EP1007539A1 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them

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Publication number
EP1007539A1
EP1007539A1 EP98933104A EP98933104A EP1007539A1 EP 1007539 A1 EP1007539 A1 EP 1007539A1 EP 98933104 A EP98933104 A EP 98933104A EP 98933104 A EP98933104 A EP 98933104A EP 1007539 A1 EP1007539 A1 EP 1007539A1
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EP
European Patent Office
Prior art keywords
polynucleotide
seq
protein
amino acid
nucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98933104A
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German (de)
French (fr)
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
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Genetics Institute LLC
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Genetics Institute LLC
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Application filed by Genetics Institute LLC filed Critical Genetics Institute LLC
Publication of EP1007539A1 publication Critical patent/EP1007539A1/en
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Definitions

  • the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 516 to nucleotide 797; the nucleotide sequence of SEQ ID NO:l from nucleotide 606 to nucleotide 797; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 773; the nucleotide sequence of the full-length protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 137 to nucleotide 1240; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 1153; the nucleotide sequence of the full-length protein coding sequence of clone bg249_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone bg249_l deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 31.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 1 to amino acid 22.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 783; the nucleotide sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 783; the nucleotide sequence of SEQ ID NO:9 from nucleotide 7 to nucleotide 462; the nucleotide sequence of the full-length protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to amino acid 112.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 112.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 645 to nucleotide 782; the nucleotide sequence of SEQ ID NO:15 from nucleotide 10 to nucleotide 773; the nucleotide sequence of the full-length protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone fhl3__10 deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid
  • 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:16;
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 from nucleotide 94 to nucleotide 216; the nucleotide sequence of SEQ ID NO:17 from nucleotide 160 to nucleotide 216; the nucleotide sequence of SEQ ID NO:17 from nucleotide 20 to nucleotide 193; the nucleotide sequence of the full-length protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 33.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 33.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2 to nucleotide 943; the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2 to nucleotide 670; the nucleotide sequence of the full-length protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 223.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • 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 223.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • nucleotide 1242 to nucleotide 1457 the nucleotide sequence of SEQ ID NO:21 from nucleotide 1326 to nucleotide 1457; the nucleotide sequence of SEQ ID NO:21 from nucleotide 869 to nucleotide 1544; the nucleotide sequence of the full-length protein coding sequence of clone hb752_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone hb752_l deposited under accession number ATCC 98482.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:22 or the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 69.
  • the polynucleotide is operably linked to an expression control sequence.
  • the invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
  • Processes are also provided for producing a protein, which comprise: (a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
  • the protein produced according to such methods is also provided by the present invention.
  • Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
  • Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier.
  • Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
  • Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • Fig. 1 is a schematic representation of the pED6 and pNOTs vectors used for deposit of clones disclosed herein.
  • nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application.
  • the nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence.
  • "Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
  • bf228 14 A polynucleotide of the present invention has been identified as clone "bf228_14".
  • bf228_14 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bf228_14 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bf228_14 protein").
  • nucleotide sequence of bf228_14 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bf228_14 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
  • Amino acids 18 to 30 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 31, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bf228__14 should be approximately 1400 bp.
  • the nucleotide sequence disclosed herein for bf228_14 was searched against the
  • bf228_14 demonstrated at least some homology with sequences identified as AA069549 (zm52e03.sl Stratagene fibroblast (#937212) Homo sapiens cDNA clone 5292763'), H83278 (yq49h09.rl Homo sapiens cDNA clone 199169 5'), and N94898 (zb31b01.sl Homo sapiens cDNA clone 305161 3' similar to contains Alu repetitive element;contains element MSR1 repetitive element). Based upon homology, bf228_14 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of bf228_14 indicates that it may contain an Alu repetitive element.
  • a polynucleotide of the present invention has been identified as clone "bg249_ l".
  • bg249_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bg249_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bg249_l protein").
  • nucleotide sequence of bg249_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bg249_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bg249_l should be approximately 2700 bp.
  • the nucleotide sequence disclosed herein for bg249_l was searched against the
  • bg249_l demonstrated at least some homology with sequences identified as AA151021 (zl47c04.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 505062 5'), AA278781 (zs79a01.rl Soares NbHTGBC Homo sapiens cDNA clone 7036565'), R75099 (MDB1032R Mouse brain, Stratagene Mus musculus cDNA 5'end), and T06990 (EST04879 Homo sapiens cDNA clone HFBEB91). Based upon homology, bg249_l proteins and each homologous protein or peptide may share at least some activity.
  • bv286 1 A polynucleotide of the present invention has been identified as clone "bv286_l".
  • bv286_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bv286_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
  • nucleotide sequence of bv286_l as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bv286_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • Amino acids 14 to 26 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 27, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bv286_l should be approximately 550 bp.
  • the nucleotide sequence disclosed herein for bv286_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols.
  • bv286_l demonstrated at least some homology with sequences identified as AA132163 (zl38c07.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 504204 5' similar to WP:F55A11.1 CE05943 EF HAND DOMAINS), AA552888 (nk57h08.sl NCI_CGAP_Pr7 Homo sapiens cDNA clone IMAGE:1017663 similar to WP:F55A11.1 CE05943 EF HAND DOMAINS), and H12316 (yjlld07.sl Homo sapiens cDNA clone 148429 3" similar to SP:JS0027 JS0027 PROBABLE CALCIUM-BINDING PROTEIN).
  • the predicted amino acid sequence disclosed herein for bv286_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bv286_l protein demonstrated at least some identity with sequences identified as L22647 (prostaglandin receptor epl subtype [Homo sapiens]). Based upon homology, bv286_l proteins and each homologous protein or peptide may share at least some activity.
  • the nucleotide sequence of bv286_l indicates that it may contain an Alu repetitive element.
  • co36_l A polynucleotide of the present invention has been identified as clone "co36_l".
  • co36_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • co36_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "co36_l protein"). The nucleotide sequence of co36_l as presently determined is reported in SEQ ID
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone co36_l should be approximately 3300 bp.
  • cpll6_l A polynucleotide of the present invention has been identified as clone "cpll6_l".
  • cpll6_l was isolated from a human adult salivary gland cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cpll6_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cpll6_l protein").
  • nucleotide sequence of cpll6_l as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cpll6_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Amino acids 3 to 15 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cpll6_l should be approximately 1600 bp.
  • nucleotide sequence disclosed herein for cpll6_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database.
  • cwll95_2 A polynucleotide of the present invention has been identified as clone "cwll95_2".
  • cwll95_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cwll95_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cwll95_2 protein").
  • nucleotide sequence of the 5' portion of cwll95_2 as presently determined is reported in SEQ ID NO:ll.
  • An additional internal nucleotide sequence from cwll95_2 as presently determined is reported in SEQ ID NO:12. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO:13.
  • Additional nucleotide sequence from the 3' portion of cwll95_2, including the polyA tail, is reported in SEQ ID NO:14.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cwll95_2 should be approximately 3300 bp.
  • cwl 195_2 demonstrated at least some homology with sequences identified as AA205460 (zq66f07.sl Stratagene neuroepithelium (#937231) Homo sapiens cDNA clone 6465973') and AA362052 (EST71451 MCF7 cell line Homo sapiens cDNA 5' end similar to EST containing Alu repeat). Based upon homology, cwll95_2 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of cwll95_2 indicates that it may contain an Alu repetitive element.
  • fhl3_10 A polynucleotide of the present invention has been identified as clone "fhl3_10".
  • fhl3_10 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • fhl3_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fhl3_10 protein").
  • the nucleotide sequence of fhl3_10 as presently determined is reported in SEQ ID
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone fhl3_10 should be approximately 1400 bp.
  • fhl3_10 The nucleotide sequence disclosed herein for fhl3_10 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. fhl3_10 demonstrated at least some homology with sequences identified as J00089 (Human Alu family interspersed repeat; clone BLUR6) and X00481 (Human non-alu family interspersed repeat). Based upon homology, fhl3_10 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of fhl3_10 indicates that it may contain a repetitive element. Clone "gc57 4"
  • a polynucleotide of the present invention has been identified as clone "gc57_4".
  • gc57_4 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • gc57_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gc57_4 protein").
  • nucleotide sequence of gc57_4 as presently determined is reported in SEQ ID NO: 17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gc57_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18.
  • Amino acids 10 to 22 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 23, or are a transmembrane domain.
  • the EcoRI / NotI restriction fragment obtainable from the deposit containing clone gc57_4 should be approximately 1900 bp.
  • gc57_4 The nucleotide sequence disclosed herein for gc57_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gc57_4 demonstrated at least some homology with sequences identified as AA095328 (13005.seq.F Fetal heart, Lambda ZAP Express Homo sapiens cDNA 5'), AA126440 (zk94e02.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 490490 3'), and Z82195 (Human DNA sequence from clone J274L71). Based upon homology, gc57_4 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of gc57_4 indicates that it may contain an Alu repetitive element.
  • hll65_3 A polynucleotide of the present invention has been identified as clone "hll65_3".
  • hll65_3 was isolated from a human adult blood (peripheral blood mononuclear cells treated with phytohemagglutinin and phorbol meristate acetate and mixed lymphocyte reaction) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • hll65_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hll65_3 protein").
  • nucleotide sequence of hll65_3 as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hll65_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone hll65_3 should be approximately 1250 bp.
  • hll65_3 demonstrated at least some homology with sequences identified as AA173098 (zp31d03.rl Stratagene neuroepithelium (#937231) Homo sapiens cDNA clone 611045 5' similar to contains element TAR1 repetitive element), AA305139 (EST176159 Colon carcinoma (Caco-2) cell line II Homo sapiens cDNA 5' end), AA426375 (zv54h02.sl Soares testis NHT Homo sapiens cDNA clone 757491 3'), and N72370 (yv38cll.rl Homo sapiens cDNA clone 245012 5').
  • the predicted amino acid sequence disclosed herein for hll65_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted hll65_3 protein demonstrated at least some identity with sequences identified as U58758 (coded for by C. elegans cDNA yk83a5.3; coded for by C.elegans cDNA yk83a5.5 [Caenorhabditis elegans]). Based upon homology, hll65_3 proteins and each homologous protein or peptide may share at least some activity.
  • hb752 1 A polynucleotide of the present invention has been identified as clone "hb752_l".
  • hb752_l was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • hb752_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
  • nucleotide sequence of hb752_l as presently determined is reported in SEQ ID NO:21. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hb752_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:22. Amino acids 16 to 28 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 29, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone hb752_l should be approximately 1800 bp.
  • hb752_l demonstrated at least some homology with sequences identified as AA100979 (zm26f09.sl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526793 3'), AA490528 (aa51gll.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone 824516 3'), H65670 (yr72gl2.rl Homo sapiens cDNA clone 2108865'), H86790 (ys72c03.sl Homo sapiens cDNA clone 220324 3'), N58917 (yy ⁇ lfll.sl Homo sapiens cDNA clone 278061 3'), and Z43307 (H.
  • Clones bf228_14, bg249_l, bv286_l, co36_l, cpll6_l, cwll95_2, fhl3_10, gc57_4, hll65_3 and hb752_l were deposited on July 2, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98482, from which each clone comprising a particular polynucleotide is 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. colt) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1.
  • the pED6dpc2 vector (“pED6" was derived from pED ⁇ dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al,
  • the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. 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 Clal site.
  • the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate.
  • the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector.
  • the cDNA may also be expressed from the vectors in which they were deposited.
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
  • oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
  • 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;
  • the oligonucleotide should preferably be labeled with g- 3 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately
  • the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
  • a third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film.
  • Other known hybridization methods can also be employed.
  • the positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R.S.
  • fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • linker For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a protein - IgM fusion would generate a decavalent form of the protein of the invention.
  • the present invention also provides both full-length and mature forms of the disclosed proteins.
  • the full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone.
  • the mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • the sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides genes corresponding to the cDNA sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which the cDNA sequences are derived and any contiguous regions of the genome necessary for the regulated expression of such genes, including but not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements.
  • the corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • 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 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 polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides .
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein.
  • stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybnd length is that anticipated for the hybridized reg ⁇ on(s) of the hybridizing polynucleotides
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by ahgning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE 0.15M NaCl, lOmM NaH,P0 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • T m melting temperature
  • each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • 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 185, 537-566 (1990).
  • 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 (transfected) with the ligated polynucleotide /expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
  • Ovary (CHO) cells human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays ⁇ ited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA im
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cytokine
  • cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol.
  • Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SOD severe combined immunodeficiency
  • 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.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention 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, my asthenia gravis, graft- versus-host disease and autoimmune inflammatory eye disease.
  • 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 may also be treatable using a protein of the present invention.
  • Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
  • tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B 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
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte 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 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 pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and 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 lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection.
  • systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • 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-l-like activity and /or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • 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.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Stiober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al, J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • 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
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • 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 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 megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York,
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not 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 tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • 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 (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
  • a protein of the invention may also exhibit angiogenic activity.
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions 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. WO95/ 16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
  • 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 characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin 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 mammals.
  • FSH follicle stimulating hormone
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • 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 by the following methods:
  • Assays for chemotactic activity consist of assays that meastire the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al, J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor /ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor /ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immime responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor /ligand interaction.
  • a protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor /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, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • 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. Cadherin malfunction is also implicated in other human diseases, 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 molecule.
  • the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the 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 cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes.
  • Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth.
  • reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the 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 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 invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
  • 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 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.
  • compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and /or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • 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 antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; 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.
  • 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.
  • 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.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.
  • the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other 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.
  • 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.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being canonic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic 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 the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question.
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of 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.
  • the addition of other known growth factors, such as IGF I (insulin like growth factor I) may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • TTGCCTTCCC TTCCCAGAGT GGAGGAGTTA GACCTGCATT GTGGGATGAG AGGAGTTGTG 660
  • GCTGCATTCA GGATTGTTCC ATCCATGGCG TGCATGTGCC AAGAAATGTG TTTTTATGGG 1380
  • Leu Glu Asp Arg lie Leu Cys His Glu Lys Thr Thr Ala Ala Leu Val 35 40 45
  • CTTACATCCA CACCTGTAGC ATCTGAGCCT GCTGCAGGGG CCCCTGTTGC AGCTGAGCCT 600
  • TTCATCTCAC TACATAGATT TGTTTGTGGT AGTTATTTCC TTGGACTTAA TTTATATTGA 960
  • TTATACAGCT ACTATAGGAT GATGTTAGTA TTGGTTTTGA GTGCAATAGG TTTTTTCCTA 1140
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:14:
  • CTGAGCTCAG GCTATCTGCC CACCTTGGCC TCCCAAAGTG CTGGGATTAC AGGTGTGAGC 120
  • GATTATCCCA TTTGTTTCTC ACAGCCGGTA TTTATTGTCT GCTCCTCTGT GCCAGGTGCT 1320 GTGCTCTGGG CAGGGGCACT GCATGGGCTG CCTGCCCTGG TGGAGCTTGT GGTCTGATGG 1380

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
- This application is a continuation-in-part of application Ser. No. 08/887,195, filed
July 2, 1997.
FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed.
SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 516 to nucleotide 797;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 606 to nucleotide 797; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:l from nucleotide 1 to nucleotide 773;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 516 to nucleotide 797; the nucleotide sequence of SEQ ID NO:l from nucleotide 606 to nucleotide 797; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 773; the nucleotide sequence of the full-length protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:l.
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:2; (b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86;
(c) fragments of the amino acid sequence of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 137 to nucleotide 1240;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 1153;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bg249_l deposited under accession number ATCC 98482; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bg249_l deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 137 to nucleotide 1240; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 1153; the nucleotide sequence of the full-length protein coding sequence of clone bg249_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone bg249_l deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4; (b) the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339;
(c) fragments of the amino acid sequence of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 26 to nucleotide 301; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 104 to nucleotide 301;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 119;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bv286_l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bv286_l deposited under accession number
ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:5 from nucleotide 26 to nucleotide 301; the nucleotide sequence of SEQ ID NO:5 from nucleotide 104 to nucleotide 301; the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 119; the nucleotide sequence of the full-length protein coding sequence of clone bv286_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone bv286_l deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 31.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:5.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 31;
(c) fragments of the amino acid sequence of SEQ ID NO:6; and (d) the amino acid sequence encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 31. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 663 to nucleotide 755;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 850; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone co36_l deposited under accession number ATCC 98482;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone co36_l deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:7 from nucleotide 663 to nucleotide 755; the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 850; the nucleotide sequence of the full-length protein coding sequence of clone co36_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone co36_l deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 1 to amino acid 22. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino acid 1 to amino acid 22;
(c) fragments of the amino acid sequence of SEQ ID NO:8; and (d) the amino acid sequence encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:8 from amino acid 1 to amino acid 22. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 783;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 783;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 7 to nucleotide 462; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity; (k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 783; the nucleotide sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 783; the nucleotide sequence of SEQ ID NO:9 from nucleotide 7 to nucleotide 462; the nucleotide sequence of the full-length protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to amino acid 112.
Other embodiments provide the gene corresponding to the cDN A sequence of SEQ ID NO:9. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 112;
(c) fragments of the amino acid sequence of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 112.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 231 to nucleotide 533; (c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482; (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482; (g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:13 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:12 from nucleotide 231 to nucleotide 533; the nucleotide sequence of the full-length protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:12, SEQ ID NO:ll or SEQ ID NO:14 . In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 13;
(b) fragments of the amino acid sequence of SEQ ID NO:13; and (c) the amino acid sequence encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:13.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 645 to nucleotide 782; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 10 to nucleotide 773;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 645 to nucleotide 782; the nucleotide sequence of SEQ ID NO:15 from nucleotide 10 to nucleotide 773; the nucleotide sequence of the full-length protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone fhl3__10 deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid
43. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:15.
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:16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 43;
(c) fragments of the amino acid sequence of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:16 or the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 43. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 94 to nucleotide 216;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 160 to nucleotide 216;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 20 to nucleotide 193;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity; (k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 from nucleotide 94 to nucleotide 216; the nucleotide sequence of SEQ ID NO:17 from nucleotide 160 to nucleotide 216; the nucleotide sequence of SEQ ID NO:17 from nucleotide 20 to nucleotide 193; the nucleotide sequence of the full-length protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 33.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:17.
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: 18; (b) the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 33;
(c) fragments of the amino acid sequence of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 33.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2 to nucleotide 943;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2 to nucleotide 670;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2 to nucleotide 943; the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2 to nucleotide 670; the nucleotide sequence of the full-length protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 223.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:19.
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:20; (b) the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 223;
(c) fragments of the amino acid sequence of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian 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 223.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 1242 to nucleotide 1457; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:21 from nucleotide 1326 to nucleotide 1457;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 869 to nucleotide 1544;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hb752_l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone hb752_l deposited under accession number
ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:22 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:21 from nucleotide 1242 to nucleotide 1457; the nucleotide sequence of SEQ ID NO:21 from nucleotide 1326 to nucleotide 1457; the nucleotide sequence of SEQ ID NO:21 from nucleotide 869 to nucleotide 1544; the nucleotide sequence of the full-length protein coding sequence of clone hb752_l deposited under accession number ATCC 98482; or the nucleotide sequence of the mature protein coding sequence of clone hb752_l deposited under accession number ATCC 98482. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid
69.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:21.
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:22;
(b) the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 69; (c) fragments of the amino acid sequence of SEQ ID NO:22; and
(d) the amino acid sequence encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:22 or the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 69.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
Processes are also provided for producing a protein, which comprise: (a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention. Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the pED6 and pNOTs vectors used for deposit of clones disclosed herein.
DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
Clone "bf228 14" A polynucleotide of the present invention has been identified as clone "bf228_14". bf228_14 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bf228_14 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bf228_14 protein").
The nucleotide sequence of bf228_14 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bf228_14 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Amino acids 18 to 30 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 31, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bf228__14 should be approximately 1400 bp. The nucleotide sequence disclosed herein for bf228_14 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bf228_14 demonstrated at least some homology with sequences identified as AA069549 (zm52e03.sl Stratagene fibroblast (#937212) Homo sapiens cDNA clone 5292763'), H83278 (yq49h09.rl Homo sapiens cDNA clone 199169 5'), and N94898 (zb31b01.sl Homo sapiens cDNA clone 305161 3' similar to contains Alu repetitive element;contains element MSR1 repetitive element). Based upon homology, bf228_14 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of bf228_14 indicates that it may contain an Alu repetitive element.
Clone "bg249 1"
A polynucleotide of the present invention has been identified as clone "bg249_ l". bg249_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bg249_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bg249_l protein").
The nucleotide sequence of bg249_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bg249_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bg249_l should be approximately 2700 bp. The nucleotide sequence disclosed herein for bg249_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bg249_l demonstrated at least some homology with sequences identified as AA151021 (zl47c04.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 505062 5'), AA278781 (zs79a01.rl Soares NbHTGBC Homo sapiens cDNA clone 7036565'), R75099 (MDB1032R Mouse brain, Stratagene Mus musculus cDNA 5'end), and T06990 (EST04879 Homo sapiens cDNA clone HFBEB91). Based upon homology, bg249_l proteins and each homologous protein or peptide may share at least some activity.
Clone "bv286 1" A polynucleotide of the present invention has been identified as clone "bv286_l". bv286_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bv286_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
"bv286_l protein").
The nucleotide sequence of bv286_l as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bv286_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino acids 14 to 26 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 27, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bv286_l should be approximately 550 bp. The nucleotide sequence disclosed herein for bv286_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bv286_l demonstrated at least some homology with sequences identified as AA132163 (zl38c07.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 504204 5' similar to WP:F55A11.1 CE05943 EF HAND DOMAINS), AA552888 (nk57h08.sl NCI_CGAP_Pr7 Homo sapiens cDNA clone IMAGE:1017663 similar to WP:F55A11.1 CE05943 EF HAND DOMAINS), and H12316 (yjlld07.sl Homo sapiens cDNA clone 148429 3" similar to SP:JS0027 JS0027 PROBABLE CALCIUM-BINDING PROTEIN). The predicted amino acid sequence disclosed herein for bv286_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bv286_l protein demonstrated at least some identity with sequences identified as L22647 (prostaglandin receptor epl subtype [Homo sapiens]). Based upon homology, bv286_l proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of bv286_l indicates that it may contain an Alu repetitive element.
Clone "co36 1"
A polynucleotide of the present invention has been identified as clone "co36_l". co36_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. co36_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "co36_l protein"). The nucleotide sequence of co36_l as presently determined is reported in SEQ ID
NO:7. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the co36_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone co36_l should be approximately 3300 bp.
The nucleotide sequence disclosed herein for co36_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. Clone "cpllβ 1"
A polynucleotide of the present invention has been identified as clone "cpll6_l". cpll6_l was isolated from a human adult salivary gland cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cpll6_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cpll6_l protein").
The nucleotide sequence of cpll6_l as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cpll6_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Amino acids 3 to 15 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cpll6_l should be approximately 1600 bp.
The nucleotide sequence disclosed herein for cpll6_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database.
Clone "cwll95 2"
A polynucleotide of the present invention has been identified as clone "cwll95_2". cwll95_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cwll95_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cwll95_2 protein").
The nucleotide sequence of the 5' portion of cwll95_2 as presently determined is reported in SEQ ID NO:ll. An additional internal nucleotide sequence from cwll95_2 as presently determined is reported in SEQ ID NO:12. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO:13. Additional nucleotide sequence from the 3' portion of cwll95_2, including the polyA tail, is reported in SEQ ID NO:14. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cwll95_2 should be approximately 3300 bp.
The nucleotide sequence disclosed herein for cwll95_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols, cwl 195_2 demonstrated at least some homology with sequences identified as AA205460 (zq66f07.sl Stratagene neuroepithelium (#937231) Homo sapiens cDNA clone 6465973') and AA362052 (EST71451 MCF7 cell line Homo sapiens cDNA 5' end similar to EST containing Alu repeat). Based upon homology, cwll95_2 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of cwll95_2 indicates that it may contain an Alu repetitive element.
Clone "fhl3 10"
A polynucleotide of the present invention has been identified as clone "fhl3_10". fhl3_10 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. fhl3_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fhl3_10 protein"). The nucleotide sequence of fhl3_10 as presently determined is reported in SEQ ID
NO:15. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the fhl3_10 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone fhl3_10 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for fhl3_10 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. fhl3_10 demonstrated at least some homology with sequences identified as J00089 (Human Alu family interspersed repeat; clone BLUR6) and X00481 (Human non-alu family interspersed repeat). Based upon homology, fhl3_10 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of fhl3_10 indicates that it may contain a repetitive element. Clone "gc57 4"
A polynucleotide of the present invention has been identified as clone "gc57_4". gc57_4 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. gc57_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gc57_4 protein").
The nucleotide sequence of gc57_4 as presently determined is reported in SEQ ID NO: 17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gc57_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18. Amino acids 10 to 22 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 23, or are a transmembrane domain. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone gc57_4 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for gc57_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gc57_4 demonstrated at least some homology with sequences identified as AA095328 (13005.seq.F Fetal heart, Lambda ZAP Express Homo sapiens cDNA 5'), AA126440 (zk94e02.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 490490 3'), and Z82195 (Human DNA sequence from clone J274L71). Based upon homology, gc57_4 proteins and each homologous protein or peptide may share at least some activity. The nucleotide sequence of gc57_4 indicates that it may contain an Alu repetitive element.
Clone "h!165 3"
A polynucleotide of the present invention has been identified as clone "hll65_3". hll65_3 was isolated from a human adult blood (peripheral blood mononuclear cells treated with phytohemagglutinin and phorbol meristate acetate and mixed lymphocyte reaction) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. hll65_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hll65_3 protein").
The nucleotide sequence of hll65_3 as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hll65_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hll65_3 should be approximately 1250 bp.
The nucleotide sequence disclosed herein for hll65_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hll65_3 demonstrated at least some homology with sequences identified as AA173098 (zp31d03.rl Stratagene neuroepithelium (#937231) Homo sapiens cDNA clone 611045 5' similar to contains element TAR1 repetitive element), AA305139 (EST176159 Colon carcinoma (Caco-2) cell line II Homo sapiens cDNA 5' end), AA426375 (zv54h02.sl Soares testis NHT Homo sapiens cDNA clone 757491 3'), and N72370 (yv38cll.rl Homo sapiens cDNA clone 245012 5'). The predicted amino acid sequence disclosed herein for hll65_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted hll65_3 protein demonstrated at least some identity with sequences identified as U58758 (coded for by C. elegans cDNA yk83a5.3; coded for by C.elegans cDNA yk83a5.5 [Caenorhabditis elegans]). Based upon homology, hll65_3 proteins and each homologous protein or peptide may share at least some activity.
Clone "hb752 1" A polynucleotide of the present invention has been identified as clone "hb752_l". hb752_l was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. hb752_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
"hb752_l protein").
The nucleotide sequence of hb752_l as presently determined is reported in SEQ ID NO:21. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hb752_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:22. Amino acids 16 to 28 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 29, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hb752_l should be approximately 1800 bp.
The nucleotide sequence disclosed herein for hb752_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hb752_l demonstrated at least some homology with sequences identified as AA100979 (zm26f09.sl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526793 3'), AA490528 (aa51gll.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone 824516 3'), H65670 (yr72gl2.rl Homo sapiens cDNA clone 2108865'), H86790 (ys72c03.sl Homo sapiens cDNA clone 220324 3'), N58917 (yyόlfll.sl Homo sapiens cDNA clone 278061 3'), and Z43307 (H. sapiens partial cDNA sequence; clone c-18g09). Based upon homology, hb752_l proteins and each homologous protein or peptide may share at least some activity.
Deposit of Clones
Clones bf228_14, bg249_l, bv286_l, co36_l, cpll6_l, cwll95_2, fhl3_10, gc57_4, hll65_3 and hb752_l were deposited on July 2, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98482, from which each clone comprising a particular polynucleotide is 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. colt) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from pEDόdpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al,
1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. 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 Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence bf228_14 SEQ ID NO:23 bg249_l SEQ ID NO:24 bv286_l SEQ ID NO:25 co36_l SEQ ID NO:26 cpll6_l SEQ ID NO:27 cwll95_2 SEQ ID NO:28 fhl3_10 SEQ ID NO:29 gc57_4 SEQ ID NO:30 hll65_3 SEQ ID NO:31 hb752_l SEQ ID NO:32
In the sequences listed above which include an N at position 2, that position is occupied in preferred probes /primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1- dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C). The oligonucleotide should preferably be labeled with g-3 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R.S.
McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the cDNA sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which the cDNA sequences are derived and any contiguous regions of the genome necessary for the regulated expression of such genes, including but not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials. Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that 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 polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
*: The hybnd length is that anticipated for the hybridized regιon(s) of the hybridizing polynucleotides When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by ahgning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. f: SSPE (lxSSPE is 0.15M NaCl, lOmM NaH,P04, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) m the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete
*TB - TR: The hybridization temperature for hybrids antiαpated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G + C bases) For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5 + 16.6(log10[Na+]) + 0.41 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions m the hybridization buffer ([Na+] for lxSSC = 0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 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 (transfected) with the ligated polynucleotide /expression control sequence.
A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
Ovary (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, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under 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® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention. USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays ςited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation /Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al, J. Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, my asthenia gravis, graft- versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent. Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting receptoπligand interactions of B lymphocyte 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 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 pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and 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 lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. 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-l-like activity and /or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β 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 immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Stiober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al, J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al, J. Immunol.
135:1564-1572, 1985; Takai et al, J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology
154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; 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, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating 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 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 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 megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Cidture of Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et 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, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes. Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not 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 tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon /ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity. A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions 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. WO95/ 16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin 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 mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin- β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that meastire the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind 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. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke). The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al, J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
Receptor /Ligand Activity
A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor /ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor /ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immime responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor /ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor /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, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin /Tumor Invasion 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. Cadherin malfunction is also implicated in other human diseases, 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 molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the 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 cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the 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 polynucleotides 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 invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and /or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and /or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by 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 antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; 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 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 therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention. When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and /or cartilage formation, the 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.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other 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 preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being canonic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
[I) GENERAL INFORMATION:
(1) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Treacy, Maurice Spauldmg, Vik i Agostino, Michael J.
(ll) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(ill) NUMBER OF SEQUENCES: 32
(IV) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vm) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger , Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1425 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
J3CTAATTTGA GAAGAAAACA AGTAGGATTT TTGTTTTGTT TTGCATTTTG CAATATGGAG 60
GAGAAATGAT TAGACCTTAG GAAGTGCCAG TGGGTTGGTC CTTTCATGAA CATGCCATCA 120
GTAAAAGCCC TGGAAACAAG GTCATACCAG AGATTCATTG TGCCTTGTCA CAACTGCAAA 180
CAATATCTGA GTGGAATATT CAAAAACTTG CTTAGAAAGA AAACTCTAGG ACAGATGGCT 240
CCACTGAAGT TATTCCAAAT ATTTAATAAA TAAAGCATAC CAGGCTTTTA TAAACTCTTC 300
TAGAAGAAAA AAGTTGGAAC TTTTCCAATT CAGTTTTTCA GGCCAGTGCA ACCTTGATAC 360
CAAAACCAAT AAAACAAACA AACAAACAAA AAACATAAAG CTATAGACCA AAGTCTCATA 420
GATTTAGATG CAAAATCCTA AAATTGAAAA AAAAAGTCTA GTCATATCCA TAAACTGTAT 480
CATCACCAAG AGATGTTTAT TAGGGCAATC AAAAGATGAT TTATTATTTT TTAAAAAATC 540
AATGTGGCCT TCCCTTCCTC TTTCTTTTGA TTCCCCTCTT TGAGTTTTTA TGTGTCTCTT 600
TTGCCTTCCC TTCCCAGAGT GGAGGAGTTA GACCTGCATT GTGGGATGAG AGGAGTTGTG 660
GCTATGTGTC TGCTGGCACC AAGAGGGCTG AGGGTGAGGT GTGGAAGGGA CAGGGGGAGG 720
AGATGGGCAG CATTGTTAAG AGATTGGTAC CACTGAGCAA ATATGTTGAG AATGATGATG 780
GCAAGGTTTC TCCCTGTTAG AGAAGGTATT TGTAGAAATA GGAATGAGGA GAGCTAGAAA 840
ACCTGGAGTG TGGGATTAGA ATAGAACTCA TATCTTTTAA ATACATAGGA ACAATAGAGA 900
AATTGTTGGG TGTGCCCATA TACATATATT TTGTGATTCA TTCTACCGAG AGGACATAAA 960
TGCAGTCACA GCTCAGTAAC AGTAAACACA CCAACTGCCA AGTTATTATT TCCTAAATAC 1020
TATCCACAAA AAAGGGGACC AGGGATGATT CCTAGTCGGA GATTGGGAGA AAAAGAAGAT 1080
GAGCCTGAAT CATTTCATGT ACCTAACAGA AAGAAAATAC TCTGGCTGGG CTCAGWGGCT 1140
CATGTTTGTA ATTCTAGCAT GTTAGGAGGT CGAGGTGGGT GTGTTGCTTG AGCCCAGGAG 1200
TTTGAGACCA GCCCAGGCAA CATGGCAAAA CTGTCTCTAC AAAAAATATA AAAGTTAGCC 1260
AGGCGTGGTG GCATGCGCCT GTCGTCCGAG ATACTCGGGA GGCAGAGAGG TGGGAGGATC 1320
ACTTGAGCCT GGGAGATTGA GACTGCATCG AGCTGTGGTC ATGCCACTGC ACTCCAGCCT 1380
GGAGGACAGA GTGAGACCCT GTCTCAGGAA AAAAAAAAAA AAAAA 1425 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met lie Tyr Tyr Phe Leu Lys Asn Gin Cys Gly Leu Pro Phe Leu Phe 1 5 10 15
Leu Leu lie Pro Leu Phe Glu Phe Leu Cys Val Ser Phe Ala Phe Pro 20 25 30
Ser Gin Ser Gly Gly Val Arg Pro Ala Leu Trp Asp Glu Arg Ser Cys 35 40 45
Gly Tyr Val Ser Ala Gly Thr Lys Arg Ala Glu Gly Glu Val Trp Lys 50 55 60
Gly Gin Gly Glu Glu Met Gly Ser lie Val Lys Arg Leu Val Pro Leu 65 70 75 80
Ser Lys Tyr Val Glu Asn Asp Asp Gly Lys Val Ser Pro Cys 85 90
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2859 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
CGCACCCAGC CGCGCCGGCG AGGACATGGG CAGCCGCGGC GCGCCCACCC CCCGCGCCGA 60
TGTGAATTAT TAAAAAGAAA ATGGCCCAAC GGAGCACTGT ATTTCCTTCT CGTGTCACCA 120
AGGAAAGGTA TAATATATGG AAAATATGCA TCTAAGGCGA GTGAGAACCA TGCCCCGACA 180
CAGCCAGTCC CTGACCATGG CACCATACTC ATCTGTAAGC CTCGTGGAGC AGCTGGAAGA 240
CAGGATCCTC TGCCATGAGA AAACCACCGC CGCCCTCGTA GAGCACGCCT TTCGGATTAA 300 AGATGACATT GTCAACAGTT TGCAGAAAAT GCAAAACAAA GGGGGAGGTG ACCGCTTGGC 360 CAGGCTTTTC TTGGAGGAGC ATATCAGAAA CATAACTGCC A AGTGAAGC AACTTAATCG 420
GGATATCGAG GTACTCCAGG AGCAGATTCG TGCTCGGGAC AACATTAGCT ATGGAACTAA 480 TTCTGCCTTA AAGACCCTGG AGATGCGCCA GCTCTCCGGT TTGGGAGATC TTCGAGGAAG 540 AGTGGCAAGA TGTGATGCCA GCATAGCTAG ACTTTCTGCA GAGCACAAAA CGACCTATGA 600 GGGGCTCCAG CACTTGAACA AAGAACAGCA GGCTGCCAAA CTTATCTTGG AAACGAAAAT 660 CAAAGATGCA GAGGGACAGA TTTCTCAGCT TTTGAACAGA GTGGACTTGT CAATATCAGA 720 GCAGAGCACC AAACTGAAGA TGTCTCACAG AGACAGTAAC CACCAGCTTC AGCTTTTGGA 780 CACTAAATTT AAAGGTACAG TTGAGGAACT CAGTAACCAG ATATTATCTG CACGGAGTTG 840 GTTGCAACAG GAACAAGAAC GGATAGAAAA AGAGCTTTTA CAGAAAATTG ATCAGCTTTC 900 CTTGATTGTT AAGGAAAACA GTGGAGCCAG TGAAAGGGAT ATGGAGAAGA AGCTCAGCCA 960
GATGTCAGCC AGGCTTGACA AAATAGAAGA GGGTCAAAAG AAGACTTTTG ATGGTCAGAG 1020
AACAAGGCAA GAAGAGGAGA AGATGCACGG GCGAATCACC AAGCTGGAGT TACAGATGAA 1080
CCAGAACATC AAGGAAATGA AAGCAGAAGT TAATGCTGGG TTTACAGCCG TCTATGAAAG 1140
CATAGGATCC CTCAGGCAAG TTCTCGAGGC CAAGATGAAG CTGGACAGGG ACCAGCTACA 1200
GAAGCAAATC CAGCTGATGC AGAAGCCAGA GACCCCCATG TGAAGGGAGC TGGGACAAGG 1260
TCCTAAAAGA CAGTTTTGCC AGTGGGGCTA GGAGCCGGAT ACCTCTGTAG CCAGGCCATC 1320
GCTGCATTCA GGATTGTTCC ATCCATGGCG TGCATGTGCC AAGAAATGTG TTTTTATGGG 1380
TCTAAATGTT TACCTTGAGT CTTGAAAATA CTCTTTTGTT AAAAGTATGA AATACAGTTT 1440
TTACCAGTTT ATTTCACTTC TCTAAATTCA ATGGAAATCC CCCGCCCTGG ATTTTGAAAG 1500
GCTTTTATCT TCTTCATTTT ACGAATGGAA AGACGACAAT TTTTCTTCAA TGCTTGATGC 1560
ACTAATGAAG ACTGTTTACT ATTTTGAAAA ATGTCATGGG GATTTTTTTT TAATTAAGAA 1620
ACTAATGAAT CATCACAGGA ATGTGTTGCT CCTCACCCTA AATTAAGAGA ATGTCCCAGT 1680
AGATTAGACT TCAACCTTTG AGTCCAATTT GGATTTTATT ATCGTTGTCT ATGCACTTCT 1740
TA ATTGGTT ATCTTCTTGT AAATCTTCTG TCTTTTGTAA GGGGAAAGGA TTTAACATTT 1800
AGAATAAACC CCACCATTTA TGTAATGGAA ATAGTTTAAA AATTGCTAAC TGCCATGTGG 1860
ATTGCAAATT AAATGGAAAC TTATTTAGAT AACGTAAGGC TCAATATCTG CGTTGACCAC 1920
CTAGATATTA CAGGTTTTAA TATTTAAAAC TATTTTTGAA TTATCCACAA CCTGTATAGT 1980 GATAGCCATA TATTTAATAA TGGAATGGTG GTTAACAGTC TATTTACTGC ACAATTAATT 2040
GTTCACTAAT CAAATAGAAT GTGGTAATTT TTCAGACTTT ATGATCTGTT TCCAAAATTG 2100
GCACAAAGTG CTAGGGTTTA TATACACTTA TCGTAACTGT ATTTTTGTGC CTTGGTTTTA 2160
TCATGTCAAT GCACTGTACT CTGTAAAAGT TTTGCAGACA AAATAGAAAG TATGATAATC 2220
CGTCAGAAGT ATGATGTAAA ACTGGAATCC TCTGTATTTT TTAAATGTTC TAAAAATTTT 2280
ATCGCTGTTA AGGTATTAAT CATTCAGTAT TACTAATGGA ATAGAAATTC ATACTTTTGT 2340
ATGGACAACA AATTGATATT GCATTTATAG CACTGTAAGA AACTTTCATC TTGAGCAACT 2400
TTGTAGATGA TGGGTGTTTT ATTTTCAATC GCCATATTTG ATCAGTCATT GAAAATTGGC 2460
CCCAGTGCTG TTTGTTCATC TCTGTATGTA AAAACTGACA GTGAGACACA ACTTTCTGAA 2520
CTGTGAGGGT GTCCCAGGAA AAAGAAAAAC AGGAATACTT TAACAATTAA AAAGAAAAAA 2580
ATGTTTTTTG TTTGCCAAGG ACTCAGGAAA ATAAAAAGCA TTTTCTATTT TTAGGACAAA 2640
TCACAAATGA AGTGTCTAAC TGGCTATTAC TGTTTACCCA TATAAAATAT GCTGCTAAAG 2700
TACATATTTT GCTGTCAATG GCTTGACAAT TTTTTTTTTC AAATTTGGAC ATGAGAGGTT 2760
ATATAGGGAC TATATTATCC AACACATATT TTCTTATTTT GCCACAAATT TCCACTTAAC 2820
AAATAAAAAA AGGCGAATGC TGTTTTGCAA AAAAAAAAA 2859 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 368 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Glu Asn Met His Leu Arg Arg Val Arg Thr Met Pro Arg His Ser
1 5 10 15
Gin Ser Leu Thr Met Ala Pro Tyr Ser Ser Val Ser Leu Val Glu Gin 20 25 30
Leu Glu Asp Arg lie Leu Cys His Glu Lys Thr Thr Ala Ala Leu Val 35 40 45
Glu His Ala Phe Arg lie Lys Asp Asp lie Val Asn Ser Leu Gin Lys 50 55 60
Met Gin Asn Lys Gly Gly Gly Asp Arg Leu Ala Arg Leu Phe Leu Glu 65 70 75 80
Glu His lie Arg Asn lie Thr Ala lie Val Lys Gin Leu Asn Arg Asp 85 90 95 lie Glu Val Leu Gin Glu Gin lie Arg Ala Arg Asp Asn He Ser Tyr 100 105 110
Gly Thr Asn Ser Ala Leu Lys Thr Leu Glu Met Arg Gin Leu Ser Gly 115 120 125
Leu Gly Asp Leu Arg Gly Arg Val Ala Arg Cys Asp Ala Ser He Ala 130 135 140
Arg Leu Ser Ala Glu His Lys Thr Thr Tyr Glu Gly Leu Gin His Leu 145 150 155 160
Asn Lys Glu Gin Gin Ala Ala Lys Leu He Leu Glu Thr Lys He Lys 165 170 175
Asp Ala Glu Gly Gin He Ser Gin Leu Leu Asn Arg Val Asp Leu Ser 180 185 190
He Ser Glu Gin Ser Thr Lys Leu Lys Met Ser His Arg Asp Ser Asn 195 200 205
His Gin Leu Gin Leu Leu Asp Thr Lys Phe Lys Gly Thr Val Glu Glu 210 215 220
Leu Ser Asn Gin He Leu Ser Ala Arg Ser Trp Leu Gin Gin Glu Gin 225 230 235 240
Glu Arg He Glu Lys Glu Leu Leu Gin Lys He Asp Gin Leu Ser Leu 245 250 255
He Val Lys Glu Asn Ser Gly Ala Ser Glu Arg Asp Met Glu Lys Lys 260 265 270
Leu Ser Gin Met Ser Ala Arg Leu Asp Lys He Glu Glu Gly Gin Lys 275 280 285
Lys Thr Phe Asp Gly Gin Arg Thr Arg Gin Glu Glu Glu Lys Met His 290 295 300
Gly Arg He Thr Lys Leu Glu Leu Gin Met Asn Gin Asn He Lys Glu 305 310 315 320
Met Lys Ala Glu Val Asn Ala Gly Phe Thr Ala Val Tyr Glu Ser He 325 330 335
Gly Ser Leu Arg Gin Val Leu Glu Ala Lys Met Lys Leu Asp Arg Asp 340 345 350 Gin Leu Gin Lys Gin He Gin Leu Met Gin Lys Pro Glu Thr Pro Met 355 360 365
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 933 base pairs
(B) TYPE: nucleic acid
(C ) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
TGCTTCGGAG ACCGTAAGGA TATTGATGAC CATGAGATCC CTGCTCAGAA CCCCCTTCCT 60
GTGTGGCCTG CTCTGGGCCT TTTGTGCCCC AGGCGCCAGG GCTGAGGAGC CTGCAGCCAG 120
CTTCTCCCAA CCCGGCAGCA TGGGCCTGGA TAAGAACACA GTGCACGACC AAGAGTACGT 180
ATTCAGCCCG GGCTGTGGTC CAGTGGCCTC CCCATCATCT GCAGCTGAGC CAGCGGCAAG 240
GGCATGCTCA GTCCTCCTTT CCTTCTTCCT GTTTCTATGG CTCCTTGACA TTCTTCAAGG 300
ATGATTCTTA TTCCTTATTG CCACCTATAA GTCAGGTATT CTTTTTTCAT CATTGTATCA 360
CAGGTGGAAG ATCTTTAGGC CCAAATGGGG CACATTACTT GTCTGAATCC GGTCTCTCCT 420
TTTTTTCACC ACAGACAGAC ACACACACAT ACAAATAGAC ACACAGGTAC ACATACACAG 480
TCATAGTAGC AGAATCCAGA AAATAGCTAA GGTTTCTTGA CTATAACAAG ACCTTTTTTA 540
AATCAACACA TTCAAACATT GAATCATTTG TTGCAGCTTT TGTCTTGGGC CAGTTAGCCT 600
CACGCATTAT ACTCGGTTAT CCTTTGTTTT TAAGGCTGGG TGCAGTGGCT CACACCTGTA 660
ATCCCAGTGC TTTGGGAGGC TGAGGCAGGT GGATTACTTG AGCCCAGGAA TTCGAGACCA 720
GCCTAGGCAA TATAGGGAAA ACCTGTCTCT AYTAAAAAAT TGCAAAAAAT TAGCTGGATG 780
TGGCAGTACA TGCCTATGGT CCCAGCTACT TGGGGGGCTG AAGTGGGAGA ATCAAMTGAG 840
CTTGGGAAGT TGAGGCTACA ATGAGCCAAG ATCACGCTCC TGCACTCCAG CCTGGGTGGC 900
AGAGTGAGAC CCTGTCTCAA AAAAAAAAAA AAA 933
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids ( B ) TYPE : amino acid
( C ) STRANDEDNESS :
( D ) TOPOLOGY : linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Thr Met Arg Ser Leu Leu Arg Thr Pro Phe Leu Cys Gly Leu Leu 1 5 10 15
Trp Ala Phe Cys Ala Pro Gly Ala Arg Ala Glu Glu Pro Ala Ala Ser 20 25 30
Phe Ser Gin Pro Gly Ser Met Gly Leu Asp Lys Asn Thr Val His Asp 35 40 45
Gin Glu Tyr Val Phe Ser Pro Gly Cys Gly Pro Val Ala Ser Pro Ser 50 55 60
Ser Ala Ala Glu Pro Ala Ala Arg Ala Cys Ser Val Leu Leu Ser Phe 65 70 75 80
Phe Leu Phe Leu Trp Leu Leu Asp He Leu Gin Gly 85 90
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2956 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGTGTGTGGT GGTTTAAGAA TGTATATCAT AGGGTCAGGT GGCCTGGGTT CATTCCCCAG 60
CTACGTAACC TTTCTATGCC TGAGTTTCCT CATCTATAAA ACAAGGATAA TAATAGTGTG 120
TACTTCTTAG GATTGTTTTG GAGACTCATA AATGAGAAAT ACGTGAAAAA CTCCCTCAAG 180
GCAGTGCTTG ACACATAATG AGCACTCAGT TATCATGGTC ATCATGGTCA TCATCACTGC 240
TACCACCACT GCTGCTGCTA TTACCACTCT ACCTCTTCCC CCTGAAACTC TAATCACTTA 300
CCCTAGAAAC AGTTAAATTA CACTTCAGTG GGAAGGATCT CAGATTTCTT AATGGCACCT 360 GCATTTATAT AATGTTGATA TTGCACGTTC CTAGAAAACA TATCAAGAAG AAACCAAAAT 420
GTGTTTCTGT ACTTTGTAAA CCTGTACAAT AGTTAGAGAT TAGAGGACCT TTATAATCTA 480
CTACTAATTA CTGTGAAAGT AAACATTGTT TAATATACCA GTTCTTAAAG AAATATTTTG 540
TCTAGTCATT AATATTCTAG TTCATCTCAA AGCTTCCATT TGACAATTTA AAATTACTTA 600
AATTTTAATA TTAAAGGAAA CAGTTTTCCT GATTCTCATG AAAGTTCCTA TTTGCACTGA 660
AGATGACTAA ACCTTTTAGT CATAGTTTTA GAAGAATTGG CTTTTTTATA GCCATTTTAT 720
TTACATATGG GTACTGCATA GCAAAGGCAG CAGATTAGCC CTGTTTGTTT TGCAGGGATG 780
AAAGGTAGCA TTCCCAGAGA TTAAGTTGTT CTTGCTATTC CCATTCTCTG CTACATTTGC 840
CTACATTCTT TGGTCCTTTC TATTATTTGT TTCTTTGGTG GAATCCCCTT GTTGCTTATG 900
GCTGGATATT GTTATTCAGC AGATGAATCA CAAGTTTAGC CTGAGGGCCC TAAAGCATCA 960
GAAATAAATT AGAGCCGAGC AAAGTTTAAC TTCTCTGGAA CTTGCACCTT TAGTTTCCAT 1020
GTATTTCTGG AACCAAGATA TTTCAAAGGC TTACTTTATT TCAGACACCT ATTATCTTCA 1080
AGTCACAGAT AACTATTGAT TCTGTAAAGT GTTTCAAAGA TTTTTGTCCA CTAGACATTT 1140
TTAAATTTGT TCAACTCCTC CTCATCATTT TAGAAATTAT TTCTGTTAGG TAAAATTAAA 1200
ACTAACAATG TATTTTAGTT TATTTTTCTA ATGATACCAG TCACCTTTCG GGGCTAACTA 1260
AACATTTTGT GCAGCATTCT CTTAGTTTAC ATCCTCCTTT CTTTCAGTCT TCCTGTTTAT 1320
TAAGGCTGTC CTGTAGCAAA CAAAAGAGTG ACTCATGTTA AAAGTATTTT AACTGCTCTA 1380
ATATATCTGA GGAAGAATAA CTTTCTAAAT TAAAGTAATG TATTTTATTA AATATTAAAA 1440
TGCATTTTTT GGCTATTCAT TTCTGTATGT AAAAGAAAAG TTAACTTTAT GGTGTTATGC 1500
AAAATATGCT AAATTTAGAT TTTAGAGCAA TATATAGGGA GATATGTCAC AAATTTCTAC 1560
ATTTTGGTTA AATTATTAGT ATTTTTTTAT ATTCAAATGT GCCTTGATAT TTAAATAATA 1620
TACTGAATGC AGAATTTATG TTATGTGAAC CATTATGGAA AATGTTAATG TTAACAAAAT 1680
GAGGTGTATT GACTTTTCAA CAATGTAAAT TAAAGATGGT ACATCTACTG TTTAAGGGCA 1740
GAGGAATTAA AAGAGTATAG ATACTGAAAT GTATCACTTA CTAGTAGTGT GGCTATAATC 1800
AAATTAATTA ATCTCTCTCT AGGCTTTAGC TTCCTCATCT TAGTTTGTTC AGGCTACTGT 1860
AACAAAATAA CATAGATTAT GTACTTTTAA ATGACAGAAG TTTATTCGGC ATGGTTTGGG 1920
AGACTAGGAA GTCTAAGATT AAAGAACCAG CAAATTTGGT GTCTGATGAG GACCCATTCC 1980
TTTGTTCACA GATGATGCCT TCTCATTGTG TTTTCAAATG TTAGAAGGAG CTAGCTAGCT 2040 TTCTGGGGTC TCTTTTGTAA AGGCACTAAT CCCAGTCATT AGGGCAAATT GGCTCCTACA 2100
GGCCCCACCT ATCTCCTAAT ACCATCACCT TGAGGATTAA GATTTCTACA TATGAATGAA 2160
GCAGGTGTTG TAGAAGGTCA GTCAGTTAGA CCATAGCACC ATCTGTAAAA TTGAATAGTA 2220
ATTTACTGCC TCATTGGATG TCAGGATTAA AGGAGATAAG ATTTTATTAG TTACTAGTTA 2280
CCATAGTGGT TTTTTTTTTA CACTATAATG TTCGTTTTTT TGTTTCATGC TTGTACCTTC 2340
AACATTTCCT TCCATTTGAA TACTTCTTTT GTCTCCTGTA GGCCTGTCTG TCCACTTAGG 2400
TGTAAGATGT GTTTTTGTGT CAGGAATGAT GGTGCAATGC TAATGTTCCA TTGCCCTATT 2460
TGGCAATACT CTGATCATTA ACTATAAAGA ATAACACCAG TGTTAACTAA CTCTCCTTGC 2520
CTGACAGTAG TGCTGCCACT ATTCCTTGTT TCTGTGGTAA TAGATGAGGT TTGTATGGTC 2580
CTGTTATTCC AGCCTCCAGA CACCATTCCA GATCAACTGG TGCCYTCWAC GCCCCCGAAG 2640
TGTATGGGGC CTCAGGTGAA GGATGAGWAC ATTTTCACTA TCATCTGGCA TTCATCTCAG 2700
ATTTTATCCT TTTCAGTTTC CATTAAATAA TATTCATGTT TTAAAATTGA TTTTTTATTA 2760
TTTAAATTTA ATTTGTTGGA GAATAAACTT TTTTTTTTCT TTTCTCCCAA GTAACGTTTT 2820
CCCCTTTAGC AACTGTATTG AGCATTTTTC TCACTGGTAT ATGGACATTT TTTTGTATAA 2880
CCTGTTGTGT CATTTTTAAA TATAGAATTG TTTTTATGTT CTCATCTTTG TATATATGTT 2940
TAAAAAAAAA AAAAAA 2956 (2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Thr Lys Pro Phe Ser His Ser Phe Arg Arg He Gly Phe Phe He 1 5 10 15
Ala He Leu Phe Thr Tyr Gly Tyr Cys He Ala Lys Ala Ala Asp 20 25 30
(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1325 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
AATCGGGAAA AAAAGCCATG TATTCTTTCG TTTCTCTCTA AAAGAAGAAA AATATAATTT 60
AAAAATACAT TGCGTATTTT CTAAAACAAT AAATTTATAG TGTTAATATT CATAGGGTCA 120
ATCAAAATGA AGCTTCTCCT TTGGGCCTGC ATTGTATGTG TTGCTTTTGC AAGGAAGAGA 180
CGGTTCCCCT TCATTGGTGA GGATGACAAT GACGATGGTC ACCCACTTCA TCCATCTCTG 240
AATATTCCTT ATGGCATACG GAATTTACCA CCTCCTCTTT ATTATCGCCC AGTGAATACA 300
GTCCCCAGTT ACCCTGGGAA TACTTACACT GACACAGGGT TACCTTCGTA TCCCTGGATT 360
CTAACTTCTC CTGGATTCCC CTATGTCTAT CACATCCGTG GTTTTCCCTT AGCTACTCAG 420
TTGAATGTTC CTCCTCTCCC TCCTAGGGGT TTCCCGTTTG TCCCTCCTTC AAGGTTTTTT 480
TCAGCAGCTG CAGCACCCGC TGCCCCACCT ATTGCAGCTG AGCCTGCTGC AGCTGCACCT 540
CTTACATCCA CACCTGTAGC ATCTGAGCCT GCTGCAGGGG CCCCTGTTGC AGCTGAGCCT 600
GCTGCAGAGG CACCTGTTGG AGCTGAGCCT GCTGCAGAGG CACCTGTTGC AGCTGAGCCT 660
GCTGCAGAGG CACCTGTTGG AGTGGAGCCA GCTGCAGAGG AACCTTCACC AGCTGAGCCT 720
GCTACAGCCA AGCCTGCTGC CCCAGAACCT CACCCTTCTC CCTCTCTTGA ACAGGCAAAT 780
CAGTGAAATT CTCTAGAAGA GTACCATGGG TTCATTTCTA TACTGATGCA GAAATAAGTG 840
AAATCTACAA AAGTTTTCTT TCTTTTCCAA AGACTATTTC ATTCTGTAGT ATTCAGAGTA 900
TTCATCTCAC TACATAGATT TGTTTGTGGT AGTTATTTCC TTGGACTTAA TTTATATTGA 960
AAAAACATTG ATAATTAAAT AAATAAAATA GATAATTTAG ACCAATGGTG ATAAGGTCTG 1020
GATGAAAACT ACGCTATGGA GGACTGAAAT GGCAATCATT CAGCCTAGCC TGGAGTCTGA 1080
TTATACAGCT ACTATAGGAT GATGTTAGTA TTGGTTTTGA GTGCAATAGG TTTTTTCCTA 1140
AACAAACATA TTTTGTAGTC AATGAACTTT TTGTCACAAA ACAGTAAAAC ATCTGTGTTT 1200
AACCTATGGT AAACAACATG TTAATGAACT ATGCTATCCA TGACTTAATG GACAGTTCAA 1260 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1320 AAAAA 1325
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 10 :
Met Lys Leu Leu Leu Trp Ala Cys He Val Cys Val Ala Phe Ala Arg
1 5 10 15
Lys Arg Arg Phe Pro Phe He Gly Glu Asp Asp Asn Asp Asp Gly His 20 25 30
Pro Leu His Pro Ser Leu Asn He Pro Tyr Gly He Arg Asn Leu Pro 35 40 45
Pro Pro Leu Tyr Tyr Arg Pro Val Asn Thr Val Pro Ser Tyr Pro Gly 50 55 60
Asn Thr Tyr Thr Asp Thr Gly Leu Pro Ser Tyr Pro Trp He Leu Thr 65 70 75 80
Ser Pro Gly Phe Pro Tyr Val Tyr His He Arg Gly Phe Pro Leu Ala 85 90 95
Thr Gin Leu Asn Val Pro Pro Leu Pro Pro Arg Gly Phe Pro Phe Val 100 105 110
Pro Pro Ser Arg Phe Phe Ser Ala Ala Ala Ala Pro Ala Ala Pro Pro 115 120 125
He Ala Ala Glu Pro Ala Ala Ala Ala Pro Leu Thr Ser Thr Pro Val 130 135 140
Ala Ser Glu Pro Ala Ala Gly Ala Pro Val Ala Ala Glu Pro Ala Ala 145 150 155 160
Glu Ala Pro Val Gly Ala Glu Pro Ala Ala Glu Ala Pro Val Ala Ala 165 170 175
Glu Pro Ala Ala Glu Ala Pro Val Gly Val Glu Pro Ala Ala Glu Glu 180 185 190 Pro Ser Pro Ala Glu Pro Ala Thr Ala Lys Pro Ala Ala Pro Glu Pro 195 200 205
His Pro Ser Pro Ser Leu Glu Gin Ala Asn Gin 210 215
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TGCGGGCTCA CANGAANAGT CTCACCTCAG TGCCAAGGGG TGTCAGAGAT GCTCACTGCC 60
CTCCTCTCCT TGGGGTTGCA TGNAGGCATG ATGGCGCTTG GCCGTGGCAG GGTAAGGAAC 120
CGGCGACNGA GGCCCATCAC GTGTTCACAT GCTCTCCTGC GTCNGTGCTT GGGAGATATG 180
GACTGTCNTG TCCTTAGACC ACATTTATNT CAAGGCAAGG GGAGC 225 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 533 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGTTCAAAAT GAGGCAAAGA TAGGAAAGTG CTTCTTACAG ATAATTTTCA AGGCCAGTGA 60
CTGGAGAGAG GGGTAGGTCT GTCAATCGAG TGCTTGCTGA CTGCACATAT CACAGGGCGT 120
GTGACGACTG CTGGGAGAGG AAAGCGAGAC ATCATTCCAA CCCTCCAGAA GCTAAAGATC 180
CTGGAACTCA AGGGGAAAAC TAACGTAAGT GCGAAAGCGA ACAAGCAAAC ATGTCCTCAA 240
CGGGGCAGGC AGGCTGTCGG GGTACAGAGC TGGGATCTGG GAAGGAACAG AGAGGGCCGC 300
TCAGGGAGAG GAAGCACAGT GCCACCGGAG GCACGCACTC AGCAGGCACT CGCAGGCTGG 360 GCAGAGGTAG AGAAGCAGCG CTGCACAGGC AGGCAGCTGA CCCAGGGCTC TTAGAGCCGG 420
GCAGGAGAGC TGGTGTGGGA CCTGGGAGGA GGACAGGAGC CTTCAAAGCA GCACCGCCTG 480
ATTGCAGCCA GGAGGGTAGC ATCAAGGAAG ATGGAACTGC GGCCAGGCCA CAT 533 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 101 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ser Ser Thr Gly Gin Ala Gly Cys Arg Gly Thr Glu Leu Gly Ser 1 5 10 15
Gly Lys Glu Gin Arg Gly Pro Leu Arg Glu Arg Lys His Ser Ala Thr 20 25 30
Gly Gly Thr His Ser Ala Gly Thr Arg Arg Leu Gly Arg Gly Arg Glu 35 40 45
Ala Ala Leu His Arg Gin Ala Ala Asp Pro Gly Leu Leu Glu Pro Gly 50 55 60
Arg Arg Ala Gly Val Gly Pro Gly Arg Arg Thr Gly Ala Phe Lys Ala 65 70 75 80
Ala Pro Pro Asp Cys Ser Gin Glu Gly Ser He Lys Glu Asp Gly Thr 85 90 95
Ala Ala Arg Pro His 100
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 458 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TAGGCAGTCA TCTTTGTAAA CCTCCACTGG TGCTGGCTGC GTTTAGAACA TACTCCATAT 60
AAAACAGGCC CTGGGATTAC AGGCATGAGC TACCGTGCCT GGCCCCCTTT TTTTTAATTA 120
CAGAGAAATA AGTTACACCT TAGTATCAGA TATTAATTTT CTTCAGTGTT CAGGCAATTA 180
GTATTTAGAA AGCTCTTGTC ATGAGATGGC TCTGGGATGT GATGATGATT GTTGGGATTG 240
AAAAAATGGT AGTATCATGG AGAGATCATA ATAAATTCTT AGTATTAAAA GTGGTTTTGC 300
TTTCAGTTAG GGAGAAAAAT TAGATTGTAC TATTTTTCCT CTATGATTTC CTTCAGTTAT 360
CTTCCAAATG TTGTTTTTTC CCCACAGCCC CCTTAACATT GTTCTCTATG CACTTCTCAA 420
TACATTTTCA TTTGTTTCTC AAAAAAAAAA AAAAAAAA 458 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1350 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
TTTTTTTTTT TTTTTGTAGA GACAGGGTCT TGCCATTTTG CCCAGGTTGG TCTCAAACTT 60
CTGAGCTCAG GCTATCTGCC CACCTTGGCC TCCCAAAGTG CTGGGATTAC AGGTGTGAGC 120
CACTGTGCCC GGCCTGTATT GTTTTAAGTT ACACTTATTC CTTTTAAAAT TCAGAATTTG 180
TTAAGCATTT AAAACAAATT CATAAATTAA AACCTCCTTG AGATACCATT TACCATGTAG 240
TTTGATGAAC ATAATACATG GTGCATTACA TTGGCAAAAG CAGTGGGGAA AAAGATGCTT 300
TTATAAATGT CTGGTGGGAG TTAAATTGTG TAACTTCTAT TACACTTTTG TAATAGCTAC 360
CAAAATATGT TATTTCTATC TACCTCTCTC TCTCTGACTC AACAGTTCCA TTTCTAGGTT 420
TTGTGTTGTG GATATTCTTG AACATTGTGA AATGTATACA GGGAGGCTTC ACAGCAGCAC 480
TGTTTGTTTC AAATGATTTG AAAACAACCT CTCCATAAAC GAGATAGGCT AAATCAAGCA 540
TGGCACACCT ATACAATGGA TGCGGCCATT AAAAAGAACA AGGCAGCTCA TATGCATCAA 600
TATAAAAAGG TCTATAAACT ATACTATCAA ATGAAAATAG CAAGATGCTA CCATTTATAT 660 TAAAAAGAGG ACAAAATATT AA ATATTCA TGGTTGCTTG TCTATGTGGA ATATTTCTGG 720
ATATATACAT AAGAAGTTAC ATTGGTTACC TATGGGCAGG TTACTACTGG GTGGCTTGTG 780
GGTGAGGGCA GGAAGAGCTT ACTTTCCATG GTAAACCTTT TTGTATATTT TGCAGCATTC 840
AAAAATTCTA ATTTAAAGTT TATTTTAGAA AAATGCCCCC ATGTATACAA GTGATTTCCA 900
AGTTCCTCCT TCAATATTTT TAATGATTAT GGAACACACT GAACTTCTTT TTTATTATTC 960
TAGCTGTGAA CTCTGTCTGC TGTCTACATG CACATATATA ATCTATGTAA TATTTAAATT 1020
TATATCCTTT ATATGTCAGT TGGGTGGTGA GTAAAAGAAA AATATATTTT TATCAGCAAA 1080
CTTGGTAAAT TGTTGAGGTT TCTGATATAG TCAGAGGTAG TTGCTTATCA CAACATTAGG 1140
TAAGTTTTTA AARACACCTA TTTAAAACAC ACTGATGTAT ATATATATTG GTCTGTTTTC 1200
ATGCTGCTGA TAAAGACATA TCCAAGACTG GGAAGAAAAA GAGGTTTAAT TGGGCTTATA 1260
GTTCCACATG GCTGGGGAGG CCTCAGAATC ATTGCGGGAG GCAAAAGGCA CTTCTTACAT 1320
GGCAGTGGCA AGAGAAAAAA AAAAAAAAAA 1350 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 16 :
Met Leu Pro Phe He Leu Lys Arg Gly Gin Asn He Asn He Phe Met
1 5 10 15
Val Ala Cys Leu Cys Gly He Phe Leu Asp He Tyr He Arg Ser Tyr 20 25 30
He Gly Tyr Leu Trp Ala Gly Tyr Tyr Trp Val Ala Cys Gly 35 40 45
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1598 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TCGGGACACT ACATGAAGTC CTGAAAATAA CAGAGAAACT GTTATATCTT TTTAATGATT 60
TATTTGCAAG TATTGAGGTT GACCTGAAAA ACAATGAAAC ACATGAACAC ACTTCCGATT 120
TTCTCCTCGC TGATTAGCTT CCTGCCTGCT GTCAGTGCTG GACGAAGTGC TATAACTACT 180
TTATGTAACA TTACAGAACA GCTAGAGGTC CTGGGGTAAG AGAAAAAAAG CACATCACAA 240
CAAATGTGAA AGCCTTCATT ATTACACGTT CCAGTTTGTC TCGCTGTGTA GGCATAAGCT 300
AATGGTTTAT TTTCAGAAAG CTGCCTGAAA CGTTGCTTTG TATTCTTCTA GGAAGAACTT 360
TAATTCCTCC TGAGGAACTC TACTTTCTGA GCCAAACTGC TAATTTTCTG CGGAACTGTC 420
TAGAAGATCA TTCAAGAGAC CCTGCAGTTG CACTTTCTCG TAAAAGTTAA AAAAAAAAAA 480
AAAAAAAAAG GTTTTTCCCG GCCTTTGAAC ATTTTGCCTA TGAGAGTTTT GCATATATTT 540
TATACTTGAG TAGACAACTT TAATAATCCA TATTTATACT ATCGCAGAAG TAAGCATTTG 600
GCAAACGTTC AGCCATTAGC ACTCATTTAA CCCTGTTAGC AATATTCTTT TGAAAAAAGT 660
GCCAGTCCTT ATGTGATAAA CTAAGAAGCC CATTGAATAT AAAANTGTGT NGGACTGAAA 720
CNGTGACCTT ATATTATTGC TAAGGGAATA TGAGATTAAC TTCCTACAGG GGCCANAACC 780
ANANAAAGGC TTCCAGCAAC TTCGATNAAA NTANTTTGGC CACATNTCAA GCCAATTGTT 840
TGTACTATTT ATGTACCTTT TTCATAACTG GAATTGCCAA ATAAGCATGG AGATCTAAAT 900
GRAAAAAAAA AAAAAAAAAA AAAGCGGCCG CAGGTCTAGA ATTCAATCGG AAAAAACAAA 960
GAGAAGAAAC ATACTGCCCC ATCTTGTTTG CATGAAACTC TAGAATCTGG TGTTTCTCTA 1020
TTTATCTGCT CCCTCTTTGC CTACCTTGGN ATTTCTTTTT TTTTTTCTTT GTAACTATGG 1080
TTTTTACCTA AAGTTTAAAC TTTTTATTAT TATTTTCTCT CTAAATTCTT GCTAGTTAAT 1140
AACATTATTA ACTTCAAGAT TTTAGAAGAG CAGTGATGAT AGTAATGATC GATAACTAGA 1200
CTATCGAGTT TCAGAAGAAA CTTCCAAGTA TATATAATGT TTGACATAGC CTTTATTTCT 1260
ACAAATCTAC TACCTGTAAA CTAACATTTT AAAATACCTG TATATGGCTG GGTGTGGTGG 1320
CTTACACCTG TAATCCCAGC AGTTTGGGAG CCTGAGGTGG GCAGATTGCT TGAGCCCAGG 1380
AGTTGGAGAC AAGCCTGGAC AAAATAGACC TCTCTCTACA AAAAGTACAA AAAATTGGCT 1440 GGGTGTGGTG GCACACGCCT GTGGTCCCAG CTACTCGGGA GGCTGAGGTG GGAGGATTGC 1500
CTGAGCCCGG GAGATGGTGG TTGCAGTGAG CTGAGATCAC CCCATTGCAC TCCAGCCTGG 1560
ATAACAGAAT AAGATGCTGT CTTAAAAAAA AAAAAAAA 1598 (2) INFORMATION FOR SEQ ID NO: 18:
(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: 18:
Met Lys His Met Asn Thr Leu Pro He Phe Ser Ser Leu He Ser Phe 1 5 10 15
Leu Pro Ala Val Ser Ala Gly Arg Ser Ala He Thr Thr Leu Cys Asn 20 25 30
He Thr Glu Gin Leu Glu Val Leu Gly 35 40
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1257 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CATGGCGTCC AGGTCTAAGC GGCGTGCCGT GGAAAGTGGG GTTCCGCAGC CGCCGGATCC 60
CCCAGTCCAG CGCGACGAGG AAGAGGAAAA AGAAGTCGAA AATGAGGATG AAGACGATGA 120
TGACAGTGAC AAGGAAAAGG ATGAAGAGGA CGAGGTCATT GACGAGGAAG TGAATATTGA 180
ATTTGAAGCT TATTCCCTAT CAGATAATGA TTATGACGGA ATTAAGAAAT TACTGCAGCA 240
GCTTTTTCTA AAGGCTCCTG TGAACACTGC AGAACTAACA GATCTCTTAA TTCAACAGAA 300 CCATATTGGG AGTGTGATTA AGCAAACGGA TGTTTCAGAA GACAGCAATG ATGATATGGA 360
TGAAGATGAG GTTTTTGGTT TCATAAGCCT TTTAAATTTA ACTGAAAGAA AGGGTACCCA 420
GTGTGTTGAA CAAATTCAAG AGTTGGTTMT ACGCTTCTGT GAGAAGAACT GTGAAAAGAG 480
CATGGTTGAA CAGCTGGACA AGTTTTTAAA TGACACCACC AAGCCTGTGG GCCTTCTCCT 540
AAGTGAAAGA TTCATTAATG TCCCTCCACA GATCGCTCTG CCCATGTACC AGCAGCTTCA 600
GAAAGAACTG KCGGGGGCAC ACAGAACCAA TAAGCCATGT GGGAAGTGCT ACTTTTACCT 660
TCTGATTAGT AAGACATTTG TGGAAGCAGG AAAAAACAAT TCCAAAAAGA AACCTAGCAA 720
CAAAAAGAAA GCTGCGTTAA TGTTTGCAAA TGCAGAGGAA GAATTTTTCT ATGAGAAGGC 780
AATTCTCAAG TTCAACTACT CAGTGCAGGA GGAGAGCGAC ACTTGTCTGG GAGGCAAATG 840
GTCTTTTGAT GACGTACCAA TGACGCCCTT GCGAACTGTG ATGTTAATTC CAGGCGACAA 900
GATGAACGAA ATCATGGATA AACTGAAAGA ATATCTATCT GTCTAACCCA TTTCCAATGG 960
ACAGTGATGG GCTTGTTTTT GTAAAATTAC CAGAAAACTC AGTGGAGATT TACTGAAAAA 1020
CTCAGACTTT ATTCAGATTA AGTTCCTCTA CAAAAAGTAG GGTTCTGTCC CATGTGTYTC 1080
TGACACATTT ACAAAATACC AGTTTTTTAA AATTTTGGTC AAATTATGAG TGGTTGATTT 1140
AAAAACTTTT CCAAGAAGAA GAAAAGCATG GAGTAGTAAT TTAAAGAACT CWTAAAAAC 1200
TTCTATTTTT TATTTTAAAA TAATAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 1257 (2) INFORMATION FOR SEQ ID NO: 20
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH. 314 ammo acids
(C) STRANDEDNESS
(D) TOPOLOGY linear
(n) MOLECULE TYPE, protein
(xi) SEQUENCE DESCRIPTION. SEQ ID NO.20
Met Ala Ser Arg Ser Lys Arg Arg Ala Val Glu Ser Gly Val Pro Gin 1 5 10 15
Pro Pro Asp Pro Pro Val Gin Arg Asp Glu Glu Glu Glu Lys Glu Val 20 25 30
Glu Asn Glu Asp Glu Asp Asp Asp Asp Ser Asp Lys Glu Lys Asp Glu
35 40 45 Glu Asp Glu Val He Asp Glu Glu Val Asn He Glu Phe Glu Ala Tyr 50 55 60
Ser Leu Ser Asp Asn Asp Tyr Asp Gly He Lys Lys Leu Leu Gin Gin 65 70 75 80
Leu Phe Leu Lys Ala Pro Val Asn Thr Ala Glu Leu Thr Asp Leu Leu 85 90 95
He Gin Gin Asn His He Gly Ser Val He Lys Gin Thr Asp Val Ser 100 105 110
Glu Asp Ser Asn Asp Asp Met Asp Glu Asp Glu Val Phe Gly Phe He 115 120 125
Ser Leu Leu Asn Leu Thr Glu Arg Lys Gly Thr Gin Cys Val Glu Gin 130 135 140
He Gin Glu Leu Val Xaa Arg Phe Cys Glu Lys Asn Cys Glu Lys Ser 145 150 155 160
Met Val Glu Gin Leu Asp Lys Phe Leu Asn Asp Thr Thr Lys Pro Val 165 170 175
Gly Leu Leu Leu Ser Glu Arg Phe He Asn Val Pro Pro Gin He Ala 180 185 190
Leu Pro Met Tyr Gin Gin Leu Gin Lys Glu Leu Xaa Gly Ala His Arg 195 200 205
Thr Asn Lys Pro Cys Gly Lys Cys Tyr Phe Tyr Leu Leu He Ser Lys 210 215 220
Thr Phe Val Glu Ala Gly Lys Asn Asn Ser Lys Lys Lys Pro Ser Asn 225 230 235 240
Lys Lys Lys Ala Ala Leu Met Phe Ala Asn Ala Glu Glu Glu Phe Phe 245 250 255
Tyr Glu Lys Ala He Leu Lys Phe Asn Tyr Ser Val Gin Glu Glu Ser 260 265 270
Asp Thr Cys Leu Gly Gly Lys Trp Ser Phe Asp Asp Val Pro Met Thr 275 280 285
Pro Leu Arg Thr Val Met Leu He Pro Gly Asp Lys Met Asn Glu He 290 295 300
Met Asp Lys Leu Lys Glu Tyr Leu Ser Val 305 310
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1544 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
GCGGTCCTGC CACACAAGCT GGGCGGCGGA GGCCACGCAG CCGGGCCTTC TTCTCTCTGG 60
GACCCTCCGC CAGCGCATAG CCGCAGGCCG GTGTGACTTC TGCACCCTCG GTTCTGAGGG 120
TACGGTGACC CCTAGTGGGC AGTTTGCAAA ATGTGATTCC TTCTTCCCAA CTCCCCATCC 180
CCCCTTCCCT TCCCGTCACG TCCTGTTTGG GGGTTAATTC GGTTTTTTCT CTGTTGCATC 240
GCGCCTACTG TGCGTGTGCG ATARCGTGTG TGGGGGTGAG AGTTTGTTTT CTGGAATGGT 300
AGGTGCTGGG AGGAGGAGTT TGATGGAGGG CTTCCTGGCT GCTTCTGGCC CTCACCTCGT 360
GGAGGCCTTC ACAGAGACCC TGTGGGCCCT GGCCCTGTGC TGGCACTGTG CCAGTCATGA 420
GGCAGCTCTG ATCACTTCCC CACTGTGGAA ACAGGACTGA CCCAGCCTTC AGTGTGGGCT 480
GCTGAAGCTA TCCTCCTCAG GCCTCAGGGA TGACCTCCTG CCTGAGCCTC TCACAGGCTG 540
GCTGTGGGCC AGTTTCATCT GCTTTCCTGT TGGGGGTCCC GGGCCTCTGC TGTCCTTGAC 600
CCACTGGTGT TCTGTGCAAG GCTTCTTCCC ATTCACCAAG TGCACACCTT GCATCTGCCG 660
CTCGGCATGC ACCAGTTCCA CACACCATCC CATTTTACAG ACAAGGACGC TGAGGCCTGC 720
AGCAGCAGTG TGACTTGCTC AAGGTCCAGT GAGTGACCTC ATTCCCCAGA AAAGGCTCCT 780
CCCACACCAG AGTACAGCCT GGGTAGGGGG AAAATCAGTT CTTTCAGCTA CCACCCATCC 840
AACCTTTGGG CCTATGTGAA AAGAAAGGAA CTAAGCTGGG TGTGTTCTGT CTGGACCTGG 900
GGAGGCCCCT GAAGGCAAAG AGGGAAACTG TCCCAGCTGT TCTGTCCTAG GGGAGGGGGA 960
CATAGCCCTA GCAGGAGCTC CCAGCCCCTC TTGGCACTCT GACACACAAG TACACCCATC 1020
TGGGGCCCGC TTTGCCACGA AGAGCTGGGC AGGCCTGCAG GGTGTGGGGA AGGAGGACAC 1080
AACCTCAAGA AAGGAAGCGT GAACCCCAGG GAACAGCGGG TCCCTTCCCT CCTCAGACAC 1140
AAGCCACCTC AGCTTGTGGC TCTTGGCCCC CAGCCCCACC AACCCACCTG TTCATTTATT 1200
CAACAGACAA TGACAGCTGA TATTTATTGG ACATTTGCAC CATGCCAAGC ATTCGGCTTG 1260
GATTATCCCA TTTGTTTCTC ACAGCCGGTA TTTATTGTCT GCTCCTCTGT GCCAGGTGCT 1320 GTGCTCTGGG CAGGGGCACT GCATGGGCTG CCTGCCCTGG TGGAGCTTGT GGTCTGATGG 1380
GTGAGGCTGA CCCAAGCCCA CCCCATTGCC AACAGGGCCA GGGCAAGAGT ACACACAGGG 1440
GCCTCATACC ATATGTCTAA ATATTTAAAA GTTATCAATC AAGCTAACAA CTGTTAAATA 1500
AAATATGTTC TATTCTCCTA CTTTGAAAAA AAAAAAAAAA AAAA 1544 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Pro Ser He Arg Leu Gly Leu Ser His Leu Phe Leu Thr Ala Gly 1 5 10 15
He Tyr Cys Leu Leu Leu Cys Ala Arg Cys Cys Ala Leu Gly Arg Gly 20 25 30
Thr Ala Trp Ala Ala Cys Pro Gly Gly Ala Cys Gly Leu Met Gly Glu 35 40 45
Ala Asp Pro Ser Pro Pro His Cys Gin Gin Gly Gin Gly Lys Ser Thr 50 55 60
His Arg Gly Leu He Pro Tyr Val 65 70
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CNATGCAGGTC TAACTCCTCC ACTCTGGG 29 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TNAGTTTGGTG CTCTGCTCTG ATATTGAC 29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GNCATCAATAT CCTTACGGTC TCCGAAGC 29
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GNAAATAGGAA CTTTCATGAG AATCAGGA 29
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ANACAATGCAG GCCCAAAGGA GAAGCTTC 29
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GNTTGCTTGTT CGCTTTCGCA CTTACGTT 29
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucelotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ANTGGTAGCAT CTTGCTATTT TCATTTGA 29
(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TNGGAAGTGTG TTCATGTGTT TCATTGTT 29
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GNCCTCGTCCT CTTCATCCTT TTCCTTGT 29
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ANCACCTGGCA CAGAGGAGCA GACAATAA 29

Claims

What is claimed is:
1. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 516 to nucleotide 797;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 606 to nucleotide 797;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 773;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bf228_14 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
2. A composition of claim 1 wherein said polynucleotide is operably linked to an expression control sequence.
3. A host cell transformed with a composition of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein, which comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and
(b) purifying the protein from the culture.
6. A protein produced according to the process of claim 5.
7. The protein of claim 6 comprising a mature protein.
8. 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:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86;
(c) fragments of the amino acid sequence of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone bf228_14 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
9. The composition of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
10. The composition of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 86.
11. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.
12. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition of claim 11.
13. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:l.
14. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 137 to nucleotide 1240;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 1153;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bg249_l deposited under accession number ATCC 98482;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bg249_l deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
15. 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:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 339;
(c) fragments of the amino acid sequence of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone bg249_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
16. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
17. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 26 to nucleotide 301;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 104 to nucleotide 301;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 119;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bv286_l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bv286_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
18. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 31;
(c) fragments of the amino acid sequence of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone bv286_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
19. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:5.
20. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 663 to nucleotide 755;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 850;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone co36_l deposited under accession number ATCC 98482;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone co36_l deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
21. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino a id 1 to amino acid 22;
(c) fragments of the amino acid sequence of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone co36_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
22. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
23. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 783; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 783;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 7 to nucleotide 462;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cpll6_ l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cpll6_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
24. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10;
(b) the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to amino acid 112;
(c) fragments of the amino acid sequence of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone cpll6_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
25. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
26. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 231 to nucleotide 533;
(c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cwll95_2 deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
27. 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: 13; b) fragments of the amino acid sequence of SEQ ID NO:13; and (c) the amino acid sequence encoded by the cDNA insert of clone cwll95_2 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
28. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:12, SEQ ID NO:ll or SEQ ID NO:14 .
29. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 645 to nucleotide 782;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 10 to nucleotide 773;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone fhl3_10 deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
30. 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: 16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 43;
(c) fragments of the amino acid sequence of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone fhl3_10 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
31. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:15.
32. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 94 to nucleotide 216;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 160 to nucleotide 216;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 20 to nucleotide 193;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone gc57_4 deposited under accession number ATCC 98482; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone gc57._4 deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
33. 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: 18;
(b) the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 33;
(c) fragments of the amino acid sequence of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone gc57_4 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
34. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:17.
35. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2 to nucleotide 943;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2 to nucleotide 670; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone hll65_3 deposited under accession number ATCC 98482;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
36. 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:20;
(b) the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 223;
(c) fragments of the amino acid sequence of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone hll65_3 deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
37. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:19.
38. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 1242 to nucleotide 1457;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 1326 to nucleotide 1457;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 869 to nucleotide 1544;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hb752_l deposited under accession number ATCC 98482;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone hb752_l deposited under accession number ATCC 98482;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:22 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
39. 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:22; (b) the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 69;
(c) fragments of the amino acid sequence of SEQ ID NO:22; and
(d) the amino acid sequence encoded by the cDNA insert of clone hb752_l deposited under accession number ATCC 98482; the protein being substantially free from other mammalian proteins.
40. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:21.
EP98933104A 1997-07-02 1998-07-01 Secreted proteins and polynucleotides encoding them Withdrawn EP1007539A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US88719597A 1997-07-02 1997-07-02
US887195 1997-07-02
US95830497A 1997-10-27 1997-10-27
US958304 1997-10-27
PCT/US1998/013813 WO1999001466A1 (en) 1997-07-02 1998-07-01 Secreted proteins and polynucleotides encoding them

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JP (1) JP2002503634A (en)
AU (1) AU8284498A (en)
CA (1) CA2295212A1 (en)
WO (1) WO1999001466A1 (en)

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Publication number Priority date Publication date Assignee Title
US6331413B1 (en) * 1999-03-17 2001-12-18 Zymogenetics, Inc. Secreted salivary ZSIG63 Polypeptide

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Publication number Priority date Publication date Assignee Title
GB9214857D0 (en) * 1992-07-13 1992-08-26 Medical Res Council Human nucleic acid fragments and their use

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* Cited by examiner, † Cited by third party
Title
See references of WO9901466A1 *

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