Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 08/742,973, filed
November 1, 1996.
FIELD OF THE INVENTION
2 0 The present invention provides novel proteins, along with therapeutic, diagnostic
and research utilities for these proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g., cytokines,such as lymphokines, inl~ rol,s, 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 hybridization cloning; activity of the protein in the case of expression
3 0 cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which
isolates DNA sequences based on the presence of a now well-recognized secretory leader
sequence motif, as well as various PCR-based or low stringency hybri~ n cloning
techniques, have advanced the state of the art by making available large numbers of
DNA/amino acid sequences for proteins that are known to have biological activity by
3 5 virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these proteins that the
present invention is directed.
SUMMARY OF THE INVENIION
4 0 In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
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(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:25;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:25 from nucleotide 73 to nucleotide 702;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:25 from nucleotide 118 to nucleotide 702;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AE648_1i deposited under accession
number ATCC 98237;
1 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AE648_1i deposited under accession number ATCC 98237;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AE648_1i deposited under acrrs~ion number
ATCC 98237;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AE648_1i deposited under ~ r~s.sion number ATCC 98237;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:26;
(i) a polynudeotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID NO:26 having biological activity;
(J) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:25 from nucleotide 73 to nucleotide 702; the nucleotide sequence of SEQ ID NO:25
from nucleotide 118 to nucleotide 702; the nucleotide sequence of the full-length protein
coding sequence of clone AE648_1i deposited under accession number ATCC 98237; o}
the nucleotide sequence of the mature protein coding sequence of clone AE648_li
3 0 deposited under ~cre.s.sinn number ATCC 98237. In other ~le~:lr~d embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of
clone AE648_1i deposited under accession number ATCC 98237. In yet other ~lerell~d
embodiments, such polynucleotide encodes a protein comprising the amino acid sequence
of SEQ ID NO:26 from amino acid 1 to amino acid 34.
.,.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:26;
(b) the amino acid sequence of SEQ ID NO:26 from amino acid 1 to
amino acid 34;
(c) fragments of the amino acid sequence of SEQ ID NO:26; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AE648_1i deposited under ac cP.csion number ATCC 98237;
1 0 the protein being substantially free from other mammalian proteins. Preferably such
protein comprises the amino acid sequence of SEQ ID NO:26 or the amino acid sequence
of SEQ ID NO:26 from amino acid 1 to amino acid 34.
In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:4;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:4 from nucleotide 92 to nucleotide 268;
(c) a polynucleotide ~:O~ ,illg the nucleotide sequence of the full
2 0 length protein coding sequence of clone AE693_1i deposited under ~cc~ n
number ATCC 98237;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AE693_1i deposited under accession number ATCC 98237;
(e) a polynucleotide comprising the nucleotide sequence of the mature
2 5 protein coding sequence of clone AE693_1i deposited under accession number
ATCC 98237;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AE693_1i deposited under accession number ATCC 98237;
(g) a polynucleotide encoding a protein comprising the amino acid
3 0 sequence of SEQ ID NO:5;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:5 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above; and
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(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleoffde sequence of SEQ ID
NO:4 from nucleotide 92 to nucleotide 268; the nucleotide sequence of the full length
5 protein coding sequence of clone AE693_1i deposited under accession number ATCC
98237; or the nucleoffde sequence of the mature protein coding sequence of cloneAE693_1i deposited under accession number ATCC 98237. In other pl~r~ . ed
embodiments, the polynucleotide encodes the full length or mature protein encoded by
the cDNA insert of clone AE693_1i deposited under accession number ATCC 98237.
1 0In other embodiments, the present invenffon provides a composition comprisinga protein, wherein said protein comprises an amino acid sequence selectcd from the group
consisffng of:
(a) the amino acid sequence of SEQ ID NO:5;
(b) fragments of the amino acid sequence of SEQ ID NO:5; and
15(c) the amino acid sequence encoded by the cDNA insert of clone
AE693_li deposited under accession number ATCC 98237;
the protein being substanffally free from other n ~rnm~ n proteins. Preferably such
protein comprises the amino acid sequence of SEQ ID NO:5.
In one embodiment, the present invenffon provides a composition comprising an
2 0 isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleoffde 137 to nucleotide 412;
(c) a polynucleotide comprising the nucleoffde sequence of the full
length protein coding sequence of clone AK438_1i deposited under accession
number ATCC 98237;
(d) a polynucleoffde encoding the full length protein encoded by the
cDNA insert of clone AK438_1i deposited under ~rrf S.ci~n number ATCC 98237;
3 0 (e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AK438_li deposited under accession number
ATCC 98237;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AK438_1i deposited under arrr-~ir~n number ATCC 98237;
,.
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(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;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a~-(f) above; and
(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:7 from nucleotide 137 to nucleotide 412; the nucleotide sequence of the full length
protein coding sequence of clone AK438_1i deposited under A~ inn number ATCC
98237; or the nucleotide sequence of the mature protein coding sequence of cloneAK438_1i deposited under ~cc~ n number ATCC 98237. In other preferred
embodiments, the polynucleotide encodes the full length or mature protein encoded by
the cDNA insert of clone AK438_1i deposited under Ac~ n number ATCC 98237.
In other embo~lim~nt~, 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;
2 0 (b) fragments of the amino acid sequence of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AK438_1i deposited under accession number ATCC 98237;
the protein being substantially free from other m;lmm~ n proteins. Preferably such
protein comprises the amino acid sequence of SEQ ID NO:8.
2 5 In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide c~ lg the nucleotide sequence of SEQ ID
NO:10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 NO:10 from nucleotide to nucleotide 285;
(c) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone AK609_1i deposited under accession
number ATCC 98237;
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(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AK609_1i deposited under accession number ATCC 98Z37;
(e~ a polynucleotide ~olllplisil lg the nucleotide sequence of the mature
protein coding sequence of clone AK609_1i deposited under accession number
ATCC 98237;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AK609_li deposited under ~ c~ssion number ATCC 98237;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:11;
1 0 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:11 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above; and
(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:10 from nucleotide to nucleotide 285; the nucleotide sequence of the full length
protein coding sequence of clone AK609_1i deposited under acc~s~si~n number ATCC98237; or the nucleotide sequence of the mature protein coding sequence of clone2 0 AK6û9_1i deposited under accession number ATCC 98237 In other ~ f~lled
embodiments, the polynucleotide encodes the full length or mature protein encoded by
the cDNA insert of clone AK609_1i deposited under ~c~ssi~n number ATCC 98237.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein col~ ises an amino acid sequence selected from the group
2 5 consisting of:
(a~ the amino acid sequence of SEQ ID NO:11;
(b) fragments of the amino acid sequence of SEQ ID NO:lli and
(c) the arnino acid sequence encoded by the cDNA insert of clone
AK609_1i deposited under accession number ATCC 98237;
3 0 the protein being substantially free from other mAmmAli~n proteins. Preferably such
protein comprises the amino acid sequence of SEQ ID NO:11.
In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
-
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(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13;
~ 'b) a polynucleotide comprising the nucleotide sequence of SEQ ID
~ NO:13 from nucleotide 43 to nucleotide 282;
(c) a polynucleotide comprising the nucleotide se~luence of SEQ ID
NO:13 from nucleotide 118 to nucleotide 282;
(d) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone AM1060_1i deposited under accession
number ATCC 98237;
1 0 (e) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone AM1060_1i deposited under acc~ssion number ATCC 98237;
(f) a polynucleotide ~:ollL~liSillg the nucleotide sequence of the mature
protein coding sequence of clone AM1060_li deposited under accession number
ATCC 98237;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AM1060_li deposited under acce~ion number ATCC 98237;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID NO:14 having biological activit~r;
(J) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; and
(k) a polynucleotide which encodes a species homolo~ue of the protein
of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:13 from nucleotide 43 to nucleotide 282; the nucleotide sequence of SEQ ID NO:13
from nucleotide 118 to nucleotide 282; the nucleotide sequence of the full length protein
coding sequence of clone AM1060_1i deposited under ~ xi~ number ATCC 98237; or
the nucleotide sequence of the mature protein coding sequence of clone AM1060_1i3 0 deposited under accession number ATCC 98237. In other pl~relled embodiments, the
polynucleotide encodes the full leng,th or mature protein encoded by the cDNA insert of
clone AM1060_1i deposited under acc~xxi~. n number ATCC 98237.
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In other embodirnents, 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:14;
(b) fragments of the amino acid sequence of SEQ ID NO:14; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AM1060_1i deposited under accession number ATCC 98237;
the protein being substantially free from other n ~mmAIi~n proteins. Preferably such
protein comprises the amino acid secluence of SEQ ID NO:14.
1 0 In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:16 from nucleotide 316 to nucleotide 438;
(c) a polynucleotide comprising the nucleotide sec~uence of the full
length protein coding sequence of clone AQ2_1i deposited under A~ ssion
number ATCC 98237;
(d) a polynucleotide encoding the full length protein encoded by the
2 0 cDNA insert of clone AQ2_li deposited under accession number ATCC 98237;
(e) a pol,vnucleotide com~ g the nucleotide sequence of the mature
protein coding sequence of clone AQ2_1i deposited under ac. ~ssion number
ATCC 98237;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AQ2_1i deposited under A~c~.ss;on number ATCC 98237;
(g) a pol,vnucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:17;
~h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:17 having biological activity;
3 0 ~i) a polynucleotide which is an allelic variant of a polynucleotide of
a)-(fl above; and
(j) a polynucleotide which encodes a species homologue of the protein
of (g) or (h) above.
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:16 from nucleotide 316 to nucleotide 438; the nucleotide sequence of the full length
protein coding sequence of clone AQ2_1i deposited under ~r(~5.~inn number ATCC 98237;
- or the nucleotide sequence of the mature protein coding sequence of clone AQ2_1i
5 deposited under accession number ATCC 98237. In other ~Lef~lled embodiments, the
~ polynucleotide encodes the full length or mature protein encoded by the cDNA insert of
clone AQ2_1i deposited under ~ inn number ATCC 98237. In yet other preferred
embodiments, such polynucleotide encodes a protein ~:OllL~ g the amino acid sequence
of SEQ ID NO:17 from amino acid 1 to amino acid 25.
1 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:17;
(b) the amino acid sequence of SEQ ID NO:17 from amino acid 1 to
amino acid 25;
(c) fragments of the amino acid sequence of SEQ ID NO:17; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AQ2_1i deposited under accession number ATCC 98237;
the protein being substantially free from other m:~mm~ n proteins. Preferably such
2 0 protein coll.~.ise~ the amino acid sequence of SEQ ID NO:17 or the amino acid sequence
of SEQ ID NO:17 from amino acid 1 to amino acid 25.
In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 142 to nucleotide 285;
(c) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone K433_1i deposited under ~fcf~ssinn
3 0 number ATCC g8237;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone K433_1i deposited under accession number ATCC 98237;
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(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone K433_1i deposited under accession number
ATCC 98237;
(fl a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone K433_1i deposited under ~c~-~s.ci-)n number ATCC 98237;
~g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:20;
(h) a polynudeotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:20 having biological activity;
1 0 (i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above; and
a) 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:19 from nucleotide 142 to nucleotide 285; the nucleotide sequence of the full length
protein coding sequence of clone K433_1i deposited under Acc~ ion number ATCC 98237;
or the nucleotide sequence of the mature protein coding sequence of clone K433_li
deposited under accession number ATCC 98237. In other ~ d embodiments, the
polynucleotide encodes the full length or mature protein encoded by the cDNA insert of
2 0 clone K433_li deposited under accession number ATCC 98237. In yet other pie~llèd
embodiments, such polynucleotide encodes a protein comprising the amino acid sequence
of SEQ ID NO:20 from amino acid 1 to amino acid 30.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from the group
2 5 consisting of:
(a) the amino acid sequence of SEQ ID NO:20;
(b) the amino acid sequence of SEQ ID NO:20 from amino acid 1 to
amino acid 30i
(c) fragments of the amino acid sequence of SEQ ID NO:20; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
K433_1i deposited under accession number ATCC g8237;
the protein being substantially free from other m:lmm~ n proteins. Preferably such
protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence
of SEQ ID NO:20 from amino acid 1 to amino acid 30.
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In one embodiment, the present invention provides a composition comprising an
isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:22;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
- NO:22 from nucleoffde 47 to nucleotide 517;
(c) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone L256_1i deposited under accession
number ATCC 98237;
(d) a polynucleotide encoding the full length protein encoded by the
cDNA insert of clone L256_1i deposited under accession number ATCC 98237;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone L256_1i deposited under a(c-o~.cion number
ATCC 98237;
(fl a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone L256_1i deposited under ~cc~c~inn number ATCC 98237;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:23;
(h) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID NO:23 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above; and
(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 from nucleotide 47 to nucleotide 517; the nucleotide sequence of the full length
protein coding sequence of c~one L256_1i deposited under accession number ATCC 98237;
or the nucleotide sequence of the mature protein coding sequence of clone L256_1i
deposited under ;~cc~ n number ATCC 98237. In other l~rer~ d embodiments, the
3 0 polynucleotide encodes the full length or mature protein encoded by the cDNA insert of
clone L256_1i deposited under accession number ATCC 98237. In yet other pl~f~ edemboriim~n~, such polynucleotide encodes a protein ~vl~ ,ing the amino acid sequence
of SEQ ID NO:23 from amino acid 8 to amino acid 157.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:23;
~b) the amino acid sequence of SEQ ID NO:23 from amino acid 8 to
amino acid 157;
(c) fragments of the amino acid sequence of SEQ ID NO:23; and
(d) the amino acid sequence encoded by the cDNA insert of clone
L256_1i deposited under accession number ATCC 98237;
the protein being substantially free from other mammalian proteins. Preferably such
protein comprises the amino acid sequence of SEQ I~ NO:23 or the amino acid sequence
of SEQ ID NO:23 from amino acid 8 to amino acid 157.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present invention.
Methods are also provided for ~l~v~lLLillg, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a therapeuticallyeffective amount of a composition comprising a protein of the present invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the pED6 and pNOTs vectors used for
deposit of clones disclosed herein.
2 5 DETAILED DESCRIPTION
ISOE~TED PROTEINS
Nucleotide and amino acid sequences, as ~re~ lly determined, are reported
below for each clone and protein disclosed in the present application. The nucleotide
sequence of each clone can readily be det~rmin~r1 by sequencing of the deposited clone
3 0 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 acidsequence of the protein encoded by a particular clone can also be determined by
expression of the clone in a suitable host cell, collecting the protein and determining its
sequence. For each disclosed protein applicants have identified what they have
12
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determined to be the reading frame best identifiable with sequence information available
at the time of filing.
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
5 sequences in its amino acid sequence. "Secreted" proteins include without limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors~ from the cell
in which they are expressed. "Secreted" proteins also include without limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
Protein"AE648 li"
One protein of the present invention has been identified as protein "AE648_1i".
A partial cDNA clone encoding AE648_1i was first isolated from a murine adult spleen
cDNA library using methods which are selective for cDNAs encoding secreted proteins
(see U.S. Pat. No. 5,536,637),or was identified as encoding a secreted or transmembrane
15 protein on the basis of computer analysis of the amino acid sequence of the encoded
protein. The nucleotide sequence of such partial cDNA was determined and searched
against the GenBank and GeneSeq databases using BLASTN/BLASTX and FASTA search
protocols. The search revealed at least some identity with sequences identified as
AA156847 (zll8dO5.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 5022812 0 5' similar to TR G624778 G624778 E25), AA269908 (va64alO.rl Soares mouse 3NME12 5
Mus musculus cDNA clone 736122 5' similar to TR G624778 G624778 E25), AF017985
(Gallus gallus putative transmembrane protein E3-16 mRNA, complete cds), D45302
(Human brain cDNA), H62690 (yr45hO4.rl Homo sapiens cDNA clone 208279 5'), N51010
(yv29blO.sl Homo sapiens cDNA clone 244123 3'), R62686 (yil2gO6.rl Homo sapiens
cDNA), and W66895 ~mel8bO8.rl Soares mouse embryo NbME13.5 14.5 Mus musculus
cDNA clone 387831 5'). The predicted amino acid sequence disclosed herein for AE648_1i
was searched against the GenPept and GeneSeq amino acid sequence databases using the
BLASTX search protocol. The predicted AE648_li protein demonstrated at least some
identity with sequences identified as AF017985 (putative transmembrane protein E3-16
3 0 [Gallus gallus]) and L38971 (putative [Mus musculus]) The human cDNA clone
corresponding to an EST database entry was ordered from Genome Systems, Inc., St.
Louis, Mo, a distributor of the I.M.A.G.E. Consortium library. The clone received from
the distributor was examined and ~l~t~rmin~i to be a full-length clone, including a 5' end
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.
and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as
"AE648_li".
Applicants' methods identified clone AE648_1i as encoding a secreted protein.
The nucleotide sequence of AE648_li as presently determined is reported in SEQ
ID NO:25. What applicants believe is the proper reading frame and the predicted amino
acid sequence of the AE648_1i protein corresponding to the foregoing nucleotide sequence
is reported in SEQ ID NO:26. Amino acids 3 to 15 are a predictcd leader/signal sequence,
with the predicted mature amino acid sequence beginning at amino acid 16, or are a
transmembrane domain.
1 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
AE648_li should be approximately 900 bp.
Protein"AE693 li"
One protein of the present invention has been identified as protein "AE693_1i".
A partial cDNA clone encoding AE693_1i was first isolated from a murine adult spleen
cDNA library using methods which are selective for cDNAs encoding secreted proteins.
The nucleotide sequence of such partial cDNA was determined and searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. The search
revealed at least some identity with ESTs identified as "yv56gll.sl Homo sapiens cDNA
2 0 clone 246788 3"' (N53173, BlastN), "ysO2dO1.rl Homo sapiens cDNA clone 213601 5"'
(H72198, BlastN) and "human adult lung 3' direct" (D45666, Fasta). The human cDNA
clone corresponding to the EST fl~h~ entry was ordered from Genome Systems, Inc.,
St. Louis, Mo, a distributor of the I.M.A.G.E. Consortiurn library. The clone received from
the distributor was examined and rl~t~rmined to be a full length clone, including a 5' end
2 5 and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as
"AE693_li".
Applicants' methods identified cIone AE693_1i as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AE693_li as presently ~f~rmine-l isreported in SEQ ID NO:4. What applicants believe is the proper reading frame and the
3 0 predicted amino acid sequence of the AE693_1i protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:5. Additional nucleotide sequence from
the 3' portion of AE693_1i, including the polyA tail, is reported in SEQ ID NO:6.
The EcoRI/NotI restriction fragment obtainable from the deposit c~ ldini~lg clone
AE693_1i should be ap~Lo~imately 1200 bp.
14
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Protein "AK438 1 i"
One protein of the present invention has been identified as protein "AK438_1i".
A partial cDNA clone encoding AK438_1i was first isolated from a human fetal kidney
cDNA library using methods which are selective for cDNAs encoding secreted proteins.
5 The nucleotide sec~uence of such partial cDNA was determined and searched against the
- GenBank database using BLASTA/BLASTX and FASTA search protocols. The search
revealed at least some identity with ESTs identified as "yf29dll.sl Homo sapiens cDNA
clone 128277 3"' (R11522, BlastN) and "yf29dll.rl Homo sapiens cDNA clone 128277 5"'
(R10447, BlastN). The human cDNA clone corresponding to the EST database entry was
10 ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E.
Consortium library. The clone received from the distributor was examined and
determined to be a full length clone, including a 5' end and 3' UTR (including a polyA
tail). This full-length clone is also referred to herein as "AK438_1i".
Applicants' methods identified clone AK438_1i as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AK438_1i as presently determined isreported in SEQ ID NO:7. What applicants believe is the proper reading frame and the
predicted amino acid sequence of the AK438_1i protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:8. Additional nucleotide sequence from
the 3' portion of AK438_li, including the polyA tail, is reported in SEQ ID NO:9.
Protein"AK609 li"
One protein of the present invention has been identified as protein "AK609_1i".
A partial cDNA clone encoding AK609_1i was first isolated from a human fetal kidney
cDNA library using methods which are selective for cDNAs encoding secreted proteins.
2 5 The nucleotide sequence of such partial cDNA was ~ rmin~ and searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. The search
revealed at least some identity with ESTs identified as "yilOeO6.sl Homo sapiens cDNA
clone 138850 3"' (R63679, BlastN) and "yilOeO6.rl Homo sapiens cDNA clone 138850 5"'
(R62724, BlastN). The human cDNA clone corresponding to the EST database entry was
30 ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E.
Consortium library. The clone received from the distributor was examined and
determined to be a full length clone, including a 5' end and 3' UTR (including a polyA
tail). This full-length clone is also referred to herein as "AK609_li".
Applicants' methods identified clone AK609_1i as encoding a secreted protein.
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The partial nucleotide sequence of AK609_1i, including its 5' end, as presently
deterrnined is reported in SEQ ID NO:10. What applicants believe is the proper reading
frame and the predicted amino acid sequence of the AK609_1i protein corresponding to
the foregoing nudeotide sequence is reported in SEQ ID NO:11. Additional nucleotide
5 sequence from the 3' portion, including any identified polyA tail, of AK609_li is reported
in SEQ ID NO:12.
Protein"AM1060 li"
One protein of the present invention has been i~ ntifie~ as protein "AMl060_1i".1 0 A partial cDNA clone encoding AM1060_1i was first isolated from a human fetal kidney
cDNA library using methods which are selective for cDNAs encoding secreted proteins.
The nucleotide sequence of such partial cDNA was determined and searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. The search
revealed at least some identity with ESTs identified as "yv74dll.sl Homo sapiens cDNA
done 248469 3"' (N58740, BlastN) and "H. spaiens partial cDNA sequence; clone c-13el2"
(Z43110, BlastN). The hurnan cDNA clone corresponding to the EST database entry was
ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E.
Consortium library. The clone received from the distributor was examined and
determined to be a full length clone, including a 5' end and 3' UTR (including a polyA
2 0 tail). This full-length clone is also referred to herein as "AM1060_li".
Applicants' methods id~onhfiefl clone AM1060_1i as encoding a secreted protein.
The nudeotide sequence of the 5' portion of AM1060_1i as presently determined
is reported in SEQ ID NO:13. What applicants believe is the proper reading frame and the
predicted arnino acid sequence of the AM1060_li protein corresponding to the foregoing
2 5 nucleotide sequence is reported in SEQ ID NO:14. Amino acids 1 to 25 are the predicted
leader/signal sequence, with the predicted mature amino acid sequence beginning at
amino acid 26. Additional nucleotide sequence from the 3' portion of AM1060_1i,
in- In~7ing the polyA tail, is reported in SEQ ID NO:15.
Protein "AQ2 1 i"
One protein of the present invention has been i-1~ntifie~ as protein "AQ2_1i". A -
partial cDNA clone encoding AQ2_1i was first isolated from a human ovary (PA-1
teratocarcinoma) cDNA library using methods which are selective for cDNAs encoding
16
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W O 98120130 PCT~US97/19857 _ -
secreted proteins. The nucleotide sequence of such partial cDNA was determined and
searched against the GenBank database using BLASTA/BLASTX and FASTA search
protocols. The search revealed at least some identity with ESTs identified as "yhl3bO6.rl
~ Homo sapiens cDNA clone 42937 5"' (R60437, BlastN) and "yjl2hlO.rl Homo sapiens
cDNA clone 148579 5"' ~H12590, BlastN). The human cDNA clone corresponding to the
EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor
of the I.M.A.G.E. Consortium library. The clone received from the distributor was
examined and det~rmined to be a full length clone, including a 5' end and 3' UTR(including a polyA tail). This full-length clone is also referred to herein as "AQ2_li".
1 0 Applicants' methods identified clone AQ2_1i as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AQ2_1i as presently determined is
reported in SEQ ID NO:16. What applicants believe is the proper reading frame and the
predicted amino acid sequence of the AQ2_1i protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:17. Additional nucleotide sequence from
the 3' portion of AQ2_1i, including the polyA tail, is reported in SEQ ID NO:18.
Protein"K433 li"
One protein of the present invention has been identified as protein "K433_1i". A2 0 partial cDNA clone encoding K433_1i was first isolated from a murine bone marrow
(stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs
encoding secreted proteins. The nucleotide sequence of such partial cDNA was
determined and searched against the GenBank database using BLASTA/BLASTX and
FASTA search protocols. The search revealed at least some identity with ESTs identified
as "yw69bll.sl Homo sapiens cDNA clone 257469 3"' (N27196, BlastN) and "ys86fO8.rl
Homo sapiens cDNA clone 221703 5"' (H92432, BlastN). The human cDNA clone
corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
Louis, Mo, a distributor of the I.M.A.G.E. Consortium library. The clone received from
the distributor was examined and ~et~rmined to be a full length clone, including a 5' end
3 0 and 3' UTR ~including a polyA tail). This full-length clone is also referred to herein as
"K433_1i".
Applicants' methods i-lentifie~l clone K433_1i as encoding a secreted protein.
The nucleotide sequence of the 5' portion of K433_li as presently determined is
reported in SEQ ID NO:19. What applicants believe is the proper reading frame and the
17
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W O 98/20130 PCT~US97119857 _ -
predicted amino acid sequence of the K433_1i protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:20. Additional nucleotide sequence from
the 3' portion of K433_1i, including the polyA tail, is reported in SEQ ID NO:21.
Protein"L256 li"
One protein of the present invention has been identified as protein "L256_1i". Apartial cDNA clone encoding L256_li was first isolated from a murine adult thymus
cDNA library using methods which are selective for cDNAs encoding secreted proteins.
The nucleotide sequence of such partial cDNA was determined and searched against the
O GenBank database using BLASTA/BLASTX and FASTA search protocols. The search
revealed at least some identity with ESTs identified as "yg53dO8.sl Homo sapiens cDNA
clone 36674 3"7 (R46636, BlastN) and "yf55eO4.rl Homo sapiens cDNA clone 25775 5"'
(R12334, BlastN). The human cDNA clone corresponding to the EST database entry was
ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E.
1 5 Consortium library. The clone received from the distributor was examined anddetermined to be a full length clone, including a 5' end and 3' UTR (including a polyA
tail). This full-length clone is also referred to herein as "L256_li".
Applicants' methods identified clone L256_1i as encoding a secreted protein.
The nucleotide sequence of the 5' portion of L256_1i as presently determined is
2 0 reported in SEQ ID NO:22. What applicants believe is the proper reading frame and the
predicted amino acid sequence of the L256_1i protein corresponding to the rol~goillg
nucleotide sequence is reported in SEQ ID NO:23. Additional nucleotide sequence from
the 3' portion of L256_1i, including the polyA tail, is reported in SEQ ID NO:24.
2 5 Deposit of Clones
Clones AE648_li, AE693_1i, AK438_li, AK609_1i, AM1060_li, AQ2_li, K433_1i
and L256_1i were deposited on October 31, 1996 with the American Type Culture
Collection as an original deposit under the Budapest Treaty and were given the accession
number ATCC 98237, from which each clone comprising a particular polynucleotide is
3 0 obtainable. All restrictions on the availability to the public of the deposited material will
be irrevocably removed upon the granting of the patent, except for the requirements
specified in 37 C.F.R. 1.808(b). Each clone has been transfected into separate bacterial
cells (E. coli) in this composite deposit.
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Each clone can be removed from the vector in which it was deposited by
performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the
appropriate fragment for such clone. Each clone was deposited in either the pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
5 pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman ef ~l.,
1991, Nucleic Acids Res. 19: 4485~490); the pNOTs vector was derived from pMT2
(Kaufman et al., 1989, Mol. Ccll. Biol. 9: 946-958) by deletion of the DHFR sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication in the ClaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the reverse
10 orientation) in the deposited isolate. In such instances, the cDNA insert can still be
isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' site
and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for
e,c~le~ion in a suitable vector. The cDNA may also be expressed from the vectors in
which they were deposited.
Bacterial cells containing a particular clone can be obtained from the compositedeposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the sequencesprovided herein, or from a combination of those sequences. In the sequences listed above
2 0 which include an N at position 2, that position is occupied in yr~r~lled probes/primers
by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for
example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy-2-~N-
biotinyl~aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-diiso~ yl)-phosphoramadite)
(Glen Research, cat. no. 10-1953)).
2 5 The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ~ C (assuming 2~ for each
3 0 A or T and 4 degrees for each G or C~.
The oligonucleotide should preferably be labeled with g-3ZP ATP (specific activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated
label should preferably be removed by gel filtration chromatography or other established
19
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WO 98/20130 PCTAUS97/19857 _ -
methods. The amount of r~ tivity incorporated into the probe should be quantitated
by measurement in a scintillation counter. PreÇerably, specific activity of the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably
5 be thawed and 100 ,ul of the stock used to inoculate a sterile culture flask containing 25 ml
of sterile L-broth cul~lai~ 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. Alicluots of these dilutions should preferably be plated to determine the
dilution and volume which will yield approximately 5000 distinct and well-separated
1 0 colonies on solid bacteriological media containing L-broth c~ ldi~ g ampicillin at 100
llg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37~C. Other
known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and Iyse, denature and bake them.
The filter is then preferably incubated at 65~C for 1 hour with gentle agitation in
6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with
NaOH) C~l ILd~ Ig 0.5% SDS, 100,ug/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
2 0 incubated at 65~C with gentle A~itAtinn overnight. The filter is then preferably washed in
500 mL of 2X SSC/0.5% SDS at room l~ pe~ldtUre without agitation, preferably followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
A third wash with 0.1X SSC/0.5% SDS at 65~C for 30 minutes to 1 hour is optional. The
filter is then preferably dried and subjected to autoradiography for sufficient time to
2 5 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,
hybri(li7~tion analysis, or DNA secluf~n~ing
3 0 Fragments of the proteins of the present invention which are capable of exhibiting
biological activity are also encompassed by the present invention. Fragrnents of the
protein may be in linear form or they may be cyclized using known methods, for example,
as described in H.U. Saragovi, et al., Bio/Technology :LQ, 773-778 (1992) and in iR.S.
McDowell, et al., ~. Amer. Chem. Soc. ~,9245-9253 (1992), both of which are incorporated
-
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W O 98/20130 PCTAUS97/19857_ -
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein binding
sites. For example, fragments of the protein may be fused through "linker" sequences to
~ the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion
5 could be to the Fc portion of an IgG m~ Other immunoglobulin isotypes may also
- be used to generate such fusions. For example, a protein - IgM fusion would generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in the sequence
10 listing by translation of the nucleotide sequence of each ~ lc)sefl 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 mammalian cell or other host cell.
The sequence of the mature form of the protein may also be de~rmin~hle from the amino
acid sequence of the full-length form.
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 intr~ r and tr~ncm~mhrane domains of the protein are deleted such that
the protein is fully secreted from the cell in which it is expressed. The intracellular and
transmembrane domains of proteins of the invention can be identified in accordance with
2 0 known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most
preferably at least 75~/O) of the length of a disclosed protein and have at least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90% or 95%
2 5 identity) with that disclosed protein, where sequence identity is determined by comparing
the amino acid sequences of the proteins when aligned so as to maximize overlap and
identity while minimi7ing sequence gaps. Also included in the present invention are
proteins and protein fragments that contain a segment preferably comprising 8 or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino acids that
30 shares at least 75% sequence identity (more preferably, at least 85% identity; most
preferably at least 95% identity) with any such segment of any of the disclosed proteins.
Species homologs of the disclosed proteins are also provided by the present
invention. Species homologs may be isolated and identified by making suitable probes
CA 02241911 1998-06-26
W O 98/20130 PCT~US97/19857 _ -
or primers from the sequences provided herein and screening a suitable nucleic acid
source from the desired species.
The invention also encompasses allelic variants of the disclosed proteins; that is,
naturally-occurring alternative forms of the isolated proteins which are identical,
5 homologous or related to that encoded by the polynucleotides disclosed herein.The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The isolated polynucleotide endcoing the protein of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED expression vectors
disclosed in Kaufrnan et al., Nucleic Acids Res. ~, 4485 4490 (1991), in order to produce
the protein recombinantly. Many suitable expression control sequences are known in the
art. General methods of expressing recombinant proteins are also known and are
exemplified in R. Kaufman, Methods in Enzymology ~, 537-566 (1990). As defined
herein "operably linked" means that the isolated polynucleotide of the invention and an
expression control sequence are situated within a vector or cell in such a way that the
protein is expressed by a host cell which has been transformed (transfectcd~ with the
ligated polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of the
protein. l~Amm~ n host cells include, for example, monkey COS cells, Chinese Harnster
2 0 Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants, He~a cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
Alternatively, it may be possible to produce the protein in lower eukaryotes such
2~i as yeast or in prokaryotes such as bacteria. Potentially suitable yeast skains include
Sa~h~ui"yces cerevisiae, Schizosaccharomyces pombe, Kll~yveromyces strains, Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimuri1lm, or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in yeast or
3 0 bacteria, it may be necessary to modify the protein produced therein, for example by
phosphorylation or ~,lyco~ylation of the a~lu~Liate sites, in order to obtain the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
CA 02241911 1998-06-26
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The protein may also be produced by operably linking the isolated polynucleotideof the invention to suita~le control sequences in one or more insect expression vectors,
and employing an insect ~x~le~ion system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit form from,
5 e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~) kit), and such methods are
- well known in the art, as described in S~lmm~rs and Smith, Texas A~;ricultural Experiment
Staffon Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
The protein of the invention may be prepared by culturing transformed host cellsunder culture conditions suitable to express the recombinant protein. The resulting
expressed protein may then be purified from such culture (i.e., from culture medium or
cell extracts~ using known purification processes, such as gel filtration and ion exchange
chromatography. The purification of the protein may also include an affinity column
containing agents which will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~3) or Cibacrom blue 3GA
Sepharose~); one or more steps involving hydrophobic interaction chromatography using
such resins as phenyl ether, butyl ether, or propyl ether; or immurlo~fflnity
chromatography.
2 0 Alternatively, the protein of the invention may also be expressed in a form which
will t~ ilit~ purification. For example, it may be expressed as a fusion protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and
2 5 InVitrogen, respectively. The protein can also be tagged with an epitope andsubsequently purified by using a specific antibody directed to such epitope. One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant3 0 methyl or other aliphatic groups, can be employed to further purify the protein. Some or
all of the foregoing purification steps, in various combinations, can also be employed to
provide a substantially homogeneous isolated recombinant protein. The protein thus
purified is substantially free of other m:~mm:3li;1n proteins and is defined in accordance
with the present invention as an "isolated protein."
23
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W O 98/20130 PCTAUS97/19857 _
- The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which
are char~cteri7l~ by somatic or germ cells containing a nucleotide sequence encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are
known to those skilled in the art. The synthetically-constructed protein sequences, by
virtue of sharing primary, secondary or tertiary structural and/or conformational
characteristics with proteins may possess biological ~lul~lLies in common therewith,
including protein activity. Thus, they may be employed as biologically active orimmunological 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 char~ct~ri7ecl 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. Moriif~l ~tinns
of interest in the protein sequences may include the alteration, substitution, replacement,
insertion or deletion of a selected amino acid residue in the coding sequence. For
example, one or more of the cysteine residues may be deleted or replaced with another
2 0 amino acid to alter the conformation of the molecule. Techniques for such alteration,
substitution, replacement, insertion or deletion are well known to those skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration, substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and d~livdLiv~s of the sequences of proteins which would be
2 5 expected to retain protein activity in whole or in part and may thus be useful for screening
or other immunological methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are believed to be encompassed by the
present invention.
3 0 USES AND BIOLO(:~ICAL ACTIVITY
The proteins of the present invention are expected to exhibit one or more of theuses or biological activities (including those associated with assays cited herein) iri~ntifi.od
below. Uses or activities described for proteins of the present invention may be provided
by administration or use of such proteins or by administration or use of polynucleotides
24
CA 02241911 1998-06-26
W O 98/20130 PCT~US97/19857 _ -
encoding such proteins (such as, for example, in gene therapies or vectors suitable for
introduction of DNA).
~ Research Uses and Utilities
The proteins provided by the present invention can similarly be used in assay todetermine biological activity, including in a panel of multiple proteins for high-
throughput screening; to raise antibodies or to elicit another immune response; as a
reagent (including the labeled reagent) in assays designed to quantitatively determine
levels of the protein (or its receptor) in biological fluids; as markers for tissues in which
the corresponding protein is prere~ ,tially expressed (either constitutively or at a
particular stage of tissue differentiation or deve}opment or in a disease state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds or potentially
binds to another protein (such as, for example, in a receptor-ligand interaction), the
protein can be used to identify the other protein with which binding occurs or to identify
inhibitors of the binding interaction. Proteins involved in these binding interactions can
also be used to screen for pepffde 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.
2 0 Methods for perforrning 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
Molec~ r Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
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 acidsupplement, use as a carbon source, use as a nitrogen source and use as a source of
3 0 carbohydrate. In such cases the protein of the invention can be added to the feed of a
particular or~ or can be adll~ lel~d as a separate solid or liquid preparation, such
as in the form of powder, pills, solutions, suspensions or capsules. In the case of
microorganisms, the protein of the invention can be added to the medium in or on which
the microorganism is cultured.
CA 02241911 1998-06-26
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Cytokine and Cell Proliferation/Differentiation Activitv
A protein of the present invention may exhibit cytokine, cell proliferation (either
inducing or inhibiting~ or cell Llif~l~nLiation (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 acffvity of a protein of the present invention is
evidenced by any one of a number of routine factor dependent cell proliferation assays
for cell lines including, without limitation, 32D, DA2, DAlG, T10, B9, B9/11, BaF3,
1 0 MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
1 5 descri~ed 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-Tnt~rc~ nce (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
2 0 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, Iymph node
cells or thymocytes include, without limitation, those described in: Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Cllrrent Protocols in Imml~nology. J.E.e.a.
2 5 Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human I1IL~1~1O1~ ~y, Schreiber, R.D. In Current Protocols in
Imml~nology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and Iymphopoietic
cells include, without limit~hon, those described in: Measurement of ~Iuman and Murine
3 0 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Cl~rrent
Protocols in ImmunologLJ. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:69n-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Ct~rrenf Protocols in
26
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Immunology. J.E.e.a. Coligan eds. Vol 1 pp.6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human
Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols
~ in 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 Cl~rrent Protocols in Immu7zology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others,
proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring
1 0 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-lnterscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and
their cellular rect:~7Lo-~; Chapter 7, Immunologic studies in Humans); Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:40S-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988.
Immune Stimulating or Suppressin~ Activitv
A protein of the present invention may also exhibit immune stimulating or
~mmune suppressing activity, including without limitation the activities for which assays
are described herein. A protein may be useful in the treatment of various immunedeficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or 13 lymphocytes, as well
2 5 as effecting the cytolytic activity of NK cells and other cell populations. These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal
infections, or may result from autoimmune disorders. More specifically, infectious
~licf~cf~c causes by viral, bacterial, fungal or other infection may be treatable using a
protein of the present invention, including infections by HIV, hepatitis viruses,
3 0 herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections
such as car.~ 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.
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Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis, systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,
5 myasthenia gravis, graft-versus-host disease and autoimmune i~fl~mm~t ry eye disease.
Such a protein of the present invention may also to be useful in the treatment of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory
problems. Other conditions, in which immune suppression is desired (including, for
example, organ transplantation), may also be treatable using a protein of the present
1 0 invention.
Using the proteins of the invention it may also be possible to immune responses,in a number of ways. Down regulation may be in the form of inhibiting or blocking an
immune response already in progress or may involve preventing the induction of an
immune response. The functions of activated T cells may be inhibited by suppressing T
15 cell responses or by inducing specific tolerance in T cells, or both. Immunos~ ion
of T cell responses is generally an active, non-antigen-specific, process which requires
continuous exposure of the T cells to the su~les~iv~ agent. Tolerance, which involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after exposure to
2 0 the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack
of a T cell response upon reexposure to specific antigen in the absence of the tnlf ri~ing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing
2 5 high level lymphokine synthesis by activated T cells, will be useful in situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For example,
blockage of T cell function should result in reduced tissue destruction in tissue
transplantation. Typically, in tissue transplants, rejection of the transplant is initiated
through its recognition as foreign by T cells, ~ollowed by an immune reaction that destroys
3 0 the transplant. The administration of a molecule which inhibits or blocks interaction of
a B7 lymphocyte antigen with its natural ligand(s) on imrnune cells (such as a soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with a
monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the
28
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molecule to the natural ligand(s) on the immune cells without 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 toanergize the T cells, thereby inducing tolerance in a subject. lnduction of long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated
administration of these blocking reagents. To achieve sufficient immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or G~D can be A~ Re~ using animal models that are predictive of efficacy in
humans. Examples of a~ liate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as
described in Lerlschow ~t nl., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad.
Sci USA, 89:11102-11105 (19g2). In addition, murine models of GVHD (see Paul ed.,
Flln~ ntal Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to
determine the effect of blocking B lymphocyte antigen function in vivo on the development
of that disease.
2 0 Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inap~lo~liate
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 ('ii.cf~ R~ Pl~v~ lg the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 5 Administration of reagents 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 which could lead to long-term relief from
3 0 the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune
disorders can be determined using a number of well-characterized animal models of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and
29
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murine experimental myasthenia gravis (see Paul ed., Fundamental 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 rcgulating immune responses, may also be useful in therapy.
5 Upregulation of immune responses may be in the form of enhancing an existing immune
response or eliciting an initial immune response. For example, enhancing an immune
response through stimulating B lymphocyte antigen function may be useful in cases of
viral infection. In addition, systemic viral diseases such as influenza, the common cold,
and encephalitis might be aUeviated by the administration of stimulatory forms of B
10 lymphocyte antigens syst.omi~ ~lly.
Alternatively, anti-viral immune responses may be enhanced in an infected patient
by removing T cells from the patient, costimulating the T cells in vi~ro with viral antigen-
pulsed APCs either expressing a peptide of the present invention or together ~,vith a
stimulatory form of a soluble peptide of the present invention and reintroducing the in
15 vitro activated T cells into the patient. Another method of enhancing anti-viral immune
responses would be to isolate infected cells from a patient, transfect them with a nucleic
acid encoding a protein of the present invention as described herein such that the cells
express all or a portion of the protein on their surface, and reintroduce the transfected
cells into the patient. The inff~ te~l cells would now be capable of delivering a
2 0 costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B Iymphocyte antigen function~ may be useful in the induction of tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of the present
2 5 invention can be administered to a subject to overcome tumor-specific tolerance in the
subject. If desired, the tumor cell can be trAn~fecte~l to express a combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo with an
expression vector directing the expression of a peptide having B7-2-like activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity. The
3 0 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
CA 02241911 1998-06-26
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costimulation signal to T cells to induce a T cell mediated immune response against the
transfected tumor cells. In addition, tumor cells which lack Ml IC 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
5cytoplasmic-domain truncated portion) of an MHC class I a chain protein and ,(32
microglobulin protein or an MHC class II cc 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 lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated
10immune response against the transfected tumor cell. Optionally, a gene encoding an
antisense construct which blocks expression of an MHC class II associated protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated
15imm~1ne response in a human subject may be sufficient to ~v~lc~ e tumor-specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
2 0limitation, 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 I.ymphocyte Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); ~f~ nn et al., Proc. Natl. Acad. Sci.
USA 78:2488-2492, 1981; Herrmann et al., J. IIrununol. 128:1968-1974, 1982; Handa et al.,
2 ~iJ. Immunol. 135:156~1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.140:508-512, 1988; ~Ierrmann 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.,
3 0Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays l'or T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation, those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: I~2 vitro
31
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W O 98/20130 PCT~US97/198S7 _ -
antibody production, Mond, J.J. and Brunswick, M. In Cllrrent Profocols ill Imm2~nology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR~ assays (which will identify, among others,
proteins that ~;e~ dle predominantly Thl and CTL responses) include, without limit ition~
5 those described in: Current Protocols in Irnmunology, 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 Funcffon 3.1-3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays ~which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
2 0 proteins that prevent apoptosis after superantigen induction and proteins that regulate
lymphocyte hom.oost~cis) in~ , without limitation, those described in: Darzynkiewicz
et al., Cytometry 13:795-808, 1992; Gorczyca et al., T~l1kf~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 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell comm~tm~nt and
development include, without limitation, those described in: Antica et al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re~ulating Activity
A protein of the present invention may be useful in regulation of hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even
marginal biological activity in support of colony forming cells or of factor-dependent cell
32
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- W O98/20130 PCTrUS97/198~7 _ -
lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and
proliferation of erythroid progenitor cells alone or in combination with other cytokines,
thereby indicating utility, for example, in treating various anemias or for use in
conjunction with irradiation/chemotherapy to stimulate the production of erythroid
5 precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid
- cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity)
useful, for example, in conjunction with chemotherapy to prevent or treat consequent
myelo-suppression; in supporting the growth and proliferation of ml~g~k~ryocytes and
consequently of platelets thereby allowing prevention or treatment of various platelet
1 0 disorders such as thrombocytopenia, and generally for use in place of or complimentary
to platelet transfusions; and /or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the above-
mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell
disorders (such as those usually treated with transplantation, including, without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in
repopulating the stem cell compartrnent post irr~ n/chemotherapy, either irl-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells orgenetically manipulated for gene therapy.
2 0 The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines
are cited above.
Assays for embryonic stem cell difrelelLliation ~which will identify, among otherE,
2 5 proteins that influence embryonic dirrelel~liation hematopoiesis) include, without
limi~a1;on, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; Mc(~l~n~h~n et al., Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
3 0 others, proteins that regulate lympho-hematopoiesis) include, without limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Ct~lture of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York,
NY. 1994; ~irayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and
33
CA 02241911 1998-06-26
W O 98/20130 PCTrUS97/19857 _ -
Briddell, R.A. In Cultl(re of HemQ~opoietic Cells. R.I. Freshney, ef al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Cl~lture of Hema~opoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long
5 term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and
Allen, T. In Culfure of Hema~opoietic Cells. R.I. Freshney, ef al. eds. Vol pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, ef 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~cem~nt, and in the treatment of burns,
15 incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the healing of
bone fractures and cartilage damage or defects in humans and other animals. Such a
preparation employing a protein of the invention may have prophylactic use in closed as
2 0 well as open fracture reduction and also in the improved fixation of artificial joints. De
novo bone formation induced by an osteogenic agent cunLIiL,ul~s to the repair ofcongenital, trauma induced, or oncologic resection induced craniofacial defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
5 disease, and in other tooth repair processes. Such agents may provide an environment
to attract bone-forming cells, stimulate growth of bone-forming cells or induce
differentiation of prog~llilulti of bone-forming cells. A protein of the invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation
of bone and/or cartilage repair or by blocking irlfl~mm~tion or processes of tissue
30 destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to theprotein of the present invention is tendon/ligament formation. A protein of the present
invention, which induces tendon/ligament-like tissue or other tissue formation in
34
-
CA 02241911 1998-06-26
W O 98/20130 PCTrUS97/19857 _ -
circumstances where such tissue is not normally formed, has application in the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a tendon/ligament-like ffssue inducing
protein may have prophylactic use in pl~v~llLillg damage to tendon or ligament tissue, as
5 well as use in the improved fixation of tendon or ligament to bone or other tissues, and
- in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue
fnrm:~h~n induced by a composition of the present invention contributes to the repair of
congenital, trauma induced, or other tendon or ligament defects of other origin, and is also
useful in cosmetic plastic ~:iUl~g~l,y for attachrnent or repair of tendons or ligaments. The
10 compositions of the present invention may provide an environment to attract tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of
tendon/ligament cells or pro~,~luLul~ ex vivo for return in vivo to effect tissue repair. The
compositions of the invention may also be useful in the treatrnent of tendinitis, carpal
15 tunnel syndrome and other tendon or ligament defects. The compositions may also
include an d~lo~l;ate matrix and/or sequestering agent as a carrier as is well known in
the art.
The protein of the present invention may also be useful fcr proliferation of neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and
2 0 peripheral nervous system diseases and neuropathies, as well as mechanical and
LldLullaLic disorders, which involve degeneration, death or trauma to neural cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases of the
peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and
localized neuropathies, and central nervous system diseases, such as Alzheimer's,
2 5 Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome. Further ~nn~i*nns which may be treated in accordance with the present
invention include mechanical and traumatic disorders, such as spinal cord disorders, head
trauma and ce:leblovdscular diseases such as stroke. Peripheral neuropathies resulting
from chemotherapy or other medical therapies may also be treatable using a protein of the
3 0 invention.
Proteins of the invention may also be useful to promote better or faster closure of
non-healing wounds, including without limit~ti~n pressure ulcers, ulcers associated with
vascular insl-ffi- iPn~y, surgical and tr~llm~tic wounds, and the like.
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It is expected that a protein of the present invention may also exhibit activity for
generation or regeneration of other tissues, such as organs (including, for example,
pancreas, liver, intestine, kidney, skin, endothelium~, muscle (smooth, skeletal or cardiac)
and vascular (incIuding vascular endothelium) tissue, or for promoting the growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfucion injury in various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting
differentiation of tissues described above from precursor tissues or cells; or for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
tinnal Patent Publication No. W095/05846 (nerve, neuronal); International PatentPublication No. W091/07491 (skin, endothelium ).
- 2 0 Assays for wound healing activity include, without limitation, those described in:
Winter, Epiderrnal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by 1~1ct~in and Mertz, J. Invest.
Dermatol 71:382-84 (1978).
2 5 Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related
activities. Inhibins are characterized by their ability to inhibit the release of follicle
stimulating hormone (FSH), while activins and are char~ct~ri7er7 by their ability to
stimulate the release of follicle stim~ ting hormone (FSH). Thus, a protein of the present
3 0 invention, alone or in heterodimers with a member of the inhibin a family, may be useful
as a contraceptive based on the ability of inhibins to decrease fertility in female mammals
and decrease spermatogenesis in male mammals Administration of sufficient amounts
of other inhibins can induce infertility in these m~mm~lc Alternatively, the protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-
36
CA 02241911 1998-06-26
- W O 98/20130 PCTrUS97/19857 _ -
,B group, may be useful as a fertility inducing therapeutic, 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 protein of the invention may also be useful for
advancement of the onset of fertility in sexually immature rr~mm~l~, so as to increase the
5 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:
Assays for activin/inhibin activity include, without limit~tit>n, those described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
1 0 al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity15 (e.g., act as a chemokine) for m~mm~ n cells, in~ fling, for example, monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins provide
particular advantages in treatment of wounds and other trauma to tissues, as well as in
2 0 treatment of localized infections. ~or example, attraction of Iymphocytes, monocytes or
neutrophils to tumors or sites of infection may result in improved immune responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it
can stimulate, directly or indirectly, the directed orientation or movement of such cell
2 5 population. Preferably, the protein or peptide has the ability to directly stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a population
of cells can be readily determined by employing such protein or peptide in any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
3 0 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent
chemotaxis) consist of assays that measure the ability of a protein to induce the migration
of cells across a membrane as well as the ability of a protein to induce the adhesion of one
cell population to another cell population. Suitable assays for movement and adhesion
37
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include, without limitation, those described in: Current Protocols in Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; ~ind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of
Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity.1 0 As a result, such a protein is expected to be useful in treat~nent of various coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery or other
causes. A protein of the invention may also be useful for dissolving or inhibiting
formation of thromboses and for treatment and ~l~v~nlion of conditions resultingL S therefrom (such as, for example, infarction of cardiac and central nervous system vessels
(e.g., stroke3.
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 limit~h(~n, those
2 0 described in: Linet et al., J. Clin. p~rm;~col 26:131-140, 1986; Burdick et al., Thrombosis
Res.45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins
3!~:467~74, 1988.
Receptor/Ligand Acffvity
2 5 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 limit~til~n~ cytokine receptors and their
ligands, receptor kinases and their ligands, receptor phosphatases and their ligands,
le< ~ L~ involved in cell-cell interactions and their ligands (irl~lu.ling without limit~tion,
3 0 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 (including, without
38
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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 in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 198?;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670,
1995.
Anti-Inflammator,v Acffvity
Proteins of the present invention may also exhibit anti-inflAmmAtory activity. The
anti-infl~mmAt )ry activity may be achieved by providing a stimulus to cells involved in
the infl~mmAtory response, by inhibiting or promoting cell-cell interactions (such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the
irlfl~mmAtory process, inhibiting or promoting cell extravasation, or by stimulating or
~ plc~Sillg production of other factors which more directly inhibit or promote an
inflammatory response. Proteins exhibiting such activities can be used to treat
inflAmmAtory conditions including chronic or acute conditions), including without
limitAtion inflAmmAtion associated with infection ~such as septic shock, sepsis or systemic
irlflAmmAtory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or
~hf-mokin~-induced lung injury, inflAmmAtory bowel disease, Gohn'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 hyp~ .~iliviLy to an antigenic substance or m~t~riAl
3 0 Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties linked to
tumor growth and metastasis. C~A~lh~rin malfunction is also implicated in other human
39
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~lic~c~, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found in other
parts of the m~le~ The cadherin domains bind calcium to form their tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids in the
first cadherin domain provide the basis for homophilic adhesion; modification of this
recognition site can change the specificity of a cadherin so that instead of recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In addition, some
cadherins engage in heterophilic adhesion with other t :~lh~rin!;
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines with
polynucleotides expressing E-cadherin has reversed cancer-associated changes by
returning altered cell shapes to norrnal, restoring cells' adhesiveness to each other and to
their substrate, decreasing the cell growth rate, and drastically reducing anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas
to a less advanced stage. It is likely that other cadherins have the same invasion
2 0 ~;U~ OL role in carcinomas derived from other tissue types. Therefore, proteins of the
present invention with cadherin activity, and polynucleotides of the present invention
encoding such proteins, can be used to treat cancer. Introducing such proteins or
polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed
in these cells by providing normal cadherin ~k~l~s~iion.
2 5 Cancer cells have also been shown to express r:~1ht~rins of a different tissue type
than their origin, thus allowing these cells to invade and metastasize in a different tissue
in the body. Proteins of the present invention with cadherin activity, and polynucleotides
of the present invention encoding such proteins, can be substituted in these cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties and
3 0 reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention ~rith cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from
CA 02241911 1998-06-26
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forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the
cancer, the less cadherin expression there will be, and this decrease in cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-
nucleotides of the present invention encoding such protein fragments, can also be used
to block cadherin function by binding to cadherins and preventing them from binding in
ways that produce undesirable effects. Additionally, fragments of proteins of the present
1 0 invention with cadherin activity, preferably truncated soluble cadherin fragments which
have been found to be stable in the circulation of cancer patients, and polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995;
MiyaW et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, ~990.
Tumor Inhibition Activit,v
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.
2 0 A protein may inhibit tumor growth directly or indirectly (such as, for example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor
growth (such as, for exa~nple, by inhibiting angiogenesis), by causing production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing, f~limin:lting
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional
activities or effects: inhibiting the growth, infection or function of, or killing, infectious
3 0 agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or diminution,
change in bone form or shape); effecting biorhythms or ca~ricadic cycles or rhythms;
41
,
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effecting the fertility of male or female subjects; effecting the metabolism, catabolism,
anabolism, processing, utiLization, storage or ~limin~*on of dietary fat, lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s);
f~ff~t~ting behavioral characteristics, including, without limitation, appetite, libido, stress,
5 cognition (including cognitive disorders), depression (including d~r~ssiv~ disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects; promoting
differentiation and growth of embryonic stem cells in lineages other than hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies
of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative
10 disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the ability to act as an antigen
in a vaccine composition to raise an immune response against such protein or another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOS~NG
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used in a
pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
2 0 Such a composition may also contain (in arl~lition to protein and a carrier) diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other m~tPri~ls well known in the art. The term
"pharm~c~llhr~lly acceptable" means a non-toxic m~t~ri~l that does not interfere with the
effectiveness of the biological activity of the active ingredient(s). The characteristics of the
carrier will depend on the route of administration. The pharmaceutical composition of
2 5 the invention may also contain cytokines, lymphokines, or other hematopoietic factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL~, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem
cell factor, and ~:.y LlL~poietin. The pharmaceutical composition may further contain other
agents which either enhance the activity of the protein or compliment its activity or use
3 0 in treatment. Such additional factors and/or agents may be included in the
pharm~ ellti. ~l composition to produce a synergistic effect with protein of the invention,
or to minimize side effects. Conversely, protein of the present invention may be included
in formulations of the particular cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects
42
,
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of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers
or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical
compositions of the invenffon may comprise a protein of the invention in such mtllhm-~ri~
or complexed form.
The pharmaceutical 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 stim~ tory signal to both B and T Iymphocytes. B
1 0 lymphocytes will respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR) following
presentation of the antigen by MHC proteins. MHC and structurally related proteins
including those encoded by class I and class II MHC genes on host cells will serve to
present the peptide antigen(s~ to T lymphocytes. The anffgen components could also be
1 5 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.
2 0 The pharm~rP~1ti~-~l composition of the invention may be in the form of a liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist
in aggregated form as micelles, insoluble monolayers, licluid crystals, or l~m~ r layers
in aqueous solution. Suitable lipids for liposomal formulation include, without limit~ti(~n,
monoglycerides, diglycerides, s1llfati~ s, lysolecithin, phospholipids, saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or
amelioration of the relevant medical condition, or an increase in rate of treatment, healing,
prevention or amelioration of such conditions. When applied to an individual active
43
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W O 98/20130 PCTrUS97/19857 _ -
ingredient, administered alone, the term refers to that ingredient alone. When applied to
a combination, the term refers to combined amounts of the active ingredients that result
in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a
5 therapeutically effective amount of protein of the present invention is administered to a
mammal having a condition to ke treated. Protein of the present invention may beadministered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines, Iymphokines
or other hematopoietic factors. When co-administered with one or more cytokines,10 lymphokines or other hematopoietic factors, protein of the present invention may be
administered either simultaneously with the cytokine(s), Iymphokine(s~, other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially, the attending physician will decide on the appropriate
sequence of administering protein of the present invention in combination with
15 cytokine(s), Iymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried out in a
variety of conventional ways, such as oral ingestion"nhalation, topical application or
2 0 cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is plerell~d.
When a therapeutically ~e~liv~ amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a tablet,
capsule, powder, solution or elixir. When administered in tablet form, the phArmAcf~uti~ Al
2 5 composition of the invention may additionally contain a solid carrier such as a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the
present invention, and preferably from about 25 to 90% protein of the present invention.
When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils
3 0 may be added. The liquid form of the p~ArmAf~utical composition may further contain
physiological saline solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein
44
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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
ad~lLLLIisl~l~d by intravenous, cutaneous or subcutaneous injection, protein of the present
5 invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
~ The preparation of such parenterally acceptable protein solutions, having due regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A p~ dpharmAcellti~-Al composition for intravenous, cutaneous, or subcutaneous injection should
contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium
10 (~hl~ri~le Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, T ActAtPr~ 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 composition15 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.
Ultim~tPly, 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
2 0 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 c~n~ plated that the various pharmaceutical compositions used
to practice the method of the present invention should contain about 0.01 ,ug to about 100
mg (preferably about 0.1ng to about 10 mg, more preferably about 0.1 ,ug to about l mg)
2 5 of protein of the present invention per kg body weight.
The duration of LLIlldv~llous Ill~ld~Jy using the phArmAfeutical 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
col.l~llL~lated that the duration of each application of the protein of the present invention
3 0 will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the AttPn~lin~ 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
CA 02241911 1998-06-26
W O 98/20130 PCT~US97/198~7 _ -
antibodies may be obtained using either the entire protein or fragments thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for example, as in
R.P. Merrifield, J. Amer.Chem.Soc. ~, 2149-2154 (1963); J.L. Krstenansky, et ~l., FEBS Lett.
211, 10 (1987~. Monoclonal antibodies binding to the protein of the invention may be
useful diagnostic agents for the imml~nndetection of the protein. Neutralizing monoclonal
antibodies binding to the protein may also be useful therapeutics for both conditions
associated with the protein and also in the treatment of some forms of cancer where
abnormal expression of the protein is involved. In the case of cancerous cells or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in detecting
and preventing the metastatic spread of the cancerous cells, which may be r~e~ t~ by
the protein.
For compositions of the present invention which are useful for bone, cartilage,
tendon or ligament regeneration, the therapeutic method includes administering the
composition topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention is, of course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may desirably
be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or
2 0 tissue damage. Topical administration may be suitable for wound healing and tissue
repair. Therapeutically useful agents other than a protein of the invention which may also
optionally be included in the composition as described ahove, may alternatively or
additionally, be administered ~imlllt~neously or sequentially with the composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
2 5 composition would include a matrix capable of delivering the protein-containing
composition to the site of bone and/or cartilage damage, providing a structure for the
developing bone and cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other implanted medical
applications.
3 0 The choice of matrix material is based on biocompatibility, biodegradabilit,v,
mechanical properties, cosmetic appearance and interface properties. The particular
application of the compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically defined calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and
46
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W O 98/20130 PCTrUS97/19857 _ -
polyanhydrides. Other potential materials are biodegradable and biologically well-
defined, such as bone or derrnal collagen. Further matrices are comprised of pure proteins
or extracellular matrix components. Other potential matrices are nonbiodegradable and
chPmi~AIly defined, such as sintered hydroxapatite, bioglass, aluminates, or other
5 ceramics. Matrices may be comprised of combinations of any of the above mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle shape, and
biodegradability.
Presently ~.~L:lled is a 50:50 (mole weight) copolymer of lactic acid and glycolic
acid in the form of porous particles having diameters ranging from 150 to 800 microns.
In some applications, it will be useful to utilize a sequestering agent, such ascarboxymethyl cellulose or autologous blood clot, to prevent the protein compositions
from disassociating from the matrix.
A preferred family of sequestering agents is ~ sj. materials such as
alkylcelluloses (inrli~(ling hydroxyalkyl~ loses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxy~ro~yl-
methylcellulose, and carboxymethylcellulose, the most ~ .ed being cationic salts of
carboxymethylcellulose (CMC). Other p l~ d sequestering agents include hyaluronic
2 0 acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl
polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents theamount necessary to prevent desorbtion of the protein from the polymer matrix and to
provide d~n~.iate handling of the composition, yet not so much that the progenitor cells
2 5 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 b~nf~fit ~l to the treatment of the bone and/or cartilage defect, wound, or tissue in
question. These agents include various growth factors such as epicl~rm;~l growth factor
3 0 (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-c~ and
TGF-,13), 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.
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The dosage regimen of a protein-containing phArm~ utical composition to be
used in tissue regeneration will be determined by the attending physician considering
various factors which modify the action of the proteins, e.g., amount of tissue weight
desired to be formed, the site of damag, the condition of the damaged tissue, the size of
5 a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity
of any infection, time of administration and other clinical factors. The dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other proteins in
the pharmaceutical composition. For example, the addition of other known growth
factors, such as IG~ I (insulin like growth factor I), to the final composition, may also effect
10 the dosage. Progress can be monitored by periodic assessment of tissue/bone growth
and/or repair, for example, X-rays, histomorphometric determinations and tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy. Suchpolynucleotides can be introduced either in vivo or ex vivo into cells for expression in a
15 rr.~mm~ n subject. Polynucleotides of the invention may also be administered by other
known methods for introduction of nucleic acid into a cell or organism (including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or activity in such cells.
2 0 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.
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SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT: Jacob~, Kenneth
McCoy, John
LaVallie, Edward
Racie, Lisa
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS
~iii) NUMBER OF SEQUENCES: 26
(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 Rr~AnARr~r~ FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version ~1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TT~RT~coMMrlNIcATIoN INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
49
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CAAGAAGGAC GAGCCCAAGA GCAGCGAGGA GGCGCTCATC GTCCCTCCGG ATGCCGTGGC 60
GGTGGATTGC AAGGACCCGG GTGACGTGGT TCCGGTTGGA CAGAGGAGAG CGTGGTGTTG 120
GTGCATGTGT TTCGGACTGG CCTT QTGCT TGCTGGCGTC ATCCTCGGAG GGGCGTACCT l80
GTACAAGTAT TTTGCTCTTC AGCCAGATGA TGTGTACTAC TGTGGACTAA AGTACATCAA 240
AGATGACGTC ATCCTGAACG AGCCTTCTGC GGATGCCC Q GCTGCTCGCT ACCAGACAAT 300
TGAAGAGAAC ATTAAGATCT TT --- - 322
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCPIPTION: SEQ ID NO:2:
Met Cys Phe Gly Leu Ala Phe Met Leu Ala Gly Val Ile Leu Gly Gly
l 5 l0 15
Ala Tyr Leu Tyr Lys Tyr Phe Ala Leu Gln Pro Asp Asp Val Tyr Tyr
Cys Gly Leu Lys Tyr Ile Lys Asp Asp Val Ile Leu Asn Glu Pro Ser
Ala Asp Ala Pro Ala Ala Arg Tyr Gln Th~ Ile Glu Glu Asn Ile Lys
50 55 60
Ile Phe
(2) INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
O (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCCCACCCTT NACATTTTGT GCAGTGATTA 'l"l'L~'l"l"l'AAAN TNTTNTTTCA TGTAAGTAGC 60
AAACAGGGCT TTACTATNTT TTCATCTCAT TAATTCAATT AAAACCATTA CCTTAAAAAA 120
AAAAAANAAA ~AAA~AA AAAAA 145
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GTTTGACCTG GCTGGAATAA CGTGTGGGCA CTTCCTTGAA C~ l~GA ~ ~G 60
TGCAACCCTG ATTGGGAAAG CAATCATTAA AATGCATATC CAGAAAATAT TTGTTATAGT 120
AACTTTCAGC AAGCACATCG TGGAGCAGAT GGTGACTTTC ATTGGTGCTG TCCCCGGCAT 180
AG~l~C~ CTGCAGAAGC CTTTTCAAGA GTACCTGGAG GCGCAGCGGC AGAAGCTTCA 240
TCACAGAAGT GAAGCGGGCA CACCGCAG 268
(2) INFOR~ATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 amino acids
(B) TYPE: amino acid
( C ) STRANnF~nMF~ s:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met His Ile Gln Lys Ile Phe Val Ile Val Thr Phe Ser Lys His Ile
1 5 10 15
_
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Val Glu Gln Met Val Thr Phe Ile Gly Ala Val Pro Gly Ile Gly Pro
Ser Leu Gln Lys Pro Phe Gln Glu Tyr Leu Glu Ala Gln Arg Gln Lys
35 40 45
Leu His His Arg Ser Glu Ala Gly Thr Pro Gln
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
tii~ MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID MO:6:
GAGACAGTAT AAGGAAAATC TGGTTGGTGT CTNACAAGTG AGCNGACACC A~ ATT 60
~l'~'l'~'l'ATTT AGAATGAAGT CTTGAAAAAA ACTTAAAAAA GACAACTTTA ATCATTCCAA 120
AAAAAAAAA~ AAAAAAAA l38
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LEMGTH: 415 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AACAGAGAAA GAAACCACCC AAGAGTATAT CAGAATCGGG ATTTCCGAGG TCACAACAGA 60
GGCTATAGAA GGCCCTATTA TTTCCGTGGG CGTAACAGAG GCTTTTATCC ATGGGGCCAA l20
TATAACCGAG GAGGCTATGG AAACTACCGC TCAAATTGGC AGAATTACCG GCAAGCATAC l80
AGTCCTCGTC GAGGCCGTTC AAGATCCCGG MCCCCAaAAA AAAGNTCCCC TCCNCCANGG 240
TCNAGAACCC NTCCNAAAAC CNCTAATANT TCTNCTCCTA ACCGNTCANG GCCCCCCNCN 300
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CCCCCCCTTC CTCCCCCCAN CCNTACCCAA TTTAATNCTC CTAACCCCAN TTNTNCAAAG 360
~A~ATT CCCCTCCNAA GNATACCCGG CCNNCTCAGG CTNCNGGGAA TANCC 4l5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Glu Thr Thr Ala Gln Ile Gly Arg Ile Thr Gly Lys His Thr Val
l 5 l0 15
Leu Val Glu Ala Val Gln Asp Pro Gly Pro Gln Lys Lys Xaa Pro Leu
Xaa Xaa Gly Xaa Glu Pro Xaa Xaa Lys Pro Leu Ile Xaa Leu Leu Leu
Thr Xaa Xaa Gly Pro Pro Xaa Pro Pro Phe Leu Pro Pro Xaa Xaa Pro
~5 60
Asn Leu Xaa Leu Leu Thr Pro Xaa Xaa Gln Arg Lys Lys Ile Pro Leu
65 70 75 80
Xaa Xaa Ile Pro Gly Xaa Leu Arg Leu Xaa Gly Ile
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AATTTGCCGA GTGGTGCCGG GTATCAGTTT GGGAAANACC AAGGTCAGTT TGACCATGGT 60
TTTGGGTCCC NGNGTCCATC CAAAAAGNGC CCTGTGGGNA AGNGTNCACC ATCCAATGGG 120
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_ TNCAAANATG GNTNATTTCA GNAGGNGGAG NGTGCTGNTT CAGGNGGNGC AGCCTATANA l80
AAGNGGTATT TAGNAGAGCA GAAGACAGAG GATGGGAAAG ATNAGGGACA GNAACAAACN 240
AATACCGNTN AAAAAAAAaA AAAAAAAA 268
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: cDNA
(xi) ~:Qu~:N~: DESCRIPTION: SEQ ID NO:lQ:
ACAGAGCCTC TACCTGG QG GAAACAAGTC CGGGATACTT TGGCAGCAAT CTCAGAAGTT 60
CTTTATGTTG A'l~ G~l~AGA AGGGGATACA GAATGCCATG CTAGATTTAA AACTCCTGAG 120
GATGCTCAAG CAGTAATAAA TGCCTATACA GAAATTAACA AGAAACACTG CTGGAAACTC l80
GAGATCCTTT CTGGTGATCA CGAACAAAGG TATTGGCAGA AGA~Ll~ G~l~TGATAGAAAG 240
GCAANNNTTA ATCAGCCTCG GGAAAAGAAA AGAGNGGTGA AAAGTTAATC ACCAGAGC~G 300
AAAAGATTAG ACTGGCAAAG ACT 323
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Thr Glu Pro Leu Pro Gly Arg Lys Gln Val Arg Asp Thr Leu Ala Ala
l 5 l0 15
Ile Ser Glu Val Leu Tyr Val Asp Leu Leu Glu Gly Asp Thr Glu Cys
His Ala Arg Phe Lys Thr Pro Glu Asp Ala Gln Ala Val Ile Asn Ala
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_ 35 40 45
Tyr Thr Glu Ile Asn Lys Lys His Cys Trp Lys Leu Glu Ile Leu Ser
Gly Asp His Glu Gln Arg Tyr Trp Gln Lys Ile Leu Val Asp Arg Lys
65 70 75 80
Ala Xaa Xaa Asn Gln Pro Arg Glu Lys Lys Arg Xaa Val Lys Ser
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TTTTTAATTA AAAGNAANAT ~L~ C~l~ NAAATTGTAN ATAAGAATTT TTTTTAGNGA 60
CNAANATGAN GNANACCACN A~ A AANATTTTAT TTGTTGAAAT TATTTTAGAN 120
N~ ~A GGNGATTTAG TAAATAAANG'l'~ ~GAC NTTTAAAAAA A~AA~AAA~A l80
190
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGCATCTGCA AC~l~G~ TTACTTCGCC TTCTACATCA TCATGAAGCT CCGGAGTGGG 60
GAGAGGATCA AGCTCATCCC CCTGCTCTGC ATCGTTTGCA C~l~CC~l~G~l~CTGGGGCTTC 120
GCGCTCTTCT TCTTCTTCCA GGGACTCAGC ACCTGGCAGA AAACCCCTGC AGAGTCGAGG l80
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GAGCACAACC GGGACTGCAT CCTCCTCGAC TTCTTTGACG ACCACGACAT CTGGCACTTC 240
CTCTCCTCCA TCGCCATGTT TCGGGTCCTT CCTGGTGTTT GCTGACACTG GATG 294
~2~ INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Lys Leu Arg Ser Gly Glu Arg Ile Lys Leu Ile Pro Leu Leu Cys
1 5 lD 15
Ile Val Cys Thr Ser Val Val Trp Gly Phe Ala Leu Phe Phe Phe Phe
Gln Gly Leu Ser Thr Trp Gln Lys Thr Pro.Ala Glu Ser Arg Glu His
Asn Arg Asp Cys Ile Leu Leu Asp Phe Phe Asp Asp His Asp Ile Trp
b0
His Phe Leu Ser Ser Ile Ala Met Phe Arg Val Leu Pro Gly Val Cys
65 70 75 80
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 230 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ACCCCAGATG CTGAGGATGG GGGAGCTCAG GCGGGGCNTC TGCTTNGGGG ATGGGAATGT 60
~l"l"l"l"l~l~CC CA~ACTTGTT TTTATAGCTC TGCTTGAAGG GCTGGGAGAT GAGGTGGGTC 120
TGGATCTTTT CTCAGAGCGT CTCCATGCTA TGGTTGCATT TCC~ ATGAATGAAT 180
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TTGCATTCAA TAAACAACCA GACTCAAAAA AAAAAAAAAA ~AAAAAAAAA 230
(2) INFORMATION FOR SEQ ID NO:16:
i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 495 base pairs
(B) TYPE: nuclelc acid
(C) STRANDEDNESS: double
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GACCTGCCTT CCTGCTCTTC TAGGTAGTCA CACTTCACTA AAGTGTCATC CACCAGTGTG 60
TTGAATCCGA AGAATGACAA~ ACCA CTGGTGTAAA AAACAAACAT TTGAAGACCC 120
TTGTGCATTG TGTGTCACAA AGCTAAATAC ATGGAAATCG TTAATATCGC TGATATTAAG 180
TAATTTCCCC ACTCTGAGTG AATACTTTGA TGATTGCCAA CA~l~GG~l~AA TAAAATGACG 240
GCTACCACAC TCATGGGTCA CTGGGGCTGC GCAGGGCTCT TTGAGGTGGG TGGCTTCTTT 300
TGGAAAGTAC TATGAACGTC TCGAAGCAGT ATTCTAGTGA TAAGAATTCT TAACATAGCC 360
AAGCGCCCCA C~ ~ CCA~ TCCCCTTTTC TGTTTGAAAA AC~ G 420
GTAGCTCCNC AAGAGAGATG ATACTGACTT TTTAAATTTT TTA~A~A~T CTGTATTCCT 480
GATATGCCTA TATTT 495
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Thr Ser Arg Ser Ser Ile Leu Val Ile Arg Ile Leu Asn Ile Ala Lys
1 5 10 15
Arg Pro Thr Phe Val Pro His Val Cys Ser Pro Phe Leu Phe Glu Lys
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Pro Val Leu Val Ala Pro Gln Glu Arg
(2) INFORMATION FOR SEQ ID NO:18:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY. linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GA~AAA~AA AAAAAAAA 18
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 285 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CACGAAGGGT TTCAAGGTCT GTCTTAGTTC TCATTCTCAA GA~ CC AGTTGCAAGT 60
TAGAGGCAAG CCAGCTAGCT GCCCAGCCTT AA~ C AGTGCCCTGT TACTAACATT 120
TTTTAACAGA TTGGNTTCTA CATGTTTAAA GTATCCAGCG TTGGATTTTA CCTCTTGCTA 180
GTTCCATTTG TCCCTGGTGC TGCTTTTAAA GGTATAGGGC CCTGTGAAGT GGANTATGTA 240
CGCAGTTGGC CTGGTGATGT ATCTGTGCCT GTTTTATCTT CTCCC 285
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(C) sTRANn~nN~ss
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 0224l9ll l998-06-26
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(xi) SEQUENCE DESCRIPTION: SEQ ID No:20:
Met Phe Lys Val Ser Ser Val Gly Phe Tyr Leu Leu Leu Val Pro Phe
1 5 10 15
Val Pro Gly Ala Ala Phe Lys Gly Ile Gly Pro Cys Glu Val Xaa Tyr
Val Arg Ser Trp Pro Gly Asp Val Ser Val Pro Val Leu Ser Ser Pro
35 40 45
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TTCCNTATGT AAGATGTCAT ACTGCAGATT TAAAATATAG ACTATCAATA AAATGCATGA 60
AGTGATCATT~ G~ GAT CA'l~'l'~'l'C~'l''l'GG~'l"l"l"l"l'C TTTAAAAAGG GGAATCTGCT 120
ATAAAGGTTC TGTTGCTTCA AACCAATGTC AAATAGACTT GATTTTTAGA GTCATGGAAT 180
TACAGTGCAA CCTTGATTTT TATTCCCCTC ACTGNTATGA GTGTGGGCAG GTACTGGTTT 240
ATATGTTATA ACTTCCGTTT TATCTGTGTT GTGTAGTTGA ATGGCTTAAT CGTTGAGTGG 300
TAAAATAAAA GATTATATTC CAATACAAGG ~AAAAAA~AA AAAAAAAAAA 350
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 517 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
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_ CACGAGGCCT C&TGCCAACA GGAAAGTTGC 'l"l"l'~'l"ll"l'~C TTCGAGATGG CTGCGGGGAT 60
~l~N~ lGGAA CATTATCTGG ACAGTATTGA AAACCTCCCG TTTGAATTAC AGAGAAACTT 120
TCAGCTCATG AGGGACCTAG ACCAAAGGAC AGAGGACCTG AAGGCTGAAA TTGACAAGTT 180
GGCCACTGAA TATATGAGTA GC&CCCGCAG CCTGAGCTCC GAGGAGAAGC TGGCCCTTCT 240
CAGACAGATC CAGGAGGCCT ATGGCAAGTG CAAGGAATTT GGTGACGACA AGGTGCAGCT 300
GGCCATGCAG ACCTATGAGA TGGTAGACAA ACACATTCGG CGGCTGGACA CAGACCTGGC 360
CC~ ~AG GCTGATCTGA AGGAGAAACA GATCGAGTCC AGTGACTATG ACAGCTCTTC 420
TAGCAAAGGC AAAAAGAGCC GGACCCAAAA GGAGAAAAAA GCTGCCAGAG CCCGTTCCAA 480
AGGGAAAAAC TCAGATGAAG AAGCCCCCAA GGCTGCC517
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Ala Ala Gly Met Xaa Leu Glu His Tyr Leu Asp Ser Ile Glu Asn
1 5 10 15
Leu Pro Phe Glu Leu Gln Arg Asn Phe GL~ Leu Met Arg Asp Leu Asp
Gln Arg Thr Glu Asp Leu Lys Ala Glu Ile Asp Lys Leu Ala Thr Glu
Tyr Met Ser Ser Ala Arg Ser Leu Ser Ser Glu Glu Lys Leu Ala Leu
Leu Arg Gln Ile Gln Glu Ala Tyr Gly Lys Cys Lys Glu Phe Gly Asp
6S 70 75 80
Asp Lys Val Gln Leu Ala Met Gln Thr Tyr Glu Met Val Asp Lys His
Ile Arg Arg Leu Asp Thr Asp Leu Ala Arg Phe Glu Ala Asp Leu Lys
100 105 110
Glu Lys Gln Ile Glu Ser Ser Asp Tyr Asp Ser Ser Ser Ser Lys Gly
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115 120 125
Lys Lys Ser Arg Thr Gln Lys Glu Lys Lys Ala Ala Arg Ala Arg Ser
130 135 140
Lys Gly Lys Asn Ser Asp Glu Glu Ala Pro Lys Ala Ala
145 150 155
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
TC~~ G~L~G AGGGCTAGGT ~l~N~ NCN CTNTTATTCT CCATTCCCTT CCTGCTTTTT 60
TCATGGTGGG GGATCCACCA GGTCATNTAG G~~ ~GCCC TAGTTGAAGG GGCACCCCTT 120
~N~ GCC AAGAGGATTC Al~C~l~GGGAG AGGGGGCAAG GTGGAATGCA GATAACTCAC 180
ATGTAAAAGG AACTTGGGTA GGTAAATAAA AGCTATACAT GTTGAAAAAA AAAAAAAAAA 240
AAAAAA 246
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 896 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CACGAGGCGG TGGACTGCAA GGACCCAGAT GATGTGGTAC CAGTTGGCCA AAGAAGAGCC 60
TGGTGTTGGT GCATGTGCTT TGGACTAGCA TTTATGCTTG CAGGTGTTAT TCTAGGAGGA 120
GCATACTTGT ACAAATATTT TGCACTTCAA CCAGATGACG TGTACTACTG TGGAATAAAG 180
TACATCAAAG ATGATGTCAT CTTAAATGAG CCCTCTGCAG ATGCCCCAGC l~ AC 240
.
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CAGACAATTG AAGAAAATAT TAAAATCTTT GAAGAAGAAG AAGTTGAATT TATCAGTGTG 300
CCTGTCCCAG AGTTTGCAGA TAGTGATCCT GCCAACATTG TTCATGACTT TAACAAGAAA 360
CTTACAGCCT ATTTAGATCT TAACCTGGAT AAGTGCTATG TGA~1~C~ GAACACTTCC 420
ATTGTTATGC CACCCAGAAA CCTACTGGAG TTACTTATTA ACATCAAGGC TGGAACCTAT 480
TTGCCTCAGT CCTATCTGAT TCATGAGCAC ATGGTTATTA CTGATCGCAT TGAAAACATT 540
GATCACCTGG ~'1"1~1'~'1"1"1'AT TTATCGACTG TGTCATGACA AGGAAACTTA CAAACTGCAA 600
CGCAGAGAAA CTATTAAAGG TATTCAGAAA CGTGAAGCCA GCAATTGTTT CGCAATTCGG 660
CATTTTGAAA ACAAATTTGC CGTGGAAACT TTAATTTGTT CTTGAACAGT CAAGAAAAAC 720
ATTATTGAGG AAAATTAATA TCACAGCATA ACCCCACCCT TTACATTTTG TGCAGTGATT 780
A~ AAA GTCTTCTTTC ATGTAAGTAG CAAACAGGGC TTTACTATCT TTTCATCTCA 840
TTAATTCAAT TAAAACCATT ACCTTAAAAA AAAAAAAAAA AAAAAAAAA~ AAAAAA 896
(2) IMFORMATION EOR SEQ ID No:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids
(B) TYPE: amino acid
(C~ STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID No:26:
Met Cys Phe Gly Leu Ala Phe Met Leu Ala Gly Val Ile Leu Gly Gly
l 5 lO 15
Ala Tyr Leu Tyr Lys Tyr Phe Ala Leu Gln Pro Asp Asp Val Tyr Tyr
Cys Gly Ile Lys Tyr Ile Lys Asp Asp Val Ile Leu Asn Glu Pro Ser
Ala Asp Ala Pro Ala Ala Leu Tyr Gln Thr Ile Glu Glu Asn Ile Lys
Ile Phe Glu Glu Glu Glu Val Glu Phe Ile Ser Val Pro Val Pro Glu
Phe Ala Asp Ser Asp Pro Ala Asn Ile Val His Asp Phe Asn Lys Lys
62
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Leu Thr Ala Tyr Leu Asp Leu Asn Leu Asp Lys Cys Tyr Val Ile Pro
100 105 110
Leu Asn Thr Ser Ile Val Met Pro Pro Arg Asn Leu Leu Glu Leu Leu
115 12 0 ~2 5
Ile Asn Ile Lys Ala Gly Thr Tyr Leu Pro Gln Ser Tyr Leu Ile His
130 135 140
Glu His Met Val Ile Thr Asp Arg Ile Glu Asn Ile Asp His Leu Gly
145 150 155 160
Phe Phe Ile Tyr Arg Leu Cys His Asp Lys Glu Thr Tyr Lys Leu Gln
165 170 175
Arg Arg Glu Thr Ile Lys Gly Ile Gln Lys Arg Glu Ala Ser Asn Cys
180 185 190
Phe Ala Ile Arg E~is Phe Glu Asn Lys Phe Ala Val Glu Thr Leu Ile
195 200 205
Cys Ser
210
63
