WO1998037094A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998037094A2
WO1998037094A2 PCT/US1998/003595 US9803595W WO9837094A2 WO 1998037094 A2 WO1998037094 A2 WO 1998037094A2 US 9803595 W US9803595 W US 9803595W WO 9837094 A2 WO9837094 A2 WO 9837094A2
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Prior art keywords
amino acid
polynucleotide
seq
protein
nucleotide
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PCT/US1998/003595
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French (fr)
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WO1998037094A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
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Genetics Institute, Inc.
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Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to AU63373/98A priority Critical patent/AU6337398A/en
Priority to CA002281059A priority patent/CA2281059A1/en
Priority to EP98907611A priority patent/EP0971950A2/en
Priority to JP53698598A priority patent/JP2002514073A/en
Publication of WO1998037094A2 publication Critical patent/WO1998037094A2/en
Publication of WO1998037094A3 publication Critical patent/WO1998037094A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

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:
  • (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).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 65 to nucleotide 1270; the nucleotide sequence of SEQ ID NO:l from nucleotide 1139 to nucleotide 1270; the nucleotide sequence of SEQ ID NO:l from nucleotide 1011 to nucleotide 1216; the nucleotide sequence of the full-length protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 326 to amino acid 384.
  • 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 326 to amino acid 384.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 555;
  • a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 418 to nucleotide 582; the nucleotide sequence of SEQ ID NO:3 from nucleotide 508 to nucleotide 582; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 555; the nucleotide sequence of the full-length protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333.
  • 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 46.
  • 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 46.
  • 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:5 from nucleotide 191 to nucleotide 1756; the nucleotide sequence of SEQ ID NO:5 from nucleotide 254 to nucleotide 1756; the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 604; the nucleotide sequence of the full-length protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333.
  • 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 138.
  • 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:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 1 to amino acid 138.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising the amino acid sequence from amino acid 115 to amino acid 124 of SEQ ID NO:8;
  • (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).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 226 to nucleotide 948; the nucleotide sequence of SEQ ID NO:7 from nucleotide 1128 to nucleotide 1601; the nucleotide sequence of the full-length protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO: 1
  • 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;
  • 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
  • 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
  • polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333.
  • 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:ll from nucleotide 494 to nucleotide 973; the nucleotide sequence of SEQ ID NO:ll from nucleotide 611 to nucleotide 973; the nucleotide sequence of SEQ ID NO: 11 from nucleotide 521 to nucleotide 940; the nucleotide sequence of the full-length protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • amino acid sequence of SEQ ID NO:12 comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO:12; and (d) the amino acid sequence encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
  • protein comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising the amino acid sequence from amino acid 45 to amino acid 54 of SEQ ID NO:14;
  • (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).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 2295 to nucleotide 2594; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1867 to nucleotide 2372; the nucleotide sequence of the full-length protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 26.
  • 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:
  • (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).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 394 to nucleotide 522; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 476; the nucleotide sequence of the full-length protein coding sequence of clone D019_l deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone D019_l deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333.
  • 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 27.
  • 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:17 from nucleotide 262 to nucleotide 654; the nucleotide sequence of SEQ ID NO:17 from nucleotide 322 to nucleotide 654; the nucleotide sequence of SEQ ID NO: 17 from nucleotide 1 to nucleotide 618; the nucleotide sequence of the full-length protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333.
  • 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 119.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
  • 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;
  • 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 119.
  • 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:19 from nucleotide 74 to nucleotide 310; the nucleotide sequence of SEQ ID NO:19 from nucleotide 125 to nucleotide 310; the nucleotide sequence of SEQ ID NO:19 from nucleotide 1 to nucleotide 338; the nucleotide sequence of the full-length protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333.
  • 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 75.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • fragments of the amino acid sequence of SEQ ID NO:20 comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID NO:20; and (d) the amino acid sequence encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
  • 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 75.
  • 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. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
  • Processes are also provided for producing a protein, which comprise:
  • 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.
  • Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, 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.
  • BOH4_l A polynucleotide of the present invention has been identified as clone "BOH4_l".
  • BOH4J. was isolated from a human adult retina 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.
  • BOH4_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BO114_l protein").
  • BOH4_l should be approximately 1600 bp.
  • BOH4_l demonstrated at least some similarity with sequences identified as AA430329 (zw20e04.rl Soares ovary tumor NbHOT Homo sapiens cDNA clone 7698545' similar to contains element MER22 repetitive element), H00825 (yj31b05.rl Homo sapiens cDNA clone 150321 5'), H12557 (yjl2c09.rl Homo sapiens cDNA clone 148528 5'), W53899 (md09c01.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA), and Z82202 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 34P24). Based upon sequence similarity, BOH4_l proteins and each similar
  • CD311 2 A polynucleotide of the present invention has been identified as clone "CD311_2".
  • CD311_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.
  • CD311_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CD311_2 protein").
  • CD311_2 The nucleotide sequence of CD311_2 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 CD311_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. 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 CD311_2 should be approximately 2400 bp.
  • the nucleotide sequence disclosed herein for CD311_2 was searched against the
  • CD311_2 demonstrated at least some similarity with sequences identified as H17421 (ym40dl2.sl Homo sapiens cDNA clone 50810 3'), H20618 (ym47b01.rl Homo sapiens cDNA clone 51411 5'), and U70476 (Rattus norvegicus cationic amino acid transporter-1 (CAT-1) mRNA, complete cds). Based upon sequence similarity,
  • CD311_2 proteins and each similar protein or peptide may share at least some activity. Clone "CG279 1"
  • CG279_1 A polynucleotide of the present invention has been identified as clone "CG279_1".
  • CG279_1 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.
  • CG279_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CG279_1 protein").
  • nucleotide sequence of CG279_1 as presently determined is reported in SEQ ID NOS. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the CG279_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • Amino acids 9 to 21 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 22, or are a transmembrane domain.
  • Amino acids 43 to 55 are a possible leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 56, or are a transmembrane domain
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone CG279_1 should be approximately 3940 bp.
  • nucleotide sequence disclosed herein for CG279_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
  • CG279_1 demonstrated at least some similarity with sequences identified as AA568111 (nfl3c05.sl NCI_CGAP_Prl Homo sapiens cDNA clone IMAGE:913640), D63222 (Human placenta cDNA 5'-end GEN-508F12), H64777 (yu62h09.rl Homo sapiens cDNA clone 238433 5' similar to contains Alu repetitive element;contains TAR1 repetitive element), Ml 7262 (Human prothrombin (F2) gene, complete cds, and Alu and Kpnl repeats), Q39724 (Expressed Sequence Tag human gene marker EST00316), U14685 (Gorilla gorilla Alu-Sb2 repeat, clone GO-14), U14691 (Gorilla gorilla Alu-Sb2 repeat, clone GOI2-11), and W15458 (zcl9h02.sl Soares parat
  • the predicted amino acid sequence disclosed herein for CG279_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted CG279_1 protein demonstrated at least some similarity to sequences identified as D25215 (KIAA0032 [Homo sapiens]), M15530 (B-cell growth factor [Homo sapiens]), and R95913 (Neural thread protein). Based upon sequence similarity, CG279_1 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts three potential transmembrane domains within the CG279_1 protein sequence, centered around amino acids 30, 250, and 390 of SEQ ID NO:6, respectively.
  • the nucleotide sequence of CG279_1 indicates that it may contain one or more Alu repetitive elements.
  • CJ424_9 A polynucleotide of the present invention has been identified as clone "CJ424_9".
  • CJ424_9 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.
  • CJ424_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CJ424_9 protein").
  • the nucleotide sequence of CJ424_9 as presently determined is reported in SEQ ID NO: A polynucleotide sequence of CJ424_9 as presently determined is reported in SEQ ID NOSEQ ID NOSEQ ID NOSEQ ID NOSEQ ID NOSEQ ID NOSEQ ID NO
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone CJ424_9 should be approximately 1650 bp .
  • CJ424_9 The nucleotide sequence disclosed herein for CJ424_9 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CJ424_9 demonstrated at least some similarity with sequences identified as AB000215 (Rattus norvegicus ccal mRNA, complete cds) and R83763 (ypl6f06.sl Homo sapiens cDNA clone 187619 3'). The predicted amino acid sequence disclosed herein for CJ424_9 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted CJ424_9 protein demonstrated at least some similarity to sequences identified as AB000215 (CCA1 protein [Rattus norvegicus]) and M59465 (A20 [Homo sapiens]). Based upon sequence similarity, CJ424_9 proteins and each similar protein or peptide may share at least some activity.
  • CR930_1 A polynucleotide of the present invention has been identified as clone "CR930_1".
  • CR930_1 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.
  • CR930_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CR930_1 protein").
  • CR930_1 should be approximately 2400 bp.
  • CR930_1 The nucleotide sequence disclosed herein for CR930_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CR930_1 demonstrated at least some similarity with sequences identified as AA058338 (zk82e08.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 489350 3'), N54229 yz03f05.rl Homo sapiens cDNA clone), R89733 (ym99el0.rl Homo sapiens cDNA clone 167082 5'), and W60141 (zc94f06.sl Pancreatic Islet Homo sapiens cDNA clone 338819 3').
  • the predicted amino acid sequence disclosed herein for CR930_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted CR930_1 protein demonstrated at least some similarity to sequences identified as a C. elegans ORF (open reading frame) (U21324) that is weakly similar to S. cerevisiae CBP3 protein precursor (SP:CBP3_YEAST, P21560), a mitochondrial membrane protein. Based upon sequence similarity, CR930_1 proteins and each similar protein or peptide may share at least some activity.
  • a polynucleotide of the present invention has been identified as clone "DA306_4".
  • DA306_4 was isolated from a human adult placenta 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.
  • DA306_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "DA306_4 protein").
  • the nucleotide sequence of DA306_4 as presently determined is reported in SEQ ID NO:ll.
  • amino acids 27 to 39 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 40; or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone DA306_4 should be approximately 2800 bp.
  • DA306_4 demonstrated at least some similarity with sequences identified as AA397398 (nc65a07.rl NCI CGAP Prl Homo sapiens cDNA clone 771444), AL008629 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 197017; HTGS phase 1), D78769 (Human placenta cDNA 5'-end GEN-512A03), M19364 (Human gamma-B-crystallin (gamma 1-2) and gamma-C-crystallin (gamma 2-1) genes, complete cds), N29380 (yw97f09.sl Homo sapiens cDNA clone 260201 3'), N47928 (yw97f09.
  • the predicted amino acid sequence disclosed herein for DA306_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted DA306_4 protein demonstrated at least some similarity to sequences identified as M12140 (envelope protein [Homo sapiens]), M19051 (pol protein [Mus musculus]), R75189 (Osteoinductive retrovirus RFB-14 pol gene product), U88902 (integrase [Homo sapiens]).
  • M12140 envelope protein [Homo sapiens]
  • M19051 poly protein [Mus musculus]
  • R75189 Oleductive retrovirus RFB-14 pol gene product
  • U88902 integratedase [Homo sapiens]
  • DA306_4 proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of DA306_4 indicates that it may contain a CpG island repeat region.
  • DG76_1 was isolated from a human adult placenta 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.
  • DG76_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "DG76_1 protein").
  • nucleotide sequence of DG76_1 as presently determined is reported in SEQ ID NO:13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the DG76_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone DG76_1 should be approximately 3300 bp.
  • DG76_1 The nucleotide sequence disclosed herein for DG76_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. DG76_1 demonstrated at least some similarity with sequences identified as AA044352 (zk54c01.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 486624 5') and N57171 (yw90f09.rl Homo sapiens cDNA clone 259529 5'). Based upon sequence similarity, DG76_1 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts two potential transmembrane domains within the DG76_1 protein sequence, centered amino acids 15 and 80 of SEQ ID NO:14, respectively.
  • the nucleotide sequence of DG76_1 indicates that it may contain a MER repeat region.
  • D019_l A polynucleotide of the present invention has been identified as clone "D019_l”.
  • D019_l 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.
  • D019_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "D019_l protein").
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone D019_l should be approximately 700 bp.
  • the nucleotide sequence disclosed herein for D019_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols.
  • D019_l demonstrated at least some similarity with sequences identified as AA339440 (EST44546 Fetal brain I Homo sapiens cDNA 5' end). Based upon sequence similarity, D019_l proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of D019_l indicates that it may contain one or more MER20 repetitive elements.
  • EQ219_1 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.
  • EQ219_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "EQ219_1 protein").
  • nucleotide sequence of EQ219_1 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 EQ219_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18.
  • Amino acids 8 to 20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 21, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone EQ219_1 should be approximately 800 bp.
  • the nucleotide sequence disclosed herein for EQ219_1 was searched against the
  • EQ219_1 demonstrated at least some similarity with sequences identified as AA400429 (zu62a09.sl Soares testis NHT Homo sapiens cDNA clone 742552 3'). Based upon sequence similarity, EQ219_1 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts an additional potential transmembrane domain within the EQ219_1 protein sequence, centered around amino acid 90 of SEQ ID NO:18. Clone "FG340 1"
  • a polynucleotide of the present invention has been identified as clone "FG340_1".
  • FG340_1 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.
  • FG340_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "FG340_1 protein").
  • nucleotide sequence of FG340_1 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 FG340_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 5 to 17 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
  • FG340_1 should be approximately 900 bp.
  • FG340_1 The nucleotide sequence disclosed herein for FG340_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. FG340_1 demonstrated at least some similarity with sequences identified as N59424 (yv51g05.sl Homo sapiens cDNA clone 246296 3'). The FG340_1 nucleotide sequence has an interesting simple "TG" nucleotide repeat from basepair 96 to basepair 131 of SEQ ID NO:19. This region encodes a Cys-Val repeat in the FG340_1 protein.
  • Clones BOH4_l, CD311_2, CG279_1, CJ424_9, CR930_1, DA306_4, DG76_1, DO19_l, EQ219_1, and FG340_1 were deposited on February 20, 1997 with the American
  • Each clone has been transfected into separate bacterial cells ⁇ E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' 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., 1991, Nucleic Acids Res.
  • 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 NotI. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for 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 oligonucleotide should preferably be labeled with g- 32 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 10 mL per 150 mm filter).
  • 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.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • 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 polynucleotide sequences disclosed herein.
  • Corresponding genes are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are 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.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
  • the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1- 39; all of which are incorporated by reference herein).
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s).
  • Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos.
  • 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.
  • a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art.
  • 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.
  • species homologs are those isolated from mammalian 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 hybrid length is that anticipated for the hybridized reg ⁇ on(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 aligning the sequences of the polynucleohdes and identifying the region or regions of optimal sequence complementarity f SSPE (lxSSPE is 0 15M NaCl, lOmM NaH 2 P0 4 , and 1 25mM EDTA, pH 7 4) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete *T B - T R
  • the hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the 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 ⁇ ombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • bacterial strains include Escherichia colt, 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 cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • 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 (SCID)), 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.
  • SCID 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, myasthenia 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 receptor: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 MR /lprApr 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
  • Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.
  • 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 a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • 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 a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • 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 Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al, J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
  • 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 craniof acial 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 measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
  • 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 immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention (including, without 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 imCurrent 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 cationic 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. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- and TGF- ⁇ ), and insulin-like growth factor (IGF).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • 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.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF 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.
  • GGCCATGGGT GCCCAGGCTT CCCAGGACCT CGGAGATGAG TTGCTGGATG ACGGGGAGGA 120
  • Pro Gin lie Pro Tyr Pro Glu Pro Thr Trp Pro Pro Pro Leu Ser Ala 65 70 75 80
  • Val lie Ala Ala Asn Tyr Pro Asp Leu Pro Ser Ala Tyr Gin Pro Gly 130 135 140 lie Leu Ser Val Ser His Ser Ala Gin Pro Pro Ala His Gin Pro Pro 145 150 155 160
  • Thr Gin Leu Pro lie lie Pro Thr Ala Gin Pro Ser Leu Thr Thr Thr 225 230 235 240
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 3 :
  • CTGCCTCAGG ACCTCTGCTG AAGAGGGGGA TGAAGTGGGT CTCCAGGGAG GCAGTGGGGG 240
  • CAGGATCCAA TACGAGTCCA GGGGTGACCG CAGGATGGTG GGGGCAGCGG GCTTCTCCAC 660
  • CTACCCCAGC CACCAAGGCC
  • CTGACGCACT GCCTCCTGCA CCTTCAGCAC ATCCCTGTGC 720
  • GGTCATTCTA CAAATGTGCT GAGTGCCAGC TGAAAACCCC ACAGGAGATG GAGTACCTTG 1740
  • AAAAAAAAAA AAA 2473 INFORMATION FOR SEQ ID NO : 4 :
  • Val lie Asp Ala Phe Ser Pro Gly Cys Leu Arg Lys Val Ala Lys Cys 1 5 10 15 lie Leu Leu Ser Glu Leu Leu Arg Ser Pro Pro Thr Phe Gly Phe Cys 20 25 30
  • CTGTCATGTC CCTGTATCCT CATGCTCATC ATCTCAGCAG GCCTGAGAGG CTGGGTCAGT 3480
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 :
  • Tyr Lys lie Arg Thr lie Ala Pro Gly Lys Thr His Thr Ala Ala lie 100 105 110
  • Glu Gly Leu lie Ser Pro Thr Glu Ala Met Gly Asn Ser Asn Gly Ala 290 295 300
  • Gin Gin Gin Glu Asn Leu Gin lie Phe Thr Gin Leu Gin Lys Leu Asn Lys 420 425 430
  • ACTCTCTGTA TCTCCTCTTA AGCTTCGTCC AGGGTTCTTT ATTTTTGTCT GCTGCCAATG 1320
  • GCCACAGACC CCTTGCTCGG GCCTCACCCA AGGCCAAACT CCAAACACAA TCAGAACCAG 1440 CCAAAGAAGC ACTTCCTGGG CACGGCCACC AGCTCTCCCG CCTCCAGTGT GGGCCGGCTC 1500
  • Lys Pro Trp Ala lie Gly Thr Leu Leu Asp Thr Asn Gly Leu Tyr Asp 130 135 140
  • TACATTCAAT TCATGGTTTC TTATAACCCT ACTCCACGTC TGGATGTGTC TAGTCCGAAT 480
  • CAGTGCGGGT CATCTCTTGG GGGATTTTCT TAAAGTGAAG AAAGGGGGTG GGGAACCATA 1920
  • GGCACAATTC AACCTGGATT CTTTTTACTT CCGAAGCACG CGGGCAACAC CCTCAGAGTC 1620
  • AACTTGCACC AGCAGCAAGA TAGCAGAGGC AGGAAGAGAG CTGGCTGGAA GACACGTACC 2520
  • MOLECULE TYPE protein
  • AAACCATCTC ATTAAATTTT GTTGCATTTT AGATAATCGT CCCCAGATGC CATGTTACCC 300
  • ARCCACTGGA RGGGTTTAAG CCGATAATTG ATGTCACATA ATTTATGTTG TAATGGAACC 480

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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 Ser. No. 60/XXX,XXX (converted to a provisional application from non-provisional application 08/804,561), filed February 24, 1997, which is incorporated by reference herein.
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 65 to nucleotide 1270;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1139 to nucleotide 1270; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:l from nucleotide 1011 to nucleotide 1216;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BOH4_l deposited under accession number
ATCC 98333; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 196 to amino acid 205 of SEQ ID NO:2;
(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 65 to nucleotide 1270; the nucleotide sequence of SEQ ID NO:l from nucleotide 1139 to nucleotide 1270; the nucleotide sequence of SEQ ID NO:l from nucleotide 1011 to nucleotide 1216; the nucleotide sequence of the full-length protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333. 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 326 to amino acid 384.
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 326 to amino acid 384;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 196 to amino acid 205 of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333; 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 326 to amino acid 384.
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 418 to nucleotide 582; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 508 to nucleotide 582;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 555; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CD311_2 deposited under accession number
ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO.4;
(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:3 from nucleotide 418 to nucleotide 582; the nucleotide sequence of SEQ ID NO:3 from nucleotide 508 to nucleotide 582; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 555; the nucleotide sequence of the full-length protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333. 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 46.
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 46;
(c) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333; 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 46.
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 191 to nucleotide 1756; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 254 to nucleotide 1756;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 604;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 256 to amino acid 265 of
SEQ ID NO:6;
(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 191 to nucleotide 1756; the nucleotide sequence of SEQ ID NO:5 from nucleotide 254 to nucleotide 1756; the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 604; the nucleotide sequence of the full-length protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333. 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 138.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NOS.
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 138;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising the amino acid sequence from amino acid 256 to amino acid 265 of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333; 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 138.
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 226 to nucleotide 948;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1128 to nucleotide 1601;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CJ424_9 deposited under accession number
ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 115 to amino acid 124 of SEQ ID NO:8; (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 226 to nucleotide 948; the nucleotide sequence of SEQ ID NO:7 from nucleotide 1128 to nucleotide 1601; the nucleotide sequence of the full-length protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333. 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) fragments of the amino acid sequence of SEQ ID NO:8 comprising the amino acid sequence from amino acid 115 to amino acid 124 of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8.
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 137 to nucleotide 895; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 1488 to nucleotide 2274;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 121 to amino acid 130 of SEQ ID NO:10;
(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:9 from nucleotide 137 to nucleotide 895; the nucleotide sequence of SEQ ID NO:9 from nucleotide 1488 to nucleotide 2274; the nucleotide sequence of the full-length protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333.
Other embodiments provide the gene corresponding to the cDNA 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) fragments of the amino acid sequence of SEQ ID NO:10 comprising the amino acid sequence from amino acid 121 to amino acid 130 of SEQ ID NO:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10.
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:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 494 to nucleotide 973;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 611 to nucleotide 973; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:ll from nucleotide 521 to nucleotide 940;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO:12; (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:ll from nucleotide 494 to nucleotide 973; the nucleotide sequence of SEQ ID NO:ll from nucleotide 611 to nucleotide 973; the nucleotide sequence of SEQ ID NO: 11 from nucleotide 521 to nucleotide 940; the nucleotide sequence of the full-length protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:ll. 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: 12;
(b) the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149;
(c) fragments of the amino acid sequence of SEQ ID NO:12 comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO:12; and (d) the amino acid sequence encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149.
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:13; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 13 from nucleotide 2295 to nucleotide 2594;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1867 to nucleotide 2372;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone DG76_1 deposited under accession number
ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising the amino acid sequence from amino acid 45 to amino acid 54 of SEQ ID NO:14; (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:13 from nucleotide 2295 to nucleotide 2594; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1867 to nucleotide 2372; the nucleotide sequence of the full-length protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 26.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:13.
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: 14; (b) the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 26;
(c) fragments of the amino acid sequence of SEQ ID NO:14 comprising the amino acid sequence from amino acid 45 to amino acid 54 of SEQ ID NO: 14; and (d) the amino acid sequence encoded by the cDNA insert of clone
DG76_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:14 or the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 26. 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 394 to nucleotide 522;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 476; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone D019_l deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone D019_l deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333; (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, the fragment comprising the amino acid sequence from amino acid 16 to amino acid 25 of SEQ ID NO:16;
(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 394 to nucleotide 522; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 476; the nucleotide sequence of the full-length protein coding sequence of clone D019_l deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone D019_l deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333. 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 27.
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 27;
(c) fragments of the amino acid sequence of SEQ ID NO: 16 comprising the amino acid sequence from amino acid 16 to amino acid 25 of SEQ ID NO:16; and (d) the amino acid sequence encoded by the cDNA insert of clone
D019_l deposited under accession number ATCC 98333; 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 27. 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 262 to nucleotide 654;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 322 to nucleotide 654;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 1 to nucleotide 618; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 60 to amino acid 69 of SEQ
ID NO:18;
(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 262 to nucleotide 654; the nucleotide sequence of SEQ ID NO:17 from nucleotide 322 to nucleotide 654; the nucleotide sequence of SEQ ID NO: 17 from nucleotide 1 to nucleotide 618; the nucleotide sequence of the full-length protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333. 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 119. 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 119;
(c) fragments of the amino acid sequence of SEQ ID NO:18 comprising the amino acid sequence from amino acid 60 to amino acid 69 of SEQ ID NO: 18; and
(d) the amino acid sequence encoded by the cDNA insert of clone EQ219J deposited under accession number ATCC 98333; 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 119.
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 74 to nucleotide 310;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 125 to nucleotide 310; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 1 to nucleotide 338;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone FG340_1 deposited under accession number
ATCC 98333; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID NO:20; (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:19 from nucleotide 74 to nucleotide 310; the nucleotide sequence of SEQ ID NO:19 from nucleotide 125 to nucleotide 310; the nucleotide sequence of SEQ ID NO:19 from nucleotide 1 to nucleotide 338; the nucleotide sequence of the full-length protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333; or the nucleotide sequence of the mature protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333. 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 75.
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 75;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID NO:20; and (d) the amino acid sequence encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333; 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 75.
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. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
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 Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, 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 "BOH4 1"
A polynucleotide of the present invention has been identified as clone "BOH4_l". BOH4J. was isolated from a human adult retina 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. BOH4_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BO114_l protein").
The nucleotide sequence of BOH4_l 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 BOH4_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Amino acids 346 to 358 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 359, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
BOH4_l should be approximately 1600 bp.
The nucleotide sequence disclosed herein for BOH4_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. BOH4_l demonstrated at least some similarity with sequences identified as AA430329 (zw20e04.rl Soares ovary tumor NbHOT Homo sapiens cDNA clone 7698545' similar to contains element MER22 repetitive element), H00825 (yj31b05.rl Homo sapiens cDNA clone 150321 5'), H12557 (yjl2c09.rl Homo sapiens cDNA clone 148528 5'), W53899 (md09c01.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA), and Z82202 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 34P24). Based upon sequence similarity, BOH4_l proteins and each similar protein or peptide may share at least some activity.
Clone "CD311 2" A polynucleotide of the present invention has been identified as clone "CD311_2".
CD311_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. CD311_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CD311_2 protein").
The nucleotide sequence of CD311_2 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 CD311_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. 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 CD311_2 should be approximately 2400 bp. The nucleotide sequence disclosed herein for CD311_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CD311_2 demonstrated at least some similarity with sequences identified as H17421 (ym40dl2.sl Homo sapiens cDNA clone 50810 3'), H20618 (ym47b01.rl Homo sapiens cDNA clone 51411 5'), and U70476 (Rattus norvegicus cationic amino acid transporter-1 (CAT-1) mRNA, complete cds). Based upon sequence similarity,
CD311_2 proteins and each similar protein or peptide may share at least some activity. Clone "CG279 1"
A polynucleotide of the present invention has been identified as clone "CG279_1". CG279_1 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. CG279_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CG279_1 protein").
The nucleotide sequence of CG279_1 as presently determined is reported in SEQ ID NOS. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the CG279_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino acids 9 to 21 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 22, or are a transmembrane domain. Amino acids 43 to 55 are a possible leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 56, or are a transmembrane domain
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone CG279_1 should be approximately 3940 bp.
The nucleotide sequence disclosed herein for CG279_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CG279_1 demonstrated at least some similarity with sequences identified as AA568111 (nfl3c05.sl NCI_CGAP_Prl Homo sapiens cDNA clone IMAGE:913640), D63222 (Human placenta cDNA 5'-end GEN-508F12), H64777 (yu62h09.rl Homo sapiens cDNA clone 238433 5' similar to contains Alu repetitive element;contains TAR1 repetitive element), Ml 7262 (Human prothrombin (F2) gene, complete cds, and Alu and Kpnl repeats), Q39724 (Expressed Sequence Tag human gene marker EST00316), U14685 (Gorilla gorilla Alu-Sb2 repeat, clone GO-14), U14691 (Gorilla gorilla Alu-Sb2 repeat, clone GOI2-11), and W15458 (zcl9h02.sl Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 322803 3'). The predicted amino acid sequence disclosed herein for CG279_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted CG279_1 protein demonstrated at least some similarity to sequences identified as D25215 (KIAA0032 [Homo sapiens]), M15530 (B-cell growth factor [Homo sapiens]), and R95913 (Neural thread protein). Based upon sequence similarity, CG279_1 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts three potential transmembrane domains within the CG279_1 protein sequence, centered around amino acids 30, 250, and 390 of SEQ ID NO:6, respectively. The nucleotide sequence of CG279_1 indicates that it may contain one or more Alu repetitive elements.
Clone "CT424 9"
A polynucleotide of the present invention has been identified as clone "CJ424_9". CJ424_9 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. CJ424_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CJ424_9 protein"). The nucleotide sequence of CJ424_9 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 CJ424_9 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 CJ424_9 should be approximately 1650 bp .
The nucleotide sequence disclosed herein for CJ424_9 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CJ424_9 demonstrated at least some similarity with sequences identified as AB000215 (Rattus norvegicus ccal mRNA, complete cds) and R83763 (ypl6f06.sl Homo sapiens cDNA clone 187619 3'). The predicted amino acid sequence disclosed herein for CJ424_9 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted CJ424_9 protein demonstrated at least some similarity to sequences identified as AB000215 (CCA1 protein [Rattus norvegicus]) and M59465 (A20 [Homo sapiens]). Based upon sequence similarity, CJ424_9 proteins and each similar protein or peptide may share at least some activity.
Clone "CR930 1"
A polynucleotide of the present invention has been identified as clone "CR930_1". CR930_1 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. CR930_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "CR930_1 protein").
The nucleotide sequence of CR930_1 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 CR930_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
CR930_1 should be approximately 2400 bp.
The nucleotide sequence disclosed herein for CR930_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CR930_1 demonstrated at least some similarity with sequences identified as AA058338 (zk82e08.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 489350 3'), N54229 yz03f05.rl Homo sapiens cDNA clone), R89733 (ym99el0.rl Homo sapiens cDNA clone 167082 5'), and W60141 (zc94f06.sl Pancreatic Islet Homo sapiens cDNA clone 338819 3'). The predicted amino acid sequence disclosed herein for CR930_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted CR930_1 protein demonstrated at least some similarity to sequences identified as a C. elegans ORF (open reading frame) (U21324) that is weakly similar to S. cerevisiae CBP3 protein precursor (SP:CBP3_YEAST, P21560), a mitochondrial membrane protein. Based upon sequence similarity, CR930_1 proteins and each similar protein or peptide may share at least some activity.
Clone "DA306 4"
A polynucleotide of the present invention has been identified as clone "DA306_4". DA306_4 was isolated from a human adult placenta 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. DA306_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "DA306_4 protein"). The nucleotide sequence of DA306_4 as presently determined is reported in SEQ ID NO:ll. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the DA306_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12. Amino acids 27 to 39 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 40; or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone DA306_4 should be approximately 2800 bp.
The nucleotide sequence disclosed herein for DA306_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. DA306_4 demonstrated at least some similarity with sequences identified as AA397398 (nc65a07.rl NCI CGAP Prl Homo sapiens cDNA clone 771444), AL008629 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 197017; HTGS phase 1), D78769 (Human placenta cDNA 5'-end GEN-512A03), M19364 (Human gamma-B-crystallin (gamma 1-2) and gamma-C-crystallin (gamma 2-1) genes, complete cds), N29380 (yw97f09.sl Homo sapiens cDNA clone 260201 3'), N47928 (yw97f09.rl Homo sapiens cDNA clone 260201 5'), Z83745 (Human DNA sequence from PAC 453A3 contains EST and STS), and Z83848 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 57A13; HTGS phase 1). The predicted amino acid sequence disclosed herein for DA306_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted DA306_4 protein demonstrated at least some similarity to sequences identified as M12140 (envelope protein [Homo sapiens]), M19051 (pol protein [Mus musculus]), R75189 (Osteoinductive retrovirus RFB-14 pol gene product), U88902 (integrase [Homo sapiens]). Based upon sequence similarity, DA306_4 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of DA306_4 indicates that it may contain a CpG island repeat region.
Clone "DG76 1" A polynucleotide of the present invention has been identified as clone "DG76_1".
DG76_1 was isolated from a human adult placenta 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. DG76_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "DG76_1 protein").
The nucleotide sequence of DG76_1 as presently determined is reported in SEQ ID NO:13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the DG76_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone DG76_1 should be approximately 3300 bp.
The nucleotide sequence disclosed herein for DG76_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. DG76_1 demonstrated at least some similarity with sequences identified as AA044352 (zk54c01.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 486624 5') and N57171 (yw90f09.rl Homo sapiens cDNA clone 259529 5'). Based upon sequence similarity, DG76_1 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts two potential transmembrane domains within the DG76_1 protein sequence, centered amino acids 15 and 80 of SEQ ID NO:14, respectively. The nucleotide sequence of DG76_1 indicates that it may contain a MER repeat region.
Clone "DQ19 1"
A polynucleotide of the present invention has been identified as clone "D019_l". D019_l 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. D019_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "D019_l protein").
The nucleotide sequence of D019_l 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 D019_l 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 D019_l should be approximately 700 bp. The nucleotide sequence disclosed herein for D019_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. D019_l demonstrated at least some similarity with sequences identified as AA339440 (EST44546 Fetal brain I Homo sapiens cDNA 5' end). Based upon sequence similarity, D019_l proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of D019_l indicates that it may contain one or more MER20 repetitive elements.
Clone "EO219 1" A polynucleotide of the present invention has been identified as clone "EQ219_1".
EQ219_1 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. EQ219_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "EQ219_1 protein").
The nucleotide sequence of EQ219_1 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 EQ219_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18. Amino acids 8 to 20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 21, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone EQ219_1 should be approximately 800 bp. The nucleotide sequence disclosed herein for EQ219_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. EQ219_1 demonstrated at least some similarity with sequences identified as AA400429 (zu62a09.sl Soares testis NHT Homo sapiens cDNA clone 742552 3'). Based upon sequence similarity, EQ219_1 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts an additional potential transmembrane domain within the EQ219_1 protein sequence, centered around amino acid 90 of SEQ ID NO:18. Clone "FG340 1"
A polynucleotide of the present invention has been identified as clone "FG340_1". FG340_1 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. FG340_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "FG340_1 protein").
The nucleotide sequence of FG340_1 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 FG340_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 5 to 17 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
FG340_1 should be approximately 900 bp.
The nucleotide sequence disclosed herein for FG340_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. FG340_1 demonstrated at least some similarity with sequences identified as N59424 (yv51g05.sl Homo sapiens cDNA clone 246296 3'). The FG340_1 nucleotide sequence has an interesting simple "TG" nucleotide repeat from basepair 96 to basepair 131 of SEQ ID NO:19. This region encodes a Cys-Val repeat in the FG340_1 protein. Similar Cys-Val stretches are found in the amino termini of X52164 (Q300 protein (AA 1-77) [Mus musculus]) and M37679 (Ig heavy chain precursor [Mus musculus]). Based upon sequence similarity, FG340_1 proteins and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones BOH4_l, CD311_2, CG279_1, CJ424_9, CR930_1, DA306_4, DG76_1, DO19_l, EQ219_1, and FG340_1 were deposited on February 20, 1997 with the American
Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98333, 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. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' 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 NotI. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for 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
B0114_l SEQ ID NO:21
CD311_2 SEQ ID NO:22 CG279_1 SEQ ID NO:23
CJ424_9 SEQ ID NO:24
CR930_1 SEQ ID NO:25
DA306_4 SEQ ID NO:26
DG76_1 SEQ ID NO:27 D019_l SEQ ID NO:28
EQ219_1 SEQ ID NO:29
FG340_1 SEQ ID NO:30
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-3-0-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)). The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest 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-32P 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 polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are 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. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1- 39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s).
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. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art. 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. Preferably, species homologs are those isolated from mammalian 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.
Figure imgf000037_0001
* The hybrid 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 aligning the sequences of the polynucleohdes and identifying the region or regions of optimal sequence complementarity f SSPE (lxSSPE is 0 15M NaCl, lOmM NaH2P04, and 1 25mM EDTA, pH 7 4) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete *TB - TR The hybridization temperature for hybrids anticipated 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+]) + 041(%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in 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 ψombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia colt, 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 cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, 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 (SCID)), 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, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, 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 receptor: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 MR /lprApr 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 chain protein and β2 microglobulin protein or an MHC class II a 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 Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al, J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 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 Culture of Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of 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 craniof acial 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 measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 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 immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without 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 imCurrent 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 cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of 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- 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
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Merberg, David Treacy, Maurice Spaulding, Vikki Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(iii) NUMBER OF SEQUENCES: 30
(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:
(viii) 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: 1551 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
GTGTTATTGT AGCGAGGGAC ATGAGCTGGA GGCTGATGGC ATCAGCTGCA GCCCTGCAGG 60
GGCCATGGGT GCCCAGGCTT CCCAGGACCT CGGAGATGAG TTGCTGGATG ACGGGGAGGA 120
TGAGGAAGAT GAAGACGAGG CCTGGAAGGC CTTCAACGGT GGCTGGACGG AGATGCCTGG 180
GATCCTGTGG ATGGAGCCTA CGCAGCCGCC TGACTTTGCC CTGGCCTATA GACCGAGCTT 240
CCCAGAGGAC AGAGAGCCAC AGATACCCTA CCCGGAGCCC ACCTGGCCAC CCCCGCTCAG 300
TGCCCCCAGG GTCCCCTACC ACTCCTCAGT GCTCTCCGTC ACCCGGCCTG TGGTGGTCTC 360
TGCCACGCGT CCCACACTGC CTTCTGCCCA CCAGCCTCCT GTGATCCCTG CCACACACCC 420
AGCTTTGTCC CGTGACCACC AGATCCCCGT GATCGCAGCC AACTATCCAG ATCTGCCTTC 480
TGCCTACCAA CCCGGTATTC TCTCTGTCTC TCATTCAGCA CAGCCTCCTG CCCACCAGCC 540
CCCTATGATC TCAACCAAAT ATCCGGAGCT CTTCCCTGCC CACCAGTCCC CCATGTTTCC 600
AGACACCCGG GTCGCTGGCA CCCAGACCAC CACTCATTTG CCTGGAATCC CACCTAACCA 660
TGCCCCTCTG GTCACCACCC TCGGTGCCCA GCTACCCCCT CAAGCCCCAG ATGCCCTTGT 720
CCTCAGAACC CAGGCCACCC AGCTTCCCAT TATCCCAACT GCCCAGCCCT CTCTGACCAC 780
CACCTCCAGG TCCCCTGTGT CTCCTGCCCA TCAAATCTCT GTGCCTGCTG CCACCCAGCC 840
CGCAGCCCTC CCCACCCTCC TGCCCTCTCA GAGCCCCACT AACCAGACCT CACCCATCAG 900
CCCTACACAT CCCCATTCCA AAGCCCCCCA AATCCCAAGG GAAGATGGCC CCAGTCCCAA 960
GTTGGCCCTG TGGCTGCCCT CACCAGCTCC CACAGCAGCC CCAACAGCCC TGGGGGAGGC 1020
TGGTCTTGCC GAGCACAGCC AGAGGGATGA CCGGTGGCTG CTGGTGGCAC TCCTGGTGCC 1080
AACGTGTGTC TTTTTGGTGG TCCTGCTTGC ACTGGGCATC GTGTACTGCA CCCGCTGTGG 1140
CCCCCATGCA CCCAACAAGC GCATCACTGA CTGCTATCGC TGGGTCATCC ATGCTGGGAG 1200
CAAGAGCCCA ACAGAACCCA TGCCCCCCAG GGGCAGCCTC AC GGGGTGC AGACCTGCAG 1260
AACCAGCGTG TGATGGGGTG CAGACCCCCC TCATGGAGTA TGGGGCGCTG GACACATGGC 1320
CGGGGCTGCA CCAGGGACCC ATGGGGGCTG CCCAGCTGGA CAGATGGCTT CCTGCTCCCC 1380
AGGCCCAGCC AGGGTCCTCT CTCAACCACT AGACTTGGCT CTCAGGAACT CTGCTTCCTG 1440 GCCCAGCGCT CGTGACCAAG GATACACCAA AGCCCTTAAG ACCTCAGGGG GCGGGTGCTG 1500 GGGTCTTCTC CAATAAATGG GGTGTCAACC TTAAAAAAAA AAAAAAAAAA A 1551
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Gly Ala Gin Ala Ser Gin Asp Leu Gly Asp Glu Leu Leu Asp Asp 1 5 10 15
Gly Glu Asp Glu Glu Asp Glu Asp Glu Ala Trp Lys Ala Phe Asn Gly 20 25 30
Gly Trp Thr Glu Met Pro Gly lie Leu Trp Met Glu Pro Thr Gin Pro 35 40 45
Pro Asp Phe Ala Leu Ala Tyr Arg Pro Ser Phe Pro Glu Asp Arg Glu 50 55 60
Pro Gin lie Pro Tyr Pro Glu Pro Thr Trp Pro Pro Pro Leu Ser Ala 65 70 75 80
Pro Arg Val Pro Tyr His Ser Ser Val Leu Ser Val Thr Arg Pro Val 85 90 95
Val Val Ser Ala Thr Arg Pro Thr Leu Pro Ser Ala His Gin Pro Pro 100 105 110
Val lie Pro Ala Thr His Pro Ala Leu Ser Arg Asp His Gin lie Pro 115 120 125
Val lie Ala Ala Asn Tyr Pro Asp Leu Pro Ser Ala Tyr Gin Pro Gly 130 135 140 lie Leu Ser Val Ser His Ser Ala Gin Pro Pro Ala His Gin Pro Pro 145 150 155 160
Met lie Ser Thr Lys Tyr Pro Glu Leu Phe Pro Ala His Gin Ser Pro 165 170 175
Met Phe Pro Asp Thr Arg Val Ala Gly Thr Gin Thr Thr Thr His Leu 180 185 190 Pro Gly lie Pro Pro Asn His Ala Pro Leu Val Thr Thr Leu Gly Ala 195 200 205
Gin Leu Pro Pro Gin Ala Pro Asp Ala Leu Val Leu Arg Thr Gin Ala 210 215 220
Thr Gin Leu Pro lie lie Pro Thr Ala Gin Pro Ser Leu Thr Thr Thr 225 230 235 240
Ser Arg Ser Pro Val Ser Pro Ala His Gin lie Ser Val Pro Ala Ala 245 250 255
Thr Gin Pro Ala Ala Leu Pro Thr Leu Leu Pro Ser Gin Ser Pro Thr 260 265 270
Asn Gin Thr Ser Pro lie Ser Pro Thr His Pro His Ser Lys Ala Pro 275 280 285
Gin lie Pro Arg Glu Asp Gly Pro Ser Pro Lys Leu Ala Leu Trp Leu 290 295 300
Pro Ser Pro Ala Pro Thr Ala Ala Pro Thr Ala Leu Gly Glu Ala Gly 305 310 315 320
Leu Ala Glu His Ser Gin Arg Asp Asp Arg Trp Leu Leu Val Ala Leu 325 330 335
Leu Val Pro Thr Cys Val Phe Leu Val Val Leu Leu Ala Leu Gly lie 340 345 350
Val Tyr Cys Thr Arg Cys Gly Pro His Ala Pro Asn Lys Arg lie Thr 355 360 365
Asp Cys Tyr Arg Trp Val lie His Ala Gly Ser Lys Ser Pro Thr Glu 370 375 380
Pro Met Pro Pro Arg Gly Ser Leu Thr Gly Val Gin Thr Cys Arg Thr 385 390 395 400
Ser Val
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2473 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
GGAGTATTGT GTCTACTTTT ATCTGTGCAC CAGCCACAAA TACCCACATT GGAAAGACCC 60
ATTTGTGATG GGTAAACATC CCTTCCTGTC TCCCACAACC CCTGTGACTG CCCTGCATGT 120
GTTCATGACC TCCGAAGGCC CAAATTCATG AAGCAGCAAA CCCAGCAGAT CTCCACCCCC 180
CTGCCTCAGG ACCTCTGCTG AAGAGGGGGA TGAAGTGGGT CTCCAGGGAG GCAGTGGGGG 240
CCTTGTTGGC AGCTGGCTCG GGAGCCGGCT TACAGGAGGG CAGCTCTGCA GTTGGGAGGG 300
GCACCGTCCG GAGGAGACCA GGCCTCTACA CACCCCCCAC TCTACTTATC ATCCCTGCTC 360
ACACACCCTT GTCCAAGGCT TTATGCATCG GATTTATTTT TCCAAATCAA GAGGACAGTG 420
ATAGATGCAT TTTCCCCAGG CTGTCTCAGA AAGGTCGCTA AATGTATACT GTTGTCAGAA 480
TTGCTGAGAT CTCCCCCCAC TTTTGGTTTT TGCAGCAGTA AAAACTCTTT CCACTGTGAC 540
TTATTTTCTC TCTCAGGCAG CCAGCCACCT GGTCCCTTGT GCTGACTCTA GCACAGTGGC 600
CAGGATCCAA TACGAGTCCA GGGGTGACCG CAGGATGGTG GGGGCAGCGG GCTTCTCCAC 660
CTACCCCAGC CACCAAGGCC CTGACGCACT GCCTCCTGCA CCTTCAGCAC ATCCCTGTGC 720
ACAGCTGGAA GGGTGCATGG CCCGCTCACC TTTGTTCAGA TGGGTGGAAA CGCTGATGAT 780
ACCAGCTCCT CCCTGCCGTG CCCCTGCCAC GGAGCAGGCA TTGTGAACTG GCTGGTGTTT 840
GCAGTCCCAC GTGGCATGGC CTCCAGCCCA ACCCACAGTG GAGACTGGAG ACAGGGCAAT 900
GAGTCTGGTC GGGGGCACGT GGACATGCCC CATAGGGGCC CCACCCAGAC TTAACAGGCA 960
AGGTCCTGGG CATTGCGCGA CGCAGGACTC AATGCTAAAG CAAGCCTGCC TGGCTCTGTG 1020
CCAGGGCCCC TCTTCTGATT TACACATCCC ATTTTTACAC AGACCCTTCC TTCTTAATAA 1080
AGGCTGACAG TTCTGTTGGC AGCCAAGAAC CCACACCATG AAGACAGGGA GTGAGGGGCC 1140
TTTGTGCCCA ACTCCAGCAC AGCTGCGTTC TGGGGTGTGT GAGAGGCATG TTCGTGTCTG 1200
TGCGCTGGTG GTCTCGTGAG ACAGTTCCGA GGACGGGGAA ATTGCAGGGT GGTGGGGGCG 1260
TGAGGCTTAT ATGTGGAACT GATGCAGAGT TCGCCTGCAG ACGGATCTGG ATATACACTA 1320
TGTATAATTG TTACGTGTAA TTTAAAATAT ATCTGTTTGC CATCGTCATG AGAAGATTAT 1380
ATGTAAGGCT CTGAAGGGAG AGGGAGATGT ACATTCTGCC AGGCTCCTGG GGACCTTATC 1440
CGAGTCATGA AATTGATGAC TGTTGATCCA GTGGTGCAAG AAGCTACACT CCATGTGTCA 1500
TCACGCTTAT GACTCCTAAT GTATTTTTAA GGCAAAAAAT GTCAGCCGAC TCCATCTTCA 1560
CCCCTCGATT CCTCGAGTCC AGCCTTTCTG TGCCAGTGCT TCACTGAGCC ACAACGCTCT 1620 CGCCATCGGG ACCCGGCTGG GCCTGGAGTC TCGGGGCACA GTTGCCATGG AGCCCTCCTG 1680
GGTCATTCTA CAAATGTGCT GAGTGCCAGC TGAAAACCCC ACAGGAGATG GAGTACCTTG 1740
GCCAAGCTTA AAGAGAAGAT TTTCTCAGGG TATTTATTAG TGTGTCCAGC AGGGTCAGGA 1800
AGCAGGATGG AAAGATGCAT TCAGACTGTT AATTTATTAA CAAGGCAAAT GATTTTGTGT 1860
TTCTTGATGA CAGACTATTA AGTTTGGGAC TTATTTTCCC ATTTGAGAAG TTATAATATA 1920
TATTTAAGAT GATAAGTTTC CTGCTTAAGT TGTGCCTTTC AGCTTCAATG AGTTTAAGGA 1980
GCACTAAGGG TAATGATACC AATGAGGGTT GGTTTATTAT CAAACCTGAA TAGCTGTGGT 2040
TTCTCCAGTA AATATTTTCT TCTACTGAAC ATGGAGCCAT TATTAAGAGT TGTGTGTTTT 2100
TTATTATGTA CATTTGTATA TTTTTTTGCT TGTTTGATGT TCTATTTTTC TAATAGTTTT 2160
CTTTTAGTTT CTTAAAGTTG TGATACTAGA TTTAGATTCT GATGCTAACT GCAAATCAGG 2220
TTGGTCTCTG CTGGGTCTCT CCTGCTTTTA TTTTACTTTA AGGACAAGTG TAGTTGTCGT 2280
CCACCACCTT TCAAAAAATG TGAAACTGCC CTGCCTCCCC TTTTTGCTGA CAACACTGTG 2340
TACATTGACC ACTTCCTACC ATACTTTATG TTGTAAAATC AAACTCTTTT GTGGTACATT 2400
ATCTCATGCT TCTGCAAATT CGAATAAATT CTATGGCTTC CAAAAAAAAA AAAAAAAAAA 2460
AAAAAAAAAA AAA 2473 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Val lie Asp Ala Phe Ser Pro Gly Cys Leu Arg Lys Val Ala Lys Cys 1 5 10 15 lie Leu Leu Ser Glu Leu Leu Arg Ser Pro Pro Thr Phe Gly Phe Cys 20 25 30
Ser Ser Lys Asn Ser Phe His Cys Asp Leu Phe Ser Leu Ser Gly Ser 35 40 45
Gin Pro Pro Gly Pro Leu Cys 50 55
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4093 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
GCTAATTTTT TGTATTTTTA GTAGAGATGG GGTTTCACCA CACTGGCCAG GCTTGTCTCG 60
CTCGAACTTC TGACCTCATG ATCCACCTGC CTCGGCCTCC CAAAGTGCTG GGATTACAGG 120
TGTGAGCCAC CGCACCTGGC AAAAGAGAAT CTTACAGAAC CTATTCACTG GGAAGGAAGC 180
CCTCATTATA ATGATTTTCA TTCTTATGTG TGTTTCAGGA CGACTGGGTT TGGATTCAGA 240
AGAGGATTAT TATACACCAC AAAAGGTGGA TGTTCCCAAG GCCTTGATTA TTGTTGCAGT 300
TCAATGTGGC TGTGATGGGA CATTTCTGTT GACCCAGTCA GGCAAAGTGC TGGCCTGTGG 360
ACTCAATGAA TTCAATAAGC TGGGTCTGAA TCAGTGCATG TCGGGAATTA TCAACCATGA 420
AGCATACCAT GAAGTTCCCT ACACAACGTC CTTTACCTTG GCCAAACAGT TGTCCTTTTA 480
TAAGATCCGT ACCATTGCCC CAGGCAAGAC TCACACAGCT GCTATTGATG AGCGAGGCCG 540
GCTGCTGACC TTTGGCTGCA ACAAGTGTGG GCAGCTGGGC GTTGGGAACT ACAAGAAGCG 600
TCTGGGAATC AACCTGTTGG GGGGACCCCT TGGTGGGAAG CAAGTGATCA GGGTCTCCTG 660
CGGTGATGAG TTTACCATTG CTGCCACTGA TGATAATCAC ATTTTTGCCT GGGGCAATGG 720
TGGTAATGGC CGCCTGGCAA TGACCCCCAC AGAGAGACCA CATGGCTCTG ATATCTGTAC 780
CTCATGGCCT CGGCCTATTT TTGGATCTCT GCATCATGTC CCGGACCTGT CTTGCCGTGG 840
ATGGCATACC ATTCTCATCG TTGAGAAAGT ATTGAATTCT AAGACCATCC GTTCCAATAG 900
CAGTGGCTTA TCCATTGGAA CTGTGTTTCA GAGCTCTAGC CCGGGAGGAG GCGGCGGGGG 960
CGGCGGTGGT GAAGAAGAGG ACAGTCAGCA GGAATCTGAA ACTCCTGACC CAAGTGGAGG 1020
CTTCCGAGGA ACAATGGAAG CAGACCGAGG AATGGAAGGT TTAATCAGTC CCACAGAGGC 1080
CATGGGGAAC AGTAATGGGG CCAGCAGCTC CTGTCCTGGC TGGCTTCGAA AGGAGCTGGA 1140 AAATGCAGAA TTTATCCCCA TGCCTGACAG CCCATCTCCT CTCAGTGCAG CGTTTTCAGA 1200
ATCTGAGAAA GATACCCTGC CCTATGAAGA GCTGCAAGGA CTCAAAGTGG CCTCTGAAGC 1260
TCCTTTGGAA CACAAACCCC AAGTAGAAGC CTCGTCACCT CGGCTGAATC CTGCAGTAAC 1320
CTGTGCTGGG AAGGGAACAC CACTGACTCC TCCTGCGTGT GCGTGCAGCT CTCTGCAGGT 1380
GGAGGTTGAG AGATTGCAGG GTCTGGTGTT AAAGTGTCTG GCTGAACAAC AGAAGCTACA 1440
GCAAGAAAAC CTCCAGATTT TTACCCAACT GCAGAAGTTG AACAAGAAAT TAGAAGGAGG 1500
GCAGCAGGTG GGGATGCATT CCAAAGGAAC TCAGACAGCA AAGGAAGAGA TGGAAATGGA 1560
TCCAAAGCCT GACTTCGATT CAGATTCCTG GTGCCTCCTG GGAACAAACT CCTGTAGACC 1620
CAGCCTCTAT TCTCCTGAGC CTATAGAGCC CCCAGGAGAC TGGGACCCAA AGAACTTCAC 1680
AGCACACTTA CCGAATGCAG AGAGCAGCTT TCCTGGCTTT GTTCACTTGC AGAAAAGGAG 1740
CGCAAGGCAG AGGCTCTGAA GCACTTTCCT TGTACATTTG GAGAGTGGCA TTGCCTTTTA 1800
GATAGGATCT AGGAGTGATT TTATTGTTTT GGAGAATGGA AGGGCCCCCA TGGCCCTGGC 1860
TTTGTCATCA GTGACTGCCA TAGCAACAGC AGCTCTGTAC CTCATCTGTT GATCCCACCT 1920
TTGAAGAGGA GACACAGTGC TCACCTTAAT TGCGCTGGTA GCAGCTTATA TCCCATGTAT 1980
CATTTTCACC ATTGATTGGA AGCTGCCTTG GGAATTCAGT ACCAGGCATT ACCCCTCTGG 2040
GTGGGAGAGG GAGAAGTGTA AAGTTGGAGT GGGCTGGAAT CAGGTGTGGC CCGCCCAGTG 2100
TCCTCTGCAG AGTGGTGAAG TAGTCTGGCC CTCTTGGGAG CCCTGAGTCC AGGAAAATAT 2160
GTCTGATGGA GTCAATCTAG GGCTTGTTTC GAAAAAGTTC AGTTACTCTG TGCAGCTAAA 2220
TGCTTTAGGA GGAAAGGTAG GCTTAGGTTG CTTTTCCTCT GAGGGTTGAT TGAAATTTCT 2280
TCAGTGAGGA ATAGAGAAAG GGCAGGACCC TCATCATCAC ACAGCTGGTG TTTCAGGCTG 2340
TGACCAATGC AGGGTGGGAT TTCCTAACTG TGGATGAGGG GATGAGGTGT CTCTGAGGGA 2400
TGAGGTGTCT CAGAGAATTG AGTCCATGGG GCAGTCAGAA TAGCCTTAAG AGAAAATCAT 2460
GAAGGAGAAG AGGTCCTCCT TTAGCTGCCT CTACTTGGTA TCTTAGAGAG GGCTTAGAGG 2520
GCTCTCAGTC TTCTGCCCAT GAAAAGACTT CTTTGAGCCT CTGCCTTCAT GGCTCTTAGG 2580
GTTCTGATCT TGATATCAGC AGCCCCAACC ACTTTCTTTC TGAATGTCTA GTCAGTATTT 2640
TTCCCCTTTT GGTGTTTTAT GAAGCCATGT GGTAACGAAT GAATCTGTAT CATTTTTCCT 2700
ACCTGAGTGG CCCAAAGCCA GCACCAAACC CTGGGAGTCC CTGAAGCCTA ACAGAACAGG 2760
TAGAACTTGC AAAAGGAATT TGGCTGAGAG CTCACTTCTA ATCCTGTACT CACTGTGTCT 2820 TTGTAGATAG AAACAAGCTA GCTTTACCAA AGAGAAATAG TGTCATAGAA GAACAAAACT 2880
TCATATAGAA AGTTCTAGGC AAGTATTTGA TGGTTTCCTT AAGGATGTGA GCTTTGTATT 2940
TCCACCTAGC CTTGTAAAAT GTTCCTGTGG TATTTTGTGT CACACATCCT ACCTTTGATG 3000
AGTCTCACAT CCCACCTTCC TATAAACAGC TAAATTAATT TTGTTTCATC TTCCCCAGAC 3060
CAAAATGTTT GATAATCTTA TCAACATTGT GGGAGGTCTT GTGCAATGGA AATTTTGCCA 3120
TTTCTCCAAA ACTGGTGGCA TAAAGGCTGA TGCTTGGGGA GAACCCCATT GCTCGGGACA 3180
GGCAACTCTG TTCAATGGGA TCTTCTTTGG TTTGATGTTC CCATTGTTTT CTCAGTTCTG 3240
GGAAGCCTAG TACATTAGTA CTAATGTAAT CACTGAAACC TTTTCTTGAA ATAAGGGAAG 3300
CAGCCAAACT TTGATTAAAG TTGCAAGTTC TGGGGACTTG CGGGGGTTGT CATAAACTGT 3360
AACAGTGGGT TTTGGTTCAG CATGTAAATG CAACTTTGAT TTTCTTGAGG ACCGATTGAC 3420
CTGTCATGTC CCTGTATCCT CATGCTCATC ATCTCAGCAG GCCTGAGAGG CTGGGTCAGT 3480
TTGGGTGTTC ATCATGAGGA TTGCTTCTGC CATGGAGCTG ATGGACGTGG GCAGGTTGCT 3540
GAGAAGGTGG GGTGAAAGTG AGTGCCGGGG GTGGGTGAGT GCCCTGGTCT TGTTCATAGG 3600
GGAGCCTTTC CCTAGCAGTG GAACGCTGTG GTCATTTTCT CTAGCATATT CCCTTGGGAA 3660
GTCTAGATTT GCTATTAATC TGGCTGAGAA TCTAAGTTCT GTGCCTTAGA GACAGTTTGC 3720
ACTTTCCCAT ATTGTGCCTG GGACAGCCAT ATGATTTTTT TTCCCACCAA ACAAGTATGC 3780
AAACAGAAAC CAGTTTCAAA GGGGGATGGA GTAAAAGATG AGGCAGTAGA AATGCCTTTG 3840
AATGGTTTTT CTGTAGCTAA TTCTCTTTAA ATTTTGTCCT GCTTTTTTTC TTTATGCAGT 3900
GCTAGGTGTT TTAAGTTTTC TAGTAGTATT GCTTTTGAGT TACAGTATAA CCTGAGTTAC 3960
TCCTCTGCTC TAACATTGTT GCAGAAGAGT AACTCAGGTT ATTGTTAGCC AGGTTGCTTG 4020
AAAGGTTGAG AGTGGAGTGG TTTGGCATTT CTGTTTTAAA TAAACATTTA AGCTCTTAAA 4080
AAAAAAAAAA AAA 4093 (2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met lie Phe lie Leu Met Cys Val Ser Gly Arg Leu Gly Leu Asp Ser 1 5 10 15
Glu Glu Asp Tyr Tyr Thr Pro Gin Lys Val Asp Val Pro Lys Ala Leu 20 25 30 lie lie Val Ala Val Gin Cys Gly Cys Asp Gly Thr Phe Leu Leu Thr 35 40 45
Gin Ser Gly Lys Val Leu Ala Cys Gly Leu Asn Glu Phe Asn Lys Leu 50 55 60
Gly Leu Asn Gin Cys Met Ser Gly lie lie Asn His Glu Ala Tyr His 65 70 75 80
Glu Val Pro Tyr Thr Thr Ser Phe Thr Leu Ala Lys Gin Leu Ser Phe 85 90 95
Tyr Lys lie Arg Thr lie Ala Pro Gly Lys Thr His Thr Ala Ala lie 100 105 110
Asp Glu Arg Gly Arg Leu Leu Thr Phe Gly Cys Asn Lys Cys Gly Gin 115 120 125
Leu Gly Val Gly Asn Tyr Lys Lys Arg Leu Gly lie Asn Leu Leu Gly 130 135 140
Gly Pro Leu Gly Gly Lys Gin Val lie Arg Val Ser Cys Gly Asp Glu 145 150 155 160
Phe Thr lie Ala Ala Thr Asp Asp Asn His lie Phe Ala Trp Gly Asn 165 170 175
Gly Gly Asn Gly Arg Leu Ala Met Thr Pro Thr Glu Arg Pro His Gly 180 185 190
Ser Asp lie Cys Thr Ser Trp Pro Arg Pro lie Phe Gly Ser Leu His 195 200 205
His Val Pro Asp Leu Ser Cys Arg Gly Trp His Thr lie Leu lie Val 210 215 220
Glu Lys Val Leu Asn Ser Lys Thr lie Arg Ser Asn Ser Ser Gly Leu 225 230 235 240
Ser lie Gly Thr Val Phe Gin Ser Ser Ser Pro Gly Gly Gly Gly Gly 245 250 255
Gly Gly Gly Gly Glu Glu Glu Asp Ser Gin Gin Glu Ser Glu Thr Pro 260 265 270
Asp Pro Ser Gly Gly Phe Arg Gly Thr Met Glu Ala Asp Arg Gly Met 275 280 285
Glu Gly Leu lie Ser Pro Thr Glu Ala Met Gly Asn Ser Asn Gly Ala 290 295 300
Ser Ser Ser Cys Pro Gly Trp Leu Arg Lys Glu Leu Glu Asn Ala Glu 305 310 315 320
Phe lie Pro Met Pro Asp Ser Pro Ser Pro Leu Ser Ala Ala Phe Ser 325 330 335
Glu Ser Glu Lys Asp Thr Leu Pro Tyr Glu Glu Leu Gin Gly Leu Lys 340 345 350
Val Ala Ser Glu Ala Pro Leu Glu His Lys Pro Gin Val Glu Ala Ser 355 360 365
Ser Pro Arg Leu Asn Pro Ala Val Thr Cys Ala Gly Lys Gly Thr Pro 370 375 380
Leu Thr Pro Pro Ala Cys Ala Cys Ser Ser Leu Gin Val Glu Val Glu 385 390 395 400
Arg Leu Gin Gly Leu Val Leu Lys Cys Leu Ala Glu Gin Gin Lys Leu 405 410 415
Gin Gin Glu Asn Leu Gin lie Phe Thr Gin Leu Gin Lys Leu Asn Lys 420 425 430
Lys Leu Glu Gly Gly Gin Gin Val Gly Met His Ser Lys Gly Thr Gin 435 440 445
Thr Ala Lys Glu Glu Met Glu Met Asp Pro Lys Pro Asp Phe Asp Ser 450 455 460
Asp Ser Trp Cys Leu Leu Gly Thr Asn Ser Cys Arg Pro Ser Leu Tyr 465 470 475 480
Ser Pro Glu Pro lie Glu Pro Pro Gly Asp Trp Asp Pro Lys Asn Phe 485 490 495
Thr Ala His Leu Pro Asn Ala Glu Ser Ser Phe Pro Gly Phe Val His 500 505 510
Leu Gin Lys Arg Ser Ala Arg Gin Arg Leu 515 520
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1601 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : CCCGACGAGG CCTTCGACCA TGAGGTCTCC GCCTTCTTCC CCGCCAACCT GGACTTCCTG 60
TGCCTGCAGG AGGTGTTTGA CAAGCGAGCA GCCACCAAAT TGAAAGAGCA GCTGCACGGC 120
TACTTCGAGT ACATCCTGTA CGACGTCGGG GTCTACGGCT GCCAGGGCTG CTGCAGCTTC 180
AAGTGTCTCA ACAGCGGCCT CCTCTTTGCC AGCCGCTACC CCATCATGGA CGTGGCCTAT 240
CACTGTTACC CCAACAAGTG TWACKACSAT GCCCTGGCCT CTAAGGGAGC TCTGTTTCTC 300
AAGGTGCAGG TGGGAAGCAC ACCTCAGGAM CAAARAATCG TCGGGTACAT CGCCTGCACA 360
CACCTGCATG CCCCGCAAGA GGACAGCGCC ATCCGGTGTG GGCAGCTGGA CCTGCTTCAG 420 GACTGGCTGG CTGATTTCCG AAAATCTACC TCCTCGTCCA GCGCAGCCAA CCCCGAGGAG 480 CTGGTGGCAT TTGACGTCGT CTGTGGAGAT TTCAACTTTG ATAACTGCTC CTCTGACGAC 540 AAGCTGGAGC AGCAACACTC CCTGTTCACC CACTACAGGG ACCCCTGCCG CCTGGGGCCT 600 GGTGAGGAGA AGCCGTGGGC CATCGGTACT CTGCTGGACA CGAACGGCCT GTACGATGAG 660 GATGTGTGCA CCCCCGACAA CCTGCAGAAG GTCCTGGAGA GTGAGGAGGG CCGCAGGGAG 720 TACCTGGCGT TTCCCACCAG CAAGAGCTCG GGCCAGAAGG GGCGGAAGGA GCTGCTGAAG 780 GGCAACGGCC GGCGCATCGA CTACATGCTG CATGCAGAGG AGGGGCTGTG CCCAGACTGG 840 AAGGCCGAGG TGGAAGAATT CAGTTTTATC ACCCAGCTGT CCGGCCTGAC GGACCACYTG 900 CCAGTAGCCA TGCGACTGAT GGTGTCTTCG GGGGAGGAGG AGGCATAGAC CGTCCGGAGC 960
AGCGGGGCYT CTGCCAGCCC TTGCAGCTGC AGCCCATCCC TGGGCCATGT CCCCTCCATC 1020
GAGTGCCCGG TGCTTGGGGG AGGAGGGCAG GGACAGGGAG GGAGCCACAG TCAGTGCCCG 1080
GGAACCTGGA AGCTGCGCTG CTCTGCGCCT CTGGGCCTCA CTGTGGSCAG AGGAGTCAGG 1140
CCCGCCCCAG GAGCCTCCAG CTGCCTAACC AGTGCCATTC TTTCACAACA CGATTTTCTA 1200
CAAATCTACA GCACAACCGA GTTTGTAACC CGTGGGTTAG TATGAGGACC GGGTTCGTGT 1260
ACTCTCTGTA TCTCCTCTTA AGCTTCGTCC AGGGTTCTTT ATTTTTGTCT GCTGCCAATG 1320
TCGTCTCGCA TGCCTGCACC CTCGCATGCA CGCTGCCCGC ATGCCACGTG CCACGCTGTA 1380
GCCACAGACC CCTTGCTCGG GCCTCACCCA AGGCCAAACT CCAAACACAA TCAGAACCAG 1440 CCAAAGAAGC ACTTCCTGGG CACGGCCACC AGCTCTCCCG CCTCCAGTGT GGGCCGGCTC 1500
CTGCAGGGTC CGAGGGCTGC ATCTCTACCA GCCAGCCCAG GGCTCTTCCC AGGGTCTCGC 1560
ATTCAAGGGC AATTACATTT TAAAAAAAAA AAAAAAAAAA A 1601 ( 2 ) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Asp Val Ala Tyr His Cys Tyr Pro Asn Lys Cys Xaa Xaa Xaa Ala 1 5 10 15
Leu Ala Ser Lys Gly Ala Leu Phe Leu Lys Val Gin Val Gly Ser Thr 20 25 30
Pro Gin Xaa Gin Xaa lie Val Gly Tyr lie Ala Cys Thr His Leu His 35 40 45
Ala Pro Gin Glu Asp Ser Ala lie Arg Cys Gly Gin Leu Asp Leu Leu 50 55 60
Gin Asp Trp Leu Ala Asp Phe Arg Lys Ser Thr Ser Ser Ser Ser Ala 65 70 75 80
Ala Asn Pro Glu Glu Leu Val Ala Phe Asp Val Val Cys Gly Asp Phe 85 90 95
Asn Phe Asp Asn Cys Ser Ser Asp Asp Lys Leu Glu Gin Gin His Ser 100 105 110
Leu Phe Thr His Tyr Arg Asp Pro Cys Arg Leu Gly Pro Gly Glu Glu 115 120 125
Lys Pro Trp Ala lie Gly Thr Leu Leu Asp Thr Asn Gly Leu Tyr Asp 130 135 140
Glu Asp Val Cys Thr Pro Asp Asn Leu Gin Lys Val Leu Glu Ser Glu 145 150 155 160
Glu Gly Arg Arg Glu Tyr Leu Ala Phe Pro Thr Ser Lys Ser Ser Gly 165 170 175
Gin Lys Gly Arg Lys Glu Leu Leu Lys Gly Asn Gly Arg Arg lie Asp 180 185 190
Tyr Met Leu His Ala Glu Glu Gly Leu Cys Pro Asp Trp Lys Ala Glu 195 200 205
Val Glu Glu Phe Ser Phe lie Thr Gin Leu Ser Gly Leu Thr Asp His 210 215 220
Leu Pro 'Val Ala Met Arg Leu Met Val Ser Ser Gly Glu Glu Glu Ala 225 230 235 240
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2274 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
GGCGTTGCTG GTGCGAGTCC TTAGGAACCA GACTAGCATT TCTCAGTGGG TTCCAGTATG 60
CAGCCGATTG ATACCTGTGT CTCCTACCCA AGGACAGGGG GACAGGGCTC TGTCTCGCAC 120
TTCCCAGTGG CCCCAGATGA GCCAGTCCCA AGCATGTGGT GGATCAGAAC AGATTCCTGG 180
AATAGACATA CAGCTGAATA GGAAGTATCA CACCACACGT AAGCTTTCTA CTACCAAAGA 240
TTCCCCACAG CCTGTTGAGG AGAAGGTTGG TGCTTTCACA AAGATAATAS AAGCCATGGG 300
ATTCACGGGA CCTTTGAAAT ACAGTAAATG GAAGATTAAG ATTGCGGCCC TGCGCATGTW 360
TACTAGCTGT GTGGAGAAAA CTGACTTCGA GGAATTCTTT CTAAGGTGTC AGATGCCTGA 420
TACATTCAAT TCATGGTTTC TTATAACCCT ACTCCACGTC TGGATGTGTC TAGTCCGAAT 480
GAAGCAGGAA GGCCGGAGTG GGAAGTACAT GTGTCGTATC ATAGTTCATT TTATGTGGGA 540
GGATGTTCAG CAGCGCGGCA GAGTCATGGG GGTTAATCCC TATATCCTGA AGAAGAACAT 600
GATCCTCATG ACAAATCATT TCTATGCAGC GATCTTGGGA TATGATGAGG GGATCCTTTC 660
AGATGATCAT GGGCTGGCCG CTGCCCTCTG GAGAACCTTC TTCAACCGGA AATGTGAAGA 720
CCCTCGACAT CTTGAATTGC TGGTAGAGTA TGTGAGGAAA CAGATACAGT ACCTGGACTC 780
CATGAACGGG GAGGATCTGC TTCTGACAGG GGAGGTGAGC TGGCGCCCTC TAGTGGAGAA 840 GAATCCTCAG AGCATCCTGA AGCCCCATTC TCCGACTTAC AACGACGAGG GACTTTGATG 900
GGCTGGGCCC TCCGCACGGC CCGCCAGCTG GCTTCGAGGA ACCTCCAGGA GAGAAGTGCC 960
TGTTGGTCCA GGACCCTGCA GAAAGTGGCC TGAACTGACC TCTGAACAGC ATCTGTCAAA 1020
TACCTGGCCC CATTTGTGTT GAGTTTCCTC TTAGTGTGCC CAGGAGTCTG ATCTGCTGGG 1080
GTACAGGGCT GGGAGAACCC CTAGCTCTCC CGGGGTGTCC TCTCCCTTAG GGGAAGCCCC 1140
GAGTGAGAGT CCCCCAGCAC ACACTCCCCA ACCCCCTCCA GCAACTACAT GTGACTGATA 1200
GCTTTTCCCA AAGGCCAAGG AAGGGATGGT GTAGGTTCAA AAGGGAAACC CCCCAGGGCC 1260
TGCTGTGGCC TAGGAGCAGA TTGTAATGCT GCCGAGTCCG GTCGGTGACC ACGCGTTGTC 1320
CCTCGGCTTT CAGCCATGGG GTTGAGTTGG CCATTAAAAG AAACAGAGAC TTCTCTCTGC 1380
CATGGCCCTT CTTTATTCCA GGGACTTAGA AACTTGCCTG AGATGGTGGA CGCAGTAATG 1440
AGGGCACCGC GCAGCTCAGT TAGAGACGGA GAAAGGGAAG AGGCTGGGAT GGTCTCTGCT 1500
GCTCTTGCCT CTAGTTCATG GAGATGTGTC TCTGTTCAGG CCAAGATACA GCCAGCCAGG 1560
CCTGTCGTCT GGGACCCAGG AGGCCTCTGA TGACCAAGGG CTTTCACATC CTAAGTCATT 1620
TGGAAGGAGG CCTTGAGAAC AAAGTCACCT TTGTCACTCC CAGTGAACTG AATGAGGAAC 1680
ATGCTGTCTC CTGTCTTGGC CTCCCCTTTC ATGAGATACT GGGGAGAAGA GAACATTCCT 1740
CCTGGCTTAG TTGTAGCAGA CCCAGACCTG TGCCCAGCTT TGGTCCCCCT TCCCAACTTC 1800
TGAAGCACGT GCTGCAGAGC CACCTTGGTC TGAGCACCTG AGGACCAGCC CCTCCTCCCT 1860
CAGTGCGGGT CATCTCTTGG GGGATTTTCT TAAAGTGAAG AAAGGGGGTG GGGAACCATA 1920
TTGCCCCTCC CTCCCCCATC AAACTTCCTT CATTTAACTT GCTATAAAAT GAGTCATATA 1980
AAGAAACTCT ATATGGGTGA GGTATATCCC ACTTCTGTGA AAACATTACA AATCAAACCG 2040
CTTCTCTCAG TTTATTTAAG ATGCTTTTGT TGCGAGCGGA GCTCTAGAGT GAAGCCTCCT 2100
GTGTGTGTGT GAGATAATAA CACCTTGTAA CTCATTACAG CTGGGCACTA TTTACATAAA 2160
CCAGAGCTGA GCCAGGCAGG AATTTGCTGA TTAATTTATT TTTAATGGAG TGAAGTATAC 2220
CATGCACCAA AATAAACTTT ACTGTGTGTA CCTAAAAAAA AAAAAAAAAA AAAA 2274 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 253 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ser Gin Ser Gin Ala Cys Gly Gly Ser Glu Gin He Pro Gly He 1 ' 5 10 15
Asp He Gin Leu Asn Arg Lys Tyr His Thr Thr Arg Lys Leu Ser Thr 20 25 30
Thr Lys Asp Ser Pro Gin Pro Val Glu Glu Lys Val Gly Ala Phe Thr 35 40 45
Lys He He Xaa Ala Met Gly Phe Thr Gly Pro Leu Lys Tyr Ser Lys 50 55 60
Trp Lys He Lys He Ala Ala Leu Arg Met Xaa Thr Ser Cys Val Glu 65 70 75 80
Lys Thr Asp Phe Glu Glu Phe Phe Leu Arg Cys Gin Met Pro Asp Thr 85 90 95
Phe Asn Ser Trp Phe Leu He Thr Leu Leu His Val Trp Met Cys Leu 100 105 110
Val Arg Met Lys Gin Glu Gly Arg Ser Gly Lys Tyr Met Cys Arg He 115 120 125
He Val His Phe Met Trp Glu Asp Val Gin Gin Arg Gly Arg Val Met 130 135 140
Gly Val Asn Pro Tyr He Leu Lys Lys Asn Met He Leu Met Thr Asn 145 150 155 160
His Phe Tyr Ala Ala He Leu Gly Tyr Asp Glu Gly He Leu Ser Asp 165 170 175
Asp His Gly Leu Ala Ala Ala Leu Trp Arg Thr Phe Phe Asn Arg Lys 180 185 ' 190
Cys Glu Asp Pro Arg His Leu Glu Leu Leu Val Glu Tyr Val Arg Lys 195 200 205
Gin He Gin Tyr Leu Asp Ser Met Asn Gly Glu Asp Leu Leu Leu Thr 210 215 220
Gly Glu Val Ser Trp Arg Pro Leu Val Glu Lys Asn Pro Gin Ser He 225 230 235 240
Leu Lys Pro His Ser Pro Thr Tyr Asn Asp Glu Gly Leu 245 250 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2711 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TATCCATTAC GTCGACTAAT ACGTACATAA GAATTCAATC GGGCCTTGGG GCTGGCCCTG 60
AAACCTGCGA GGGGCTTCCG TCCACGTCCC CAGTGGACCT ACCACCCCTC CATCTGGGAA 120
AGCAGGCCAC AGCAGCCGGA CAAAGGAAGC TCCTCAGCCT CTAGTCGCCT CTCTGTGCAT 180
GCACATCGGT CACTGATCTC GCCTACTGGC ACAGACGTGT TTATCGGCCA ACCTGACCCT 240
CACAAAAAGC TACCACCGAA GTGGACAGGC CCCTACACTG TGATACTCAG CACACCAACT 300
GCAGTGAGAG TCCGAGGACT CCCCAACTGG ATCCATCGCA CCAGGGTCAA GCTCACCCCC 360
AAGGCAGCTT CTTCCTCCAA AACATTAACA GCTAAGTGTT TGTCTGGGCC AATTTCTCCT 420
ACCAAGTTTA AATTAACCAA CATTTTTTTC TTAAAACCAA AACACAAGGA AGACTAACCA 480
CGTGCTTCCA GGAATGGCCT GTATCTACCC AACCACTTTC TATACCTCTC TTCCAACCAA 540
AAGTCTTAAT ATGGGAATAT CCCTCACCAC GATCCTAATA CTGTCAGTAG CTGTCCTGCT 600
GTCCACAGCA GCCCCTCCGA GCTGCCGTGA GTGTTATCAG TCTTTGCACT ACAGAGGGGA 660
GATGCAACAA TACTTTACTT ACCATACTCA TATAGAAAGA TCCTGTTATG GAAACTTAAT 720
CGAGGAATGT GTTGAATCAG GAAAGAGTTA TTATAAAGTA AAGAATCTAG GAGTATGTGG 780
CAGTCGTAAT GGGGCTATTT GCCCCAGAGG GAAGCAGTGG CTTTGCTTCA CCAAAATTGG 840
ACAATGGGGA GTAAACACTC AGGTGCTTGA GGACATAAAG AGAGAACAGA TTATAGCCAA 900
AGCCAAAGCC TCAAAACCAA CAACTCCCCC TGAAAATCGC CCGCGGCATT TCCATTCCTT 960
TATACAAAAA CTATAAGCAG ATGCATCCCT TCCTAAGCCA GGAAAAAATC TGTTTGTAGA 1020
TCTAGGAGAA CCATTGTGCT TACCATGAAT GTGTCCAATT GTTGGGTATG CGGGGGAGCT 1080
TTATGAGTGA ACAGTGGCTG TGGGACGGGA TAGACATTCC CCCTTACTTA CAGGCATCCC 1140
AAAACCCCAG ACTCACTTTC ACTCCTCAGG AATGCCCGCA GTCCTGGACA CTTACCAACT 1200 CAGTATGAGG GACGGTGTGC ATATCCCGCA AGTGGACTGA TAAAACCCAT CGCGCCGTAG 1260
GTGAAAACCC GTCACCAAAC CCTAACAGTC AATGCCTCCA TAGCTGAGTG GTGGCCAAGG 1320
TTACCCCCTG GAGCCTGGTC TCCTTCTAAC TTAAGCTACC TCAATTGTGT CTTGTCAAAA 1380
AAGGCCTGGT ACTGTACAAA CACCACTAAC CCTTATGCCG CATACCTCCG CCTAAGTGTA 1440
CTATGCGACA ATCCTAGGAA CACCAGCTGA CAATGGACTG CCACTGACGG ATTCCTGTGG 1500
ATATGGGGAA CCCAGGCTTA CTCACAGCTA CCTTATCACT GGCAAGGTAC TTGCTTCCTA 1560
GGCACAATTC AACCTGGATT CTTTTTACTT CCGAAGCACG CGGGCAACAC CCTCAGAGTC 1620
CCTGTGTATG ATAACCAGAG AAAAATGATC CTTGGAGGTA GGAGGGAGCC AAAGATTGTG 1680
AGAGGACGAG TGGCCTCCGC AACGGATCAT TGAATACTAT GGTCCTGCCA CTTGGGCAGA 1740
GGATGGTTCA TGGGGTTATC GCACTCCCAT ATATATGCCA AATAGAGCGA TTAGACTACA 1800
AGCTGTTCTA GAGATAATCA CTAACCAAAC TGCCTCAGCC CTAGAAATGC TCGCGCAACA 1860
AC AAACCAA ATGCGCGCGG CAATTTATCA AAACAGGCTG GCCCTAGACT ACTTATTAGC 1920
AGAAGAGGGT GCGGGCTGTG GTAAGTTTAA CATCTCCAAT TGCTGTCTTA ACATAGGCAA 1980
TAATGGAGAA GAGGTTCTGG AAATCGCTTC AAACATCAGA AAAGTAGCCC GTGTACCAGT 2040
CCAAACCTGG GAGGGATGGG ACCCAGCAAA CTTCTAGGAG GGTGGTTCTC TAATTTAGGA 2100
GGATT AAAA TGCTGGTGGG GACAGTCATT TTCATCACTG GGGTCCTCCT GTTTCTCCCC 2160
TGTGGTATCC CATTAAAACT CTTGTTGAAA CTACAGTTAA CCTCCTGACA ATCCAGATGA 2220
TGCTCCTGCT ACAGCGGCAC GATGGATACC AACCCGTCTC TCAAGAATAC CCCAAAAATT 2280
AAGTTTTTCT TTTTCCAAGG TGCCCACGCC ACCCYTATGT CACGCCTGAA GTAGTTATTG 2340
AGAAAGTCGT CCCTTTCCCC TTTTCTATAA CCAAATAGAC AGGAATGGAA GATTCTCCTC 2400
GGGGCCTGAA AGCTTGCGGG ATGAATAACT CCTCCTCCTC AGGCCCAGTC CCAAGGTACA 2460
AACTTGCACC AGCAGCAAGA TAGCAGAGGC AGGAAGAGAG CTGGCTGGAA GACACGTACC 2520
CCCTGAAGAT CAAGAGGGAG GTCGCCCTGG TACTACATAG CAGTCACGTT AGGCTGGGAC 2580
AATTCCTGTT TACAGAGGAC TATAAAACCC CTGCCCCATC CTCACTTGGG GCTGATGCCA 2640
TTTTAGGCCT CAGCCTGTCT GCATGCAGGC GCTCATTAAA ACAGCATGTT GCTCCAAAAA 2700
AAAAAAAAAA A 2711 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Ala Cys He Tyr Pro Thr Thr Phe Tyr Thr Ser Leu Pro Thr Lys 1 5 10 15
Ser Leu Asn Met Gly He Ser Leu Thr Thr He Leu He Leu Ser Val 20 25 30
Ala Val Leu Leu Ser Thr Ala Ala Pro Pro Ser Cys Arg Glu Cys Tyr 35 40 45
Gin Ser Leu His Tyr Arg Gly Glu Met Gin Gin Tyr Phe Thr Tyr His 50 55 60
Thr His He Glu Arg Ser Cys Tyr Gly Asn Leu He Glu Glu Cys Val 65 70 75 80
Glu Ser Gly Lys Ser Tyr Tyr Lys Val Lys Asn Leu Gly Val Cys Gly 85 90 95
Ser Arg Asn Gly Ala He Cys Pro Arg Gly Lys Gin Trp Leu Cys Phe 100 105 110
Thr Lys He Gly Gin Trp Gly Val Asn Thr Gin Val Leu Glu Asp He 115 120 125
Lys Arg Glu Gin He He Ala Lys Ala Lys Ala Ser Lys Pro Thr Thr 130 135 140
Pro Pro Glu Asn Arg Pro Arg His Phe His Ser Phe He Gin Lys Leu 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2892 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
TGATTATTGA GCATAACTGA TGAGAGAATG ACAGTATTGG TGACTAGCTT TTTTGTTTTT 60
AAATAACACA CTTTAAAATG CCACAGTGTT ATTTGAAAGC TTAAAGGCAT TTTCTCATCT 120
TCAGTATTTG CTTTTTATCA GTTGTAGAAA AGAACCTTAG TGTTTTCTTC TCTACCTATG 180
TATGCTTATG AAACCGCTCT ACCTACATAT GAAACTTCTC TACCTATGTA TGTTTACGAA 240
AAGAGATGTA TTTGCCAAGA AAATCTTGAT TATATAAAAG ACAAAAAGAT TATAAAAGAC 300
AGTTTCTGTT TAAGTAAAAC TGTCTTTGAA ACTTAAGAAA GCTTAAGTTT TAAAAAAATC 360
TCAGTTAAAC ATCATTGGTT TATTTGACTT ACTGTTAACT CTTGCTTCTC TTTGACTCCA 420
TGGTGTTTTT AGATAACCAT GTATGAACTT TATGTTAATG GTTTGTGGGA TACTAAAATA 480
GCTTGAAGAT GACTTGATGA CTGTGCATTT TATATAGTTT TATTCTCCTA AAATCTCAGG 540
AGGGCAGCAA GTGCTGTCAA CGATTATATA GTGATGAGAT TTTTATGGGA ATGATTTCTT 600
CTTGGTGCGT TTTACACATT TGTACTGATA GCAAAACTAA AGTTTAAAGC AGCAAAGTTT 660
AAGTTCTCCT TAAATCCTAC AGAAACCAAC CTTTTAAGGA CA AATTTCA TCTAAAACAT 720
GACGATATTT AGCACACCTT TTAATGTGGG TATATATCAA GTGTTTAAGG ACTGGCTAGT 780
ATGTGATAGA GCAAGACCTG AGACTTTATA AGTATTTGCT CGTGTCTGTT GACAGACCTC 840
TTTCTTTCAA ACTTGTTAGA AGAGTGGTAA GACATATCCA ATTGGAAAAT AAGATGCAGT 900
GTTGTATAGC ACATACATTT AAAGTGCTTG CGTTAAAATT AGTTTCTCAA TAAGATAAAA 960
TTATTTTAAA AATTTGGTTC ACTTTATTAC AATAGTGGCA ATTTAGCTTT TCAGTATTAC 1020
AGGAATTTAA AAATTGGTTT CTTGTAGGGG ACATCTCAAC TTTGGGAATA TCTTCACTTA 1080
ATTTTTAAAA AATATTTTCA TGCTTTATTG TCCAGCTATA CAATATATCG CAAAATCCTG 1140
ACAAGTTCAT TGTATTAAGG TATTAACTAT TACATGGAAA GCAATTCTGT TCATCTTTTG 1200
ATGTTTGTGT TGAAAATGCT TATCTTTGTG TTTTGATCTT CCAACAGCTG AGAGCTTGAA 1260
CTGATTTAAA CATTTGTCAA TATACTTAAG AATGCTTTAA GTAAAGAAGG GGAAAATTTT 1320
AAGTAAGTTT TTTCCCTTCT AGGAAGAAAA ACTATGATGA TGTTAAGAAA ATGTCATTAT 1380
AGAGCTTGCT CAATAATATG TTCTTTAAAT CCACCTCCAT TTGTACATTA TAGGTATCAT 1440
TCTGTTTTTG CTTAAAATAA TCTGCAACCA TTTCAGATAG TTTTACAGCA AATTGATCTA 1500
AAAGCCACTA ATAAATTCTA GGGTTTGAGT CTAGAAGCCA AGCAAACTGT CACCAATGTC 1560
AGTTGTAAAT TAGAATGCAA CATGAGGCTT CAGACTCATG ACAATGA AT ACATGAAAAC 1620 AAAAATATAA TTGTGTCTAC CTTCCTACTT TCCCTTTTGA CATATGTAGT TGGAATTTTA 1680
CATAGTCTTA AAATCCATAT TTAGAATCTT ACCTGTTTCT ATAATAATTA GTAAAATGCC 1740
AAAGTAGTGA TAGAATATTG TGGCATTGAA GTAGCCGAAA AATTGTTAGT TTTAGCATCA 1800
AAAAAGTAAA TAGATGTTGA AATGAATTTT TGTATGTGCC AGGTTGAAGA GAGTGTGCCA 1860
GTGACAGGAA GTAGTCTAAA AAATTAACAG TTATGGTTTT AATAGGATCT GAAAGACAAT 1920
CTTTAAAGAA ATGGGAGAAA TTGGGGGTAT CAGTGAACCT ATACCAACCT CTCTTTGTAC 1980
ATAAATATGG TGATGTAGCT AGATATAAAA ATCAGTGTCT TACTGGCACC ATTTACAGTT 2040
TAGAAAACAA TCTTTTTCTT AAAAATGCCC ATCTGATTTC TATTTTTAGG AGCTACTTGG 2100
ATTTGTATGT ATTTTTTCTA CGTGAAAATA TATGTACTCT TCACTTTTGT TCCAGTACTA 2160
TAATTGCTCA TGCACTCTTT CTCCCCTTTG AGAACATTCA GTGAAATACA ACTTCATCAA 2220
AGATTTGCTC AAAGGAGAAG AATCGCATGA GTGTGAAAAG TAGATGCTCG TAGCCAGAAC 2280
AGAAAAGGTT ACACATGATC ATGGCACAGA AGATAGGAGG TTTGACTTGG TGGGCCATAA 2340
TGTTTATTAT CCTTTTTGAA A AACAGGGA CCAGCAGCAG TTTTCTCAGG ATAAATGCTC 2400
TACCCCACTT CTCTATGAAC AGGTGTGGGG AGGCTTACTT TCCATTTTCA TATTTATACA 2460
CCTCTCTACA AAAGCAATTT TTAATGAAGG TTAGTGGAAT TGTTAAAAAT CTGAGAGGAA 2520
TGATGACTGG AGGTGTTTGG GGTTTTTTTC TGTATTCATT TTTTAATGAG AAAAGTTTTA 2580
AATGTAGTAC AGGTTAGACC CAACTACTAC CTTACTATTA TAGGACGATT CTATGTTTCT 2640
GTTAAAGTAT TCAAGTAGCT TTCTCTGGGG GAAAAAGTAC CACTTGGACA CTTAAAGGAA 2700
TTGGGATTTT TGTCTACTTT GGATAAGGCA GTTGACTTCT TAAGTAAAAG CAATAGTGTA 2760
AAATGTCATT TTGTTTGGAA TGTTAAGTGA GCAAATAAAA AACATGTTGA AATTGTAAAA 2820
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2880
AAAAAAAAAA AA 2892 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 100 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met He Met Ala Gin Lys He Gly Gly Leu Thr Trp Trp Ala He Met 1 5 10 15
Phe He He Leu Phe Glu He Thr Gly Thr Ser Ser Ser Phe Leu Arg 20 25 30
He Asn Ala Leu Pro His Phe Ser Met Asn Arg Cys Gly Glu Ala Tyr 35 40 45
Phe Pro Phe Ser Tyr Leu Tyr Thr Ser Leu Gin Lys Gin Phe Leu Met 50 55 60
Lys Val Ser Gly He Val Lys Asn Leu Arg Gly Met Met Thr Gly Gly 65 70 75 80
Val Trp Gly Phe Phe Leu Tyr Ser Phe Phe Asn Glu Lys Ser Phe Lys 85 90 95
Cys Ser Thr Gly 100
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 618 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ATTCGTTACA AACATGTTTG CATGCCTACC ATATCTCAGG CACTGGGGAT ACAGCAGATC 60
AAGATCCTAC CCCATGGAAC TAAAAGAGGA CAGAGTACTG AGTGGAACAT ACGATGATAG 120
ATTTACAAAT AATGTAGCAT ACTTCTACTT CATTGTATCT TAAGTTTCTT GAAATATTGC 180
TACTGGAGAT TGGAAAGAAA TCTTAATGTT ATGGGGTATT GTCTAAGAAG CTTTATTTTA 240
AAACCATCTC ATTAAATTTT GTTGCATTTT AGATAATCGT CCCCAGATGC CATGTTACCC 300
TAGTGCAGAG TTTGGGGCTG GATAAGTTTT TGTTGTAGGT GGCTATCCTG TGTTTTGTAG 360
GGTATTTAGC AGCATCCTGG CCTTAAAACA AAAATGTTTT CAGACATTGC CAAATGTCCC 420
CCGAGCGGTA AAGTCACCCC CAAGTTGAGA ACCGCTCTAT ACAAAGAGCT GTTATTAGAG 480 CTAGACATTT CTGAATTGGC ATCAATTTCT ATATTGTATC CATAAACATT AGTAGCCACG 540
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 600
AAAAAAAAAA AAAAAAAA 618 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Phe Ser Asp He Ala Lys Cys Pro Pro Ser Gly Lys Val Thr Pro 1 5 10 15
Lys Leu Arg Thr Ala Leu Tyr Lys Glu Leu Leu Leu Glu Leu Asp He 20 25 30
Ser Glu Leu Ala Ser He Ser He Leu Tyr Pro 35 40
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 772 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TGCCATACAC TTCAGCAGAG TTTGCAACTT CTCTTCTAAG TCTTTATCCT TCCCCCAAGG 60
CATGCCTAGC ACAGGACTCT TGAACAGTGA TGCCTCAATT AGAGTTGCTA GCCAATAGAT 120
TGAAGCTATG TTGGCACAAT ATCCTACATC CTCCCGATCT ACTGGCTGAG CCCAACCCCA 180
CCTAAGAAGG ACAATAAAGA TCTGTGTTCA GAGTCATACT GAATAGAGAC TTCTGGACTC 240
TATAGAACCC ACTGCCTCCT GATGAAGTCC CTACTGTTCA CCCTTGCAGT TTTTATGCTC 300 CTGGCCCAAT TGGTCTCAGG TAATTGGTAT GTGAAAAAGT GTCTAAACGA CGTTGGAATT 360
TGCAAGAAGA AGTGCAAACC TGAAGAGATG CATGTAAAGA ATGGTTGGGC AATGTGCGGC 420
AAACAAAGGG ACTGCTGTGT TCCAGCTGAC AGACGTGCTA ATTATCCTGT TTTCTGTGTC 480
CAGACAAAGA CTACAAGAAT TTCAACAGTA ACAGCAACAA CAGCAACAAC AACTTTGATG 540
ATGACTACTG CTTCGATGTC TTCGATGGCT CCTACCCGTT TCTCCCACTG GTTGAACATT 600
CCAGCCTCTG TCTCCTGCTC TAGGATCCCC GACTCATTAA AGCAAAGAGG CTTAAAAAAA 660
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 720
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 772 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 131 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Lys Ser Leu Leu Phe Thr Leu Ala Val Phe Met Leu Leu Ala Gin 1 5 10 15
Leu Val Ser Gly Asn Trp Tyr Val Lys Lys Cys Leu Asn Asp Val Gly 20 25 30
He Cys Lys Lys Lys Cys Lys Pro Glu Glu Met His Val Lys Asn Gly 35 40 45
Trp Ala Met Cys Gly Lys Gin Arg Asp Cys Cys Val Pro Ala Asp Arg 50 55 60
Arg Ala Asn Tyr Pro Val Phe Cys Val Gin Thr Lys Thr Thr Arg He 65 70 75 80
Ser Thr Val Thr Ala Thr Thr Ala Thr Thr Thr Leu Met Met Thr Thr 85 90 95
Ala Ser Met Ser Ser Met Ala Pro Thr Arg Phe Ser His Trp Leu Asn 100 105 110
He Pro Ala Ser Val Ser Cys Ser Arg He Pro Asp Ser Leu Lys Gin 115 120 125 Arg Gly Leu 130
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 875 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CCTGTTTTCA ATATTTTATA ATGAAAATTT TTGAACATTC GGAGAAGTTG AAAGAATTAC 60
ACCCAGAACA CCCATGTCTA CCATGTTACT ACATTTGTGT GTGTGTGTGT GTGTGTGTGT 120
GTGTGCGTGT GCAATTTTAA TTACAATGGT CGAGGAAGCT CTTGCTGAGA AGGTGATGTT 180
GAATAAAGAC TCTAAAGACC CTAAAGAGTT GAGAGAGAGC TGTGTGGAGT TCTGGGGCCC 240
AGGCACAGCA AGTACAAAGA TCCTGAAGCA GGAGCATTCT TGGTGTGTTC AAGGAAAGCA 300
AGGAGGCCAG TGAGGTTGGA AAAGGGAATG AGGTCAGART AATAATAGGG TGAAAGARGA 360
TGGCTGGGGG ATGGGGGGGC ARTGARGCAG GGCCTATGGT TTCTACTTGG TGARGTGGGA 420
ARCCACTGGA RGGGTTTAAG CCGATAATTG ATGTCACATA ATTTATGTTG TAATGGAACC 480
CGTGTGACTG CTCCTGGGGA ACAGACAAAA GAAAGGGTAG TAGAGACACC AGCTAGGAAG 540
CAGATTCAGC TAGAAGAGAT GATACCTTCC ACTAAGGTGT TGGAGAAGTG GTTGGATTCT 600
AGATAATTTT TGAAGGTGGA GTTGGCAGAA TTTGAAGATC ATTCCATTTC TGTTCATACA 660
GAGCTTCCTC ATTCTCTTTT GACAGCTGCC TAGAATCTCA TTGTATCATA ATGTTTTAAA 720
CTAGTCCCCG ATGATGAATA TTTAGGTTGA TGCTTTCTCT CTTGCTGCCA CAAAACAGAC 780
ATATATTTGT ATAAAATGCC ACGTGGACAT GTCATTCTTC ACATAAGTGA GTATTTGTAG 840
AATAAATTCC AAGAAGCAGA AAAAAAAAAA AAAAA 875 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Ser Thr Met Leu Leu His Leu Cys Val Cys Val Cys Val Cys Val 1 - 5 10 15
Cys Ala Cys Ala He Leu He Thr Met Val Glu Glu Ala Leu Ala Glu 20 25 30
Lys Val Met Leu Asn Lys Asp Ser Lys Asp Pro Lys Glu Leu Arg Glu 35 40 45
Ser Cys Val Glu Phe Trp Gly Pro Gly Thr Ala Ser Thr Lys He Leu 50 55 60
Lys Gin Glu His Ser Trp Cys Val Gin Gly Lys Gin Gly Gly Gin 65 70 75
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TNCACGATGCC CAGTGCAAGC AGGACCAC 29
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TNGGACAAGGG TGTGTGAGCA GGGATGAT 29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D') TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GNGCTTCCTTC CCAGTGAATA GGTTCTGT 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: ANAAACTCGGT TGTGCTGTAG ATTTGTAG 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: ANGAACTAGAG GCAAGAGCAG CAGAGACC 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: ANCTCTTTCCT GATTCAACAC ATTCCTCG 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: CNCTCATGCGA TTCTTCTCCT TTGAGCAA 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: ANCTCTGCACT AGGGTAACAT GGCATCTG 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 = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GNGTGAACAGT AGGGACTTCA TCAGGAGG 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: ANTGTAGTAAC ATGGTAGACA TGGGTGTT 29

Claims

What is claimed is:
1. 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 65 to nucleotide 1270;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1139 to nucleotide 1270;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1011 to nucleotide 1216;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BOH4_l deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 196 to amino acid 205 of SEQ ID NO:2;
(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. The polynucleotide of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with the polynucleotide of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein encoded by the polynucleotide of claim 2, which process comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and
(b) purifying said 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 protein comprising 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 326 to amino acid 384;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 196 to amino acid 205 of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone BOH4_l deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
9. The protein of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
10. The protein of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 from amino acid 326 to amino acid 384.
11. A composition comprising the protein of claim 8 and 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. 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 418 to nucleotide 582;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 508 to nucleotide 582;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 555;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CD311_2 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO:4; (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).
15. A protein comprising 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 46;
(c) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone CD311_2 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
16. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
17. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NOS;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NOS from nucleotide 191 to nucleotide 1756;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NOS from nucleotide 254 to nucleotide 1756;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NOS from nucleotide 1 to nucleotide 604;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CG279_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CG279JL deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 256 to amino acid 265 of SEQ ID NO:6;
(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 protein comprising 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 138;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising the amino acid sequence from amino acid 256 to amino acid 265 of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone CG279_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
19. An isolated gene corresponding to the cDNA sequence of SEQ ID NOS.
20. 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 226 to nucleotide 948;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1128 to nucleotide 1601;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CJ424_9 deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 115 to amino acid 124 of SEQ ID NO:8;
(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 protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8; (b) fragments of the amino acid sequence of SEQ ID NO:8 comprising the amino acid sequence from amino acid 115 to amino acid 124 of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone CJ424_9 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
22. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
23. 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 137 to nucleotide 895;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 1488 to nucleotide 2274;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CR930_1 deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 121 to amino acid 130 of SEQ ID NO:10;
(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).
24. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10 comprising the amino acid sequence from amino acid 121 to amino acid 130 of SEQ ID NO:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone CR930_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
25. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
26. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 494 to nucleotide 973;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 611 to nucleotide 973;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 521 to nucleotide 940;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone DA306_4 deposited under accession number ATCC 98333; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO:12;
(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).
27. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12;
(b) the amino acid sequence of SEQ ID NO:12 from amino acid 11 to amino acid 149;
(c) fragments of the amino acid sequence of SEQ ID NO: 12 comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO: 12; and
(d) the amino acid sequence encoded by the cDNA insert of clone DA306_4 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
28. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:ll.
29. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 2295 to nucleotide 2594; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1867 to nucleotide 2372;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone DG76_1 deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 45 to amino acid 54 of SEQ ID NO: 14;
(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 protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 14;
(b) the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 26;
(c) fragments of the amino acid sequence of SEQ ID NO:14 comprising the amino acid sequence from amino acid 45 to amino acid 54 of SEQ ID NO:14; and (d) the amino acid sequence encoded by the cDNA insert of clone DG76_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
31. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:13.
32. 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 394 to nucleotide 522;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 476;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone D019_l deposited under accession number ATCC 98333;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone D019_l deposited under accession number ATCC 98333;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 16 to amino acid 25 of SEQ ID NO:16;
(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).
33. A protein comprising 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 27;
(c) fragments of the amino acid sequence of SEQ ID NO:16 comprising the amino acid sequence from amino acid 16 to amino acid 25 of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone D019_l deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
34. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:15.
35. 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 262 to nucleotide 654;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 322 to nucleotide 654;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 1 to nucleotide 618;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone EQ219_1 deposited under accession number ATCC 98333; (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333;
(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, the fragment comprising the amino acid sequence from amino acid 60 to amino acid 69 of SEQ ID NO:18;
(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).
36. A protein comprising 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 119;
(c) fragments of the amino acid sequence of SEQ ID NO: 18 comprising the amino acid sequence from amino acid 60 to amino acid 69 of SEQ ID NO: 18; and
(d) the amino acid sequence encoded by the cDNA insert of clone EQ219_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
37. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:17.
38. 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 74 to nucleotide 310; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 125 to nucleotide 310;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 1 to nucleotide 338;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone FG340_1 deposited under accession number ATCC 98333;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID NO:20;
(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 protein comprising 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 75; (c) fragments of the amino acid sequence of SEQ ID NO:20 comprising the amino acid sequence from amino acid 34 to amino acid 43 of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone FG340_1 deposited under accession number ATCC 98333; the protein being substantially free from other mammalian proteins.
40. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:19.
PCT/US1998/003595 1997-02-24 1998-02-24 Secreted proteins and polynucleotides encoding them WO1998037094A2 (en)

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CA002281059A CA2281059A1 (en) 1997-02-24 1998-02-24 Secreted proteins and polynucleotides encoding them
EP98907611A EP0971950A2 (en) 1997-02-24 1998-02-24 Secreted proteins and polynucleotides encoding them
JP53698598A JP2002514073A (en) 1997-02-24 1998-02-24 Secreted proteins and polynucleotides encoding them

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US6541232B1 (en) 1996-06-10 2003-04-01 Immunex Corporation Polypeptides having kinase activity
US6913893B2 (en) 1996-06-10 2005-07-05 Immunex Corporation IL-1/TNF-α-activated kinase (ITAK), and methods of making and using the same
WO2000036097A3 (en) * 1998-12-18 2000-09-08 Immunex Corp Methods for regulating vascularization using gef containing nek-like kinase (gnk)
WO2000036097A2 (en) * 1998-12-18 2000-06-22 Immunex Corporation Methods for regulating vascularization using gef containing nek-like kinase (gnk)
WO2001009317A1 (en) * 1999-07-29 2001-02-08 Helix Research Institute Stomach cancer-associated gene
WO2001012818A1 (en) * 1999-08-14 2001-02-22 Memorec Stoffel Gmbh Medizinisch-Molekulare Entwicklung Neutral cerebral-sphingomyelinase
WO2002002610A2 (en) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Secretion and trafficking molecules
WO2002002610A3 (en) * 2000-06-29 2002-09-19 Incyte Genomics Inc Secretion and trafficking molecules
US7358351B2 (en) 2000-08-02 2008-04-15 The Johns Hopkins University Endothelial cell expression patterns
EP2267013A3 (en) * 2003-09-26 2011-06-08 Ganymed Pharmaceuticals AG Identification of tumour-associated cell surface antigens for diagnosis and therapy
US9139880B2 (en) 2003-09-26 2015-09-22 Biontech Ag Identification of tumor-associated cell surface antigens for diagnosis and therapy
US7875424B2 (en) * 2003-09-26 2011-01-25 Ganymed Pharmaceuticals, Ag Identification of tumour-associated cell surface antigens for diagnosis and therapy
US9910043B2 (en) 2003-09-26 2018-03-06 Biontech Ag Identification of tumor-associated cell surface antigens for diagnosis and therapy
EP2272534A3 (en) * 2003-09-26 2011-06-15 Ganymed Pharmaceuticals AG Identification of tumour-associated cell surface antigens for diagnosis and therapy
US20130280166A1 (en) * 2003-09-26 2013-10-24 Biontech Ag Identification of tumor-associated cell surface antigens for diagnosis and therapy
US9733251B2 (en) 2003-09-26 2017-08-15 Ganymed Pharmaceuticals, Ag Identification of tumour-associated cell surface antigens for diagnosis and therapy
AU2015201738B2 (en) * 2003-09-26 2016-10-13 Biontech Ag Identification of tumour-associated cell surface antigens for diagnosis and therapy
US9267177B2 (en) 2003-09-26 2016-02-23 Biontech Ag Identification of tumor-associated cell surface antigens for diagnosis and therapy
US9194004B2 (en) 2005-03-24 2015-11-24 Biontech Ag Identification of surface-associated antigens for tumor diagnosis and therapy
WO2006100089A3 (en) * 2005-03-24 2007-01-11 Ganymed Pharmaceuticals Ag Identification of surface-associated antigens for tumor diagnosis and therapy
US9090940B2 (en) 2005-03-24 2015-07-28 Ganymed Pharmaceuticals Ag Identification of surface-associated antigens for tumor diagnosis and therapy
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US10302647B2 (en) 2005-03-24 2019-05-28 Biontech Ag Identification of surface-associated antigens for tumor diagnosis and therapy

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CA2281059A1 (en) 1998-08-27

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