertility in sexually
immature m~mm~l~, so as to increase the lifetime reproductive performance of domestic
animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
CA 02261 ~ 81 1999 - 01 - 2 ~
WO 98/04694 PCT/US97/12884
Assays for activin/inhibin activity include, without limitation, those described in: Vale
et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al.,
Nature 321 :776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl.
Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chf mot~rtic 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
1 0 and chemokinetic proteins can be used to mobilize or attract a desired cell population to a
desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in
treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.
For example, attraction of Iymphocytes, monocytes or neutrophils to tumors or sites of
infection may result in improved immune responses against the tumor or infecting agent.
1 5 A protein or peptide has chemotactic activity for a particular cell population if it can
stimnl lte, 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
2 0 chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent
chemotaxis) consist of assays that measure the ability of a protein to induce the migration of
2 5 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
30 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS
103:140-146,1995;MulleretalEur.J.Immunol.25: 1744-1748;Gruberetal.J.ofImmunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
28
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CA 02261~81 1999-01-2~
WO 98~'~16!~ PCT/US97/12884
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As
a result, such a protein is expected to be useful in treatment of various coagulation disorders
(including hereditary disorders, such as hemophilias) or to enhance coagulation and other
hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein
5 of the invention may also be useful for dissolving or inhibiting forrnation of thromboses and
for llc~Llllclll and prevention of conditions resulting therefrom (such as, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis
Res. 45:413-419, 1987; Hulllplll~y et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins
35:467-474, 1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors, receptor
ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors
and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases
and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell
2 0 interactions and their ligands (inr~ ing without limitation, cellular adhesion molecules (such
as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen
presentation, antigen recognition and development of cellular and humoral immune responses).
Receptors and ligands are also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein of the present invention
2 5 (including, without limitation, fragments of receptors and ligands) may themselves be useful
as inhibitors of receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured by the
following methods:
Suitable assays for receptor-ligand activity include without limitation those described
3 0 in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W.Strober, Pub. Greene Publishing ~soci~tes and Wiley-Interscience(Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22),
Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborget
3 5 al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
29
CA 02261~81 1999-01-2~
wo 98~'a';6~q PCT/US97/12884
Anti-IIlll~.. ~tc-rv Activity
Proteins of the present invention may also exhibit anti-i-.ll~ ory activity. The anti-
infl~mm~tory activity may be achieved by providing a stimulus to cells involved in the
infl,~..-...i.loly response, by inhibiting orpromoting cell-cell interactions (such as, for example,
5 cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by stim~ ting or ~u~ g production
of other factors which more directly inhibit or promote an inflammatory response. Proteins
exhibiting such activities can be used to treat infl~mm~tQry conditions including chronic or
acute conditions), including without limitation inflammation associated with infection (such
10 as septic shock, sepsis or systemic infl:lmm~t-)ry response syndrome (SIRS)), ischemia-
reperfusion injury, endotoxin lethality, arthritis, complement-me~ ted hyperacute rejection,
nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's
disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
15 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
2 0 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, elimin~ting or inhibiting factors, agents or cell types
2 5 which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional
3 0 activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other parasites; effecting (~u~ s~ing
or enhancing) bodily characteristics, including, without limitation, height, weight, hair color,
eye color, skin, fat to lean ratio or other tissue pigm.ont~tion, or organ or body part size or shape
(such as, for example, breast augmentation or diminution, change in bone form or shape);
3 5 effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female
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CA 0226l~8l l999-0l-2~
WO ~ 1J~ PCT/US97/12884
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
5 (including depressive disorders) and violent behaviors; providing analgesic effects or other
pain reducing effects; ~ ,lllolhlg dirrelGllliation and growth of embryonic stem cells in lineages
other than hematopoietic lineages; horrnonal or endocrine activity; in the case of enzymes,
colleL;~ g deficiencies of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity
10 (such as, for example, the ability to bind antigens or complement); and the ability to act as an
antigen in a vaccine composition to raise an immune response against such protein or another
material or entity which is cross-reactive with such protein.
15 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 pharm~e~l~ic~l
composition when combined with a ph~rm~reutic:llly acceptable carrier. Such a composition
may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers,
20 solubilizers, and other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The ch~a~;~eli~lics of the carrier will depend on
the route of ~ tion The ph~lll~eu~ic~l composition of the invention may also contain
cytokines, Iymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, L-l,
IL-2,IL-3,IL-4,IL-5,IL-6,L-7,L-8,L-9,L-10,IL-11,IL-12,L-13,L-14,L-15,IFN,
TNF0, TNF1, TNF2, G-CSF, Meg-CSF, Lhl~ll,bupoietin, 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 ~leatlllent. Such additional factors
and/or agents may be included in the phallllacc~ulical composition to produce a synergistic
3 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-infl~mm~tory agent to
minimi7e side effects of the cytokine, Iymphokine, other hematopoietic factor, thrombolytic
or anti-thrombotic factor, or anti-infl~mm~tory agent.
.. ...........
CA 02261~81 1999-01-2~
WO gX/0~1691 PCT/US97/12884
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 mllltim~ric or
complexed form.
The pharrn~e-lti~l composition of the invention may be in the form of a complex of
the protein(s) of present invention along with protein or peptide antigens. The protein and/or
peptide antigen will deliver a 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
1 0 MHC proteins. MHC and structurally related proteins including those encoded by class I and
class II MHC genes on host cells will serve to present the peptide antigen(s) to T Iymphocytes.
The antigen components could also be supplied as purified MHC-peptide complexes alone or
with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able
to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to
bind the TCR and other molecules on T cells can be combined with the pharmaceutical
composition of the invention.
The ph~ ~ u~ic~l cc""po~ilion 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 amphipathic agents such as lipids which exist in aggregated form as
2 0 micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable
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
2 5 No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "ther~pel-tic~lly effective amount" means the total amount of
each active component of the ~h;~"~ l composition or method that is sufficient to show
a mP~ningful 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
3 0 such conditions. When applied to an individual active ingredient, a~mini~t~red alone, the term
refers to that ingredient alone. When applied to a combination, the term refers to combined
amounts of the active ingredients that result in the therapeutic effect, whether ~clmini~tt-red in
combination, serially or ~imlllt~neously
In practicing the method of l~tal,l,~.,l or use of the present invention, a therapeutically
3 5 effective amount of protein of the present invention is administered to a m~mm~l having a
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CA 0226 1 ~81 1 999 - 01 - 2~
WO 98/04694 PCT/US97/12884
condition to be treated. Protein of the present invention may be ~tlminict~red in accordance
with the method of the invention either alone or in combination with other therapies such as
trf ~m~ntC employing cytokines, Iymphokines or other hematopoietic factors. When co-
~r~minictered with one or more cytokines, Iymphokines or other hematopoietic factors, protein
5 of the present invention may be ~lminict~red either simultaneously with the cytokine(s),
Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If ~lminict~red sequentially, the attending physician will decide on the
appropriate sequence of ~minict~ring protein of the present invention in combination with
cytokine(s), Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
1 0 factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried out in a variety
of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, h~lla~ iluneal, p~t;llt~.~.l or intravenous injection. Intravenous ~ minictration
15 to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
~dministt~red orally, protein of the present invention will be in the form of a tablet, capsule,
powder, solution or elixir. When ~(lminict~red in tablet form, the ph:lrm~elltical composition
of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The
2 0 tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and
preferably from about 25 to 90% protein of the present invention. When ~lminict~red 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
pharnn~entic~l composition may further contain physiological saline solution, dextrose or
2 5 other s~h~ride solution, or glycols such as ethylene glycol, propylene glycol or polyethylene
glycol. When ~l.";~ red in liquid form, the pharmaceutical co.,lposi~ion contains from
about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1
to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is
3 0 ~(lminictt~red by intravenous, cutaneous or subcutaneous injection, protein of the present
invention will be in the form of a pyrogen-free, palcn~lally accel,~ble aqueous solution. The
pl~dlion of such p~Gnte-~lly 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, cut~n~onC, or s-lhcur~n.~o--c injection should contain, in addition
3 5 to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection,
CA 0 2 2 6 1 ~ 8 l 1 9 9 9 - 0 l - 2 ~
WO 98~'~16~1 PCT/US97tl2884
Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated
Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of
the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical composition of
the present invention will depend upon the nature and severity of the condition being treated,
and on the nature of prior treatments which the patient has undergone. Ultimately, the
attending physician will decide the amount of protein of the present invention with which to
treat each individual patient. Initially, the attending physician will administer low doses of
protein of the present invention and observe the patient's response. Larger doses of protein of
the present invention may be administered until the optimal therapeutic effect is obtained for
the patient, and at that point the dosage is not increased further. It is contemplated that the
various pharrnaceutical compositions used to practice the method of the present invention
should contain about 0.01 llg to about l 00 mg (preferably about O. l ~lg to about l O mg, more
preferably about O. l llg 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. It is contemplated
that the duration of each application of the protein of the present invention will be in the range
2 0 of 12 to 24 hours of continuous intravenous :lfimini~tratjon Ultimately the ~ttenrling 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
2 5 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. 21l, lO (19~7). Monoclonal
3 0 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 thtl~uLics 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 a~ainst the
34
CA 02261~81 1999-01-2~
WO ~ 91 PCT/US97/12884
protein may be useful in ~IPt~c~ing and preventing the l..r~ ic spread of the cancerous cells,
which may be me~ ed by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon
or ligament l~gen~dlion, the therapeutic method includes a-lministçring the composition
5 topically, systematically, or locally as an implant or device. When :~minisrf~red, the
therapeutic composition for use in this invention is, of course, in a pyrogen-free,
physiologically acceptable form. Further, the composition may desirably be encapsulated or
injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical
admini~tration may be suitable for wound healing and tissue repair. Therapeutically useful
10 agents other than a protein of the invention which may also optionally be included in the
composition as described above, may alternatively or additionally, be ~3rlminic~red
simultaneously or sequentially with the composition in the methods of the invention.
Preferably for bone and/or cartilage formation, the composition would include a matrix capable
of delivering the protein-containing composition to the site of bone and/or cartilage damage,
15 providing a structure for the developing bone and cartilage and optimally capable of being
resorbed into the body. Such matrices may be formed of materials presently in use for other
implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appe~ a"ce and interface properties. The particular
2 0 application of the compositions will define the appropriate formulation. Potential matrices for
the compositions may be biodegradable and chemically defined calcium sulfate,
tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
Other potential materials are biodegradable and biologically well-defined, such as bone or
dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix
2 5 components. Other potential matrices are nonbiodegradable and chemically defined, such as
sintered hydroxapatite, bioglass, ahlmin~s, or other ceramics. Matrices may be co"l~-ised
of combinations of any of the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in
cull")os;lion~ such as in calcium-~lllmin~-phosphate and processing to alter pore size, particle
3 0 size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid
in the form of porous particles having (li~m~t~rs ranging from lS0 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
3 5 matrix.
CA 02261~81 1999-01-2~
WO !~ 1 Gg 1 rcTluss7ll2ss4
A preferred family of sequestering agents is cellulosic m~ti~ri~lc such as alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulosè, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and
carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose
(CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate,
poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
The amount of sequestP-ring agent useful herein is 0.5-20 wt%, preferably 1-10 Wt~G based on
total formulation weight, which represents the amount necessary to prevent desorbtion of the
protein from the polymer matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from infiltrating the matrix, thereby
providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other agents
beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
These agents include various growth factors such as epidermal growth factor (EGF), platelet
derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-,~), 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.
2 0 The dosage regimen of a protein-containing pharrn~eutic~l composition to be used
in tissue regeneration will be determined by the :ltt~nrling 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
2 5 ~-imini~tration 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 ~cec~m~.nt of tissue/bone growth and/or repair, for example, X-rays,
3 0 histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Suchpolynucleotides can be introduced either in vivo or ex vivo into cells for expression in a
m~rnm~ n subject. Polynucleotides of the invention may also be ~-lmini~t~red by other
known methods for introduction of nucleic acid into a cell or ~ l (including, without
3 5 limitation, in the form of viral vectors or naked DNA).
r
CA 0226 1 ~81 1 999 - 01 - 2~
WO 98/04694 PCT/US97/12884
Cells may also be cultured ex vivo in the presence of proteins of the present invention
in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells
can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully
set forth.
CA 0226l~8l l999-0l-2~
W O 981~16~ PCTrUS97/12884
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John
LaVallie, Edward
Racie, Lisa
Merberg, David
Treacy, Maurice
Spaulding, Vikki
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES:16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark 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: 271 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
38
1. 1 1
CA 02261~81 1999-01-2~
W 098l'~16g4 PCTrUS97/12884
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TAATCATGCC TCTTGGAAGT NAGTTAACGG GCGTGATTGT GGAAAATGAA AATATTACCA 60
AAGAAGGTGG CTTATTGGAC ATGGCCNNNA AANAAAATGA CTTNNATGCN GAGCCCNNTT l20
TAAAGCAGAC AATTAAAGCA ACAGTNNAAA ATGGCAANAA NGATGGCNTT GCTGTTGATC l80
NTGTTGTNGG CCTGAATACN GAAAAATATG CTGAAACTGT CCAACTTNAN CNTAAAAGAA 240
CCCCNGGTNA AGTNAAAGAC ATTTCCTTGA A 27l
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 591 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATGGTNACAG GTGCAGGTGT TNTCCTGGGA GATAATGATG CACCACCAGG AACAAGTGCC 60
AGCCAAGAAG GAGATGGTTC TGTGAATGAT GGTACAGAAG GTGAGAGTGC AGTCACCAGC 120
ACGGGGATAA CAGAAGATGG AGAGGGGCCA GCAAGTTGCA CAGGTTCAGA ANATTGCNTC l80
GAAGGCTTTG CTATAAGTTC TGAATCGGAA GAAAATGGAG AGAGTGCAAT GGACAGCACA 240
GTGGCCAAAG AAGGCACTAA TGTACCATTA GTTGCTGCTG GTCCTTGTGA TGATGAAGGC 300
ATTGTGACTA GCACAGGCGC NAAAGAGGAA GACGAGGAAG GGGAGGATGT TGTGACTAGT 360
ACTGGAAGAG GAAATGAAAT TGGGCATGCT TCAACTTGTA CAGGGTTAGG AGAAGAAAGT 420
GAAGGGGTCT TGATTTGTGA AAGTGCAGAA GGGGACAGTC AGATTGGTAC TGTGGTAGAG 480
CATGTGGAAG CTGAGGCTGG AGCTGCCATC ATGAATGCAA ATGAAAATAA TGTTGACAGC 540
ATGAGTGGCA CAGAGAAAGG AAGTAAAGAC ACAGATATCT GCTCCAGTGC C 59l
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
39
. .~. . . .
CA 0226l~8l l999-0l-2~
W O 98/~5~9~ PCT~US97/12884
(C) STRANDEDNESS:
~D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Val Thr Gly Ala Gly Val Xaa Leu Gly Asp Asn Asp Ala Pro Pro
1 5 10 15
Gly Thr Ser Ala Ser Gln Glu Gly Asp Gly Ser Val Asn Asp Gly Thr
Glu Gly Glu Ser Ala Val Thr Ser Thr Gly Ile Thr Glu Asp Gly Glu
Gly Pro Ala Ser Cys Thr Gly Ser Glu Xaa Cys Xaa Glu Gly Phe Ala
Ile Ser Ser Glu Ser Glu Glu Asn Gly Glu Ser Ala Met Asp Ser Thr
Val Ala Lys Glu Gly Thr Asn Val Pro Leu Val Ala Ala Gly Pro Cys
Asp Asp Glu Gly Ile Val Thr Ser Thr Gly Ala Lys Glu Glu Asp Glu
100 105 110
Glu Gly Glu Asp Val Val Thr Ser Thr Gly Arg Gly Asn Glu Ile Gly
115 120 125
His Ala Ser Thr Cys Thr Gly Leu Gly Glu Glu Ser Glu Gly Val Leu
130 135 140
Ile Cys Glu Ser Ala Glu Gly Asp Ser Gln Ile Gly Thr Val Val Glu
145 150 155 160
His Val Glu Ala Glu Ala Gly Ala Ala Ile Met Asn Ala Asn Glu Asn
165 170 175
Asn Val Asp Ser Met Ser Gly Thr Glu Lys Gly Ser Lys Asp Thr Asp
180 185 190
Ile Cys Ser Ser Ala
195
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 289 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
r ~ .
CA 0226l~8l l999-0l-2~
W 0 98~1~91 PCT~US97/12884
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GTATGCCAAT TTCCGCCAGC ATTGACNGAC ATGAAGAGAA TCAGNTGACT GCAGACAACC 60
CAGAAGGGAA CGGTGACTTN TCAGCCACAG AAGTGAGCAA GCACAAGTTC CCCATGCCCA 120
GCTTAATTGC TGAGAATAAC TGTCGGTGTC CTGGGCCAGT CAGGGGAGGC AAAGAACTGG l80
GTCCCGTGTT GGCAGTGAGC ACCGAGGAGG GGCACAACGG GCCATCAGTC CACAAGCCNT 240
CTGCAGGGCA AGGCCATCAA GTG~T~l~ lG TGCGGAAAAA AAAAAAAAA 289
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTTGGTCCAN TTGGTTTNNT TCGNTTCCCC ~'l"l"l"l"l'~'l"l'C CCCTTGGTTT ~l~l"l"l"l"l"l"l"l~ 60
CGGGCAACAA TATTTTCCAA GGCTAATACC AAGGCANACC AATTCAACTC CCAAGGNTCG l20
GGAATTTTTA ACCTTTTAAT TNNATGGCCC CTCCCACTCC TTTTCTACGG CGATTTGTCT l80
GTGTCTGGCC CCCACCCACT GCCCATCCCC CATTGTTGTC TGGATGTGGT TCTATTTTTT 240
ATCGGTCTCC TTTCCCCTCC TCCCCGTTCT CGCCCCCGCC CCACCCCCTG CTCCCACTAC 300
CCTTTGTCTC TTGCTCTTTC TTGGGCTTCT GTACAACTCA ACTTGTATAC ACTGTGTACA 360
CACAACCAGC CAAACGAAAA CCCAACGGCR AA~UUU~AA AAAAAAAAAA AAAAAAAAAA 420
AAAAAAAAA 429
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 amino acids
41
., . , . . .. ~
CA 0226l~8l l999-0l-2~
WO 98/~1C9~ PCT/US97/12884
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Gly Pro Xaa Gly Xaa Xaa Arg Phe Pro Leu Phe Leu Pro Leu Gly
Phe Leu Phe Phe Arg Ala Thr Ile Phe Ser Lys Ala Asn Thr Lys Ala
Xaa Gln Phe Asn Ser Gln Gly Ser Gly Ile Phe Asn Leu Leu Ile Xaa
Trp Pro Leu Pro Leu Leu Phe Tyr Gly Asp Leu Ser Val Ser Gly Pro
His Pro Leu Pro Ile Pro His Cys Cys Leu Asp Val Val Leu Phe Phe
. 80
Ile Gly Leu Leu Ser Pro Pro Pro Arg Ser Arg Pro Arg Pro Thr Pro
Cys Ser His Tyr Pro Leu Ser Leu Ala Leu Ser Trp Ala Ser Val Gln
100 105 110
Leu Asn Leu Tyr Thr Leu Cys Thr His Asn Gln Pro Asn Glu Asn Pro
115 120 125
Thr Ala
130
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TCAGAAAAGA CAATNTTTTA GTAANAAAGN ANTGAGGTGT GGCAGGCTNG AGATTTGGCC 60
42
CA 0226l~8l l999-0l-2~
W O 98/04694 PCT~US97/12884
ANANACTNGA GTGACAANGA CATCCACTTT GC~'1"1"1'N'1~1' CCACAGGTGT CCACTCCCAG 120
GTCCAACTGC AGATTTNGAA TTCGGCNTTC ATGGCCTAGC CCCTTCCACC TCTTCTCCTA 180
NGACTTGGAG GANTCCTCCC TGTCCACCAA GGAGAAGGAA GCAGANTCCC AGAAGGAAAA 240
CAGANACAGC AATTTTGGCA ATAACTCTTA TCACTCCTCA AGACCCTCAT CTGGATCCAG 300
TGTGCCCACC ACCCCCACAT CATCCGTCTC ACCCCCACAG GAGGCCAGGT TGGAAAGGTC 360
ATCACCGAGT GGTCTTCTCA CATCATCCTT CAGGCAGCAC CAAGAGTCAC TGGCAAAAAA 420
AAAAAAAAA 429
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gln Lys Arg Gln Xaa Phe Ser Xaa Lys Xaa Xaa Arg Cys Gly Arg Leu
1 5 10 15
Glu Ile Trp Pro Xaa Thr Xaa Val Thr Xaa Thr Ser Thr Leu Pro Xaa
Ser Pro Gln Val Ser Thr Pro Arg Ser Asn Cys Arg Phe Xaa Ile Arg
Xaa Ser Trp Pro Ser Pro Phe His Leu Phe Ser Xaa Asp Leu Glu Xaa
Ser Ser Leu Ser Thr Lys Glu Lys Glu Ala Xaa Ser Gln Lys Glu Asn
Arg Xaa Ser Asn Phe Gly Asn Asn Ser Tyr His Ser Ser Arg Pro Ser
Ser Gly Ser Ser Val Pro Thr Thr Pro Thr Ser Ser Val Ser Pro Pro
100 105 110
Gln Glu Ala Arg Leu Glu Arg Ser Ser Pro Ser Gly Leu Leu Thr Ser
115 120 125
Ser Phe Arg Gln His Gln Glu Ser Leu Ala
130 135
43
.. .... ... . . . .
CA 02261~81 1999-01-2~
W O98~S~g1 PCTrUS97/12884
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TGGCCTACCT GACCTCAGGT GATCTGCCCG CCTCGGCCTC TGAAAGTGCT GGGATTATAG 60
GCATGAGCCN ACATGCCTGA CCTGTTATTT ATTTTAAATT ATATCAGGAA TACACACACA l20
CACACACACA CACACACACA CACACACACA ACTTATAAAG ATAATGGTCT CCTTGGCACT l80
CCCACCCACC CACCCATCCA AATTTACACA AATTAATC 2l8
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
lxi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
ATCCAAATTN ACACAAGTAA ATCTGTAATC AATTTGGTTA GAAGGGATTT ATTTTAATAT 60
TTTTGGGGAT TGCTTAGATG CAGTATAATT TTTAGTTATA TTAGTAGTAA TTGGAAATGT .l20
GTAlllll~l GACTGAAGTC ACCTTCTAAA TAATTTCTAG AATAAAATTT TTATATTGAA l80
GAAGTTGGTN TTAACCATTT 'lll~ lCAGG AGCATGCATT TTGAAATCAT TCTGTGGGAA 240
GATGAAAACA AATTTAGTTC TATGTCTCCC CTTTTTAGAG ATGTTGACAC TTTCCTTAAA 300
TGTACCATGC ATGATTTGTC TA 322
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
44
r ~ T
CA 0226l~8l l999-0l-2~
W O 98~ 4 PCTrUS97/12884
~B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Lys Thr Asn Leu Val Leu Cys Leu Pro Phe Leu Glu Met Leu Thr
1 5 10 15
Leu Ser Leu Asn Val Pro Cys Met Ile Cys Leu
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 379 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CAGTTTTGAT GTAAGTAAAT ACCCAACAGA AATGCAAACA TATACTTACC AAAACTCATG 60
TCCAAGAATA TTCGTAGAAG CACAATTCTT ATGATAGCAA AAAGGTAGAA AACAACTTAA 120
Al~lllllAA GCAGTAGCAT AAGAGTAATA CCGTGTGGTT TGTTTANACA GTGAGATCCT 180
GTACAGCCAT GTAAAAGACC AAAATATTCC CTGTAACAAT GAGAATGAAT CTCCTGTGCT 240
TGCTTCGGCA GCACATACAN TAAAATTGGA ACGATACAGA GATTANCATG NCCCCTGTGC 300
AAGGAGAATG AATTTTCGTA ATGTTCAGCA AAAGAAGCCA GATATAAATG AATATTCCAT 360
TTTATAAAAA NAAAAAAAA 379
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
~.
CA 02261~81 1999-01-2~
W O 98/~1C~ PCTrUS97/12884
(A) DESCRIPTION: /deSC = ~O1igOnUC1eOtid
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GNTGAAGCAT GCCCAATTTC ATTTCCTCT 29
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 baSe PairS
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: Sing1e
(D) TOPOLOGY 1inear
(ii) MOLECULE TYPE Other nUC1eiC aCid
(A) DESCRIPTION /deSC = ~O1igOnUC1eOti
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TNCAAGTTGA GTTGTACAGA AGCCCAAGA 29
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 29 baSe PairS
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS Sing1e
(D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: Other nUC1eiC aCid
(A) DESCRIPTION: /deSC = ~O1igOnUC1eOtide~
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GNTGTGAGAA GACCACTCGG TGATGACCT 29
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 baSe PairS
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: Sing1e
(D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: Other nUC1eiC aCid
(A) DESCRIPTION: /deSC = ~O1igOnUC1eOtide~
46
r
CA 0226l58l l999-0l-25
WO 98/04694 PCT/US97/12884
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
TNGAGTCTGG GTGGTAGACA AATCATGCA 29
47
, ~ .. .. .. . . ..
