WO1998057976A1 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998057976A1
WO1998057976A1 PCT/US1998/012516 US9812516W WO9857976A1 WO 1998057976 A1 WO1998057976 A1 WO 1998057976A1 US 9812516 W US9812516 W US 9812516W WO 9857976 A1 WO9857976 A1 WO 9857976A1
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WIPO (PCT)
Prior art keywords
seq
polynucleotide
amino acid
protein
nucleotide
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Application number
PCT/US1998/012516
Other languages
French (fr)
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
Steven H. Howes
Kim Fechtel
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Genetics Institute, Inc.
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Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to JP50468199A priority Critical patent/JP2002513294A/en
Priority to CA002294569A priority patent/CA2294569A1/en
Priority to EP98930279A priority patent/EP1003770A4/en
Priority to AU79704/98A priority patent/AU7970498A/en
Publication of WO1998057976A1 publication Critical patent/WO1998057976A1/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
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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:
  • NO:l from nucleotide 1 to nucleotide 850;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 185 to nucleotide 1600; the nucleotide sequence of SEQ ID NO:l from nucleotide 1403 to nucleotide 1600; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 850; the nucleotide sequence of the full-length protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:2, or 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 231 to amino acid 240 of SEQ ID NO:2.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:2, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2having biological activity, the fragment comprising the amino acid sequence from amino acid 231 to amino acid 240 of SEQ ID NO:2.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dx290_l deposited under accession number ATCC 98468;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 47 to nucleotide 2065; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1086 to nucleotide 1848; the nucleotide sequence of the full-length protein coding sequence of clone dx290_l deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone dx290_l deposited under accession number ATCC 98468.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 312 to amino acid 600.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or 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 331 to amino acid 340 of SEQ ID NO:4.
  • 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 312 to amino acid 600.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4having biological activity, the fragment comprising the amino acid sequence from amino acid 331 to amino acid 340 of SEQ ID NO:4.
  • 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
  • (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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 107 to nucleotide 724; the nucleotide sequence of SEQ ID NO:5 from nucleotide 218 to nucleotide 724; the nucleotide sequence of SEQ ID NO:5 from nucleotide 536 to nucleotide 866; the nucleotide sequence of the full-length protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 6 to amino acid 92.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:6, or 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 97 to amino acid 106 of SEQ ID NO:6.
  • 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 6 to amino acid 92.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6having biological activity, the fragment comprising the amino acid sequence from amino acid 97 to amino acid 106 of SEQ ID NO:6.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:8, or 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 195 to amino acid 204 of SEQ ID NO:8.
  • 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:8 or the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid 363.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8having biological activity, the fragment comprising the amino acid sequence from amino acid 195 to amino acid 204 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
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or 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 38 to amino acid 47 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:
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or 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 38 to amino acid 47 of SEQ ID NO:10.
  • 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
  • (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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:ll from nucleotide 43 to nucleotide 1671; the nucleotide sequence of SEQ ID NO:ll from nucleotide 112 to nucleotide 1671; the nucleotide sequence of SEQ ID NO:ll from nucleotide 224 to nucleotide 679; the nucleotide sequence of the full-length protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 62 to amino acid 212.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:12, or 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 266 to amino acid 275 of SEQ ID NO:12.
  • 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: 12 or the amino acid sequence of SEQ ID NO:12 from amino acid 62 to amino acid 212.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:12, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12having biological activity, the fragment comprising the amino acid sequence from amino acid 266 to amino acid 275 of SEQ ID NO:12.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468.
  • 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
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 14, or 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 79 to amino acid 88 of SEQ ID NO:14.
  • 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: 14 or the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 125.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 14, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14having biological activity, the fragment comprising the amino acid sequence from amino acid 79 to amino acid 88 of SEQ ID NO:14.
  • 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
  • (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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:16, or 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 160 to amino acid 169 of SEQ ID NO:16.
  • 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:16 or the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 167.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 16, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16having biological activity, the fragment comprising the amino acid sequence from amino acid 160 to amino acid 169 of SEQ ID NO:16.
  • 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: 18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 18;
  • a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
  • polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 from nucleotide 77 to nucleotide 616; the nucleotide sequence of SEQ ID NO:17 from nucleotide 164 to nucleotide 616; the nucleotide sequence of SEQ ID NO:17 from nucleotide 1 to nucleotide 415; the nucleotide sequence of the full-length protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468.
  • 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 113.
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:18, or 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 85 to amino acid 94 of SEQ ID NO:18.
  • 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;
  • fragments of the amino acid sequence of SEQ ID NO:18 comprising eight consecutive amino acids of SEQ ID NO:18; and (d) the amino acid sequence encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
  • 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 113.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:18, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18having biological activity, the fragment comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:18.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468;
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 78 to amino acid
  • the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or 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 370 to amino acid 379 of SEQ ID NO:20.
  • 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 eight consecutive amino acids of SEQ ID NO:20; and (d) the amino acid sequence encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
  • protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of SEQ ID NO:20 from amino acid 78 to amino acid 355.
  • the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20having biological activity, the fragment comprising the amino acid sequence from amino acid 370 to amino acid 379 of SEQ ID NO:20.
  • 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.
  • 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.
  • a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • 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 forms) 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.
  • a polynucleotide of the present invention has been identified as clone "dol5_4"_ dol5_4 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • dol5_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dol5_4 protein").
  • nucleotide sequence of dol5_4 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 dol5_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
  • Amino acids 394 to 406 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 407, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone dol5_4 should be approximately 1900 bp.
  • dol5_4 demonstrated at least some similarity with sequences identified as AA 113909 (zm80f 12.r 1 Stratagene neuroepithelium (#937231 ) Homo sapiens cDNA clone 531983 5'), AA189888 (mu55h06.rl Soares mouse lymph node NbMLN Mus musculus cDNA clone 643355 5'), and U52052 (Human S6 A-8 mRNA expressed in chromosome 6-suppressed melanoma cells). Based upon sequence similarity, dol5_4 proteins and each similar protein or peptide may share at least some activity.
  • dx290_l A polynucleotide of the present invention has been identified as clone "dx290_l".
  • dx290_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.
  • dx290_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dx290_l protein").
  • nucleotide sequence of dx290_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dx290_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone dx290_l should be approximately 2300 bp.
  • the nucleotide sequence disclosed herein for dx290_l was searched against the
  • dx290_l demonstrated at least some similarity with the sequence identified as AA064383 (ml47h02.rl Stratagene mouse testis (#937308) Mus musculus cDNA clone 515187 5'). Based upon sequence similarity, dx290_l proteins and each similar protein or peptide may share at least some activity.
  • ek390 4" A polynucleotide of the present invention has been identified as clone "ek390_4".
  • ek390_4 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.
  • ek390_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ek390_4 protein").
  • nucleotide sequence of ek390_4 as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ek390_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • Amino acids 25 to 37 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 38, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ek390_4 should be approximately 1000 bp.
  • the nucleotide sequence disclosed herein for ek390_4 was searched against the
  • ek390_4 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a potential transmembrane domain within the ek390_4 protein sequence centered around amino acid 160 of SEQ ID NO:6.
  • the nucleotide sequence of ek390_4 indicates that it may contain GGGA repeat sequences.
  • a polynucleotide of the present invention has been identified as clone "er471_7".
  • er471_7 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.
  • er471_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "er471_7 protein").
  • nucleotide sequence of er471_7 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 er471_7 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8.
  • Amino acids 74 to 86 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 87, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone er471_7 should be approximately 2250 bp.
  • er471_7 The nucleotide sequence disclosed herein for er471_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. er471_7 demonstrated at least some similarity with sequences identified as AA039137 (mi98h06.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone 474683 5'), AA066962 (mm38g05.rl Stratagene mouse melanoma
  • the predicted amino acid sequence disclosed herein for er471_7 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted er471_7 protein demonstrated at least some similarity to sequences identified as AFO 16448 (Cosmid F41E6 [Caenorhabditis elegans]) and L08407 (collagen type XVII [Mus musculus]). Based upon sequence similarity, er471_7 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts three potential transmembrane domains within the er471 7 protein sequence, centered around amino acids 40, 80, and 110 of SEQ ID NO:8, respectively.
  • fs40_3 A polynucleotide of the present invention has been identified as clone "fs40_3".
  • fs40_3 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.
  • fs40_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fs40_3 protein").
  • the nucleotide sequence of fs40_3 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 fs40_3 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 fs40_3 should be approximately 1000 bp.
  • fs40_3 demonstrated at least some similarity with sequences identified as AA411142 (zt37g01.rl Soares ovary tumor NbHOT Homo sapiens cDNA clone 724560 5'), AA412527 (zul2a03.sl Soares testis NHT Homo sapiens cDNA clone 731596 3'), AA565855 (nj32d09.sl NCI_CGAP_AA1 Homo sapiens cDNA clone IMAGE:994193), HI 7042 (ym39fl2.sl Homo sapiens cDNA clone 50584 3'), and T33280 (EST57284 Homo sapiens cDNA 3' end similar to None). Based upon sequence similarity, fs40_
  • TopPredll computer program predicts a potential transmembrane domain within the fs40_3 protein sequence at the C-terminus of SEQ ID NO:10. Clone "ga63 6"
  • ga63_6 A polynucleotide of the present invention has been identified as clone "ga63_6".
  • ga63_6 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.
  • ga63_6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ga63_6 protein").
  • the nucleotide sequence of ga63_6 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 ga63_6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ga63_6 should be approximately 2300 bp.
  • ga63_6 The nucleotide sequence disclosed herein for ga63_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ga63_6 demonstrated at least some similarity with sequences identified as AA405433 (zul3hl0.rl Soares testis NHT Homo sapiens cDNA clone
  • AA406076 (zu67c02.sl Soares testis NHT Homo sapiens cDNA clone 743042 3' similar to TR:G475021 G475021 SP32 PRECURSOR), AA424694 (zul3hl0.sl Soares testis NHT Homo sapiens cDNA clone 731779 3' similar to TR G475021 G475021 SP32 PRECURSOR; contains element TARl repetitive element), D16200 (Pig mRNA for s ⁇ 32, partial sequence), D16203 (Guinea pig mRNA for sp32, complete eds), and D 17573 (Mouse mRNA for proacrosin-binding protein (sp32), complete eds).
  • ga63_6 The predicted amino acid sequence disclosed herein for ga63_6 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted ga63_6 protein demonstrated at least some similarity to sequences identified as D 16200 (sp32 precursor
  • sp32 proacrosin-binding protein [Mus musculus]
  • the sp32 protein is found in the acrosomal vescicle of sperm, which is involved in egg-sperm fusion in fertilization. This protein is initially synthesized as a 61 -kDa precursor protein with a putative signal peptide at the amino terminus. The carboxyl-terminal half of the precursor molecule corresponds to the mature sp32 protein. Thus, sp32 is produced by post-translational modification of the precursor.
  • binding of sp32 to proacrosin may be involved in packaging the acrosin zymogen into the acrosomal matrix.
  • ga63_6 proteins and each similar protein or peptide may share at least some activity.
  • gm335 4" A polynucleotide of the present invention has been identified as clone "gm335_4"_ gm335_4 was isolated from a human adult uterus 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.
  • gm335_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gm335_4 protein").
  • nucleotide sequence of gm335_4 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 gm335_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14.
  • 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 gm335_4 should be approximately 800 bp.
  • the nucleotide sequence disclosed herein for gm335_4 was searched against the
  • gm335_4 demonstrated at least some similarity with sequences identified as AA055367 (zf20b05.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 377457 5'), AC002389 (Human DNA from chromosome 19 specific cosmid R28461, genomic sequence, complete sequence), W08522 (mb46hl0.rl Soares mouse p3NMF19.5
  • hy370_9 A polynucleotide of the present invention has been identified as clone "hy370_9".
  • hy370_9 was isolated from a human adult trachea 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.
  • hy370_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hy370_9 protein").
  • nucleotide sequence of hy370_9 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 hy370_9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16. 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 hy370_9 should be approximately 1200 bp.
  • hy370_9 The nucleotide sequence disclosed herein for hy370_9 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hy370_9 demonstrated at least some similarity with the sequence identified as AA763313 (vv89h07.rl Stratagene mouse skin (#937313) Mus musculus cDNA clone 1229629 5'). Based upon sequence similarity, hy370_9 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 hy370_9 protein sequence centered around amino acid 140 of SEQ ID NO:16.
  • a polynucleotide of the present invention has been identified as clone "ie47_4".
  • ie47_4 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.
  • ie47_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ie47_4 protein").
  • the nucleotide sequence of ie47_4 as presently determined is reported in SEQ ID NO: 17.
  • amino acids 17 to 29 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 30, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ie47_4 should be approximately 2300 bp.
  • ie47_4 demonstrated at least some similarity with sequences identified as AA071953 (mfl7h08.rl Life Tech mouse brain Mus musculus cDNA clone 405375 5' similar to TR G304421 G304421 SILENCER ELEMENT), AA207250 (zq82d05.sl Stratagene hNT neuron (#937233) Homo sapiens cDNA clone 648105 3' similar to TR G304421 G304421 SILENCER ELEMENT), L14938 (Chicken SCG10 protein mRNA, complete eds), L20260 (Mouse SCG10 gene sequence), R49053 (yg58c05.sl Homo sapiens cDNA clone 37017 3'), S82024 (S
  • SCG10 protein is considered to be a membrane-bound protein present in neural growth cones and developing neurons (Maucuer et al, 1993, /. Biol. Chem. 268: 16420-16429; Stein et al, 1988, Neuron 1:463-476; which are incorporated by reference herein). Based upon sequence similarity, ie47_4 proteins and each similar protein or peptide may share at least some activity. Clone "s!95 10"
  • a polynucleotide of the present invention has been identified as clone "sl95_10".
  • sl95_10 was isolated from a human adult neural tissue 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.
  • sl95_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "sl95_10 protein").
  • nucleotide sequence of sl95_10 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 sl95_10 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 35 to 47 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 48, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone sl95_10 should be approximately 3500 bp.
  • sl95_10 demonstrated at least some similarity with sequences identified as AA 113800 (zn65b05.sl Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone 563025 3' similar to TR:G600018 G600018 SSM4P), AA114062 (zn65b05.rl Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone 563025 5'), AA280316 (ztl0f06.sl Soares NbHTGBC Homo sapiens cDNA clone 712739 3'), AF009301 (Homo sapiens TEB4 protein mRNA, complete eds), N70344 (za ⁇ OflO.sl Homo sapiens cDNA
  • sl95_10 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts eleven additional potential transmembrane domains within the sl95_10 protein sequence, centered around amino acids 130, 170, 210, 260, 320, 470, 520, 560, 600, 650, and 690 of SEQ ID NO:20, respectively.
  • the nucleotide sequence of sl95_10 indicates that it may contain a simple GAA repeat region.
  • ie47_4 / and sl95_10 were deposited on June 19, 1997 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an original deposit under the Budapest Treaty and were given the accession number ATCC 98468, 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.
  • 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 Figures 1A and IB, respectively.
  • 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. 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.
  • 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 an oligonucleotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
  • the design of the oligonucleotide probe should preferably follow these parameters:
  • 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 Nad/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).
  • 6X SSC 20X stock is 175.3 g Nad/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH
  • SDS 100 ⁇ g/ml of yeast RNA
  • 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.
  • 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 immunoglobulm.
  • linker For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulm 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(s) 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(s) of the mature form(s) 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-
  • 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.
  • 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).
  • 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.
  • 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 homologues 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.
  • polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Species homologues 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 homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canis familiar is, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'
  • allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
  • allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides that hybridize 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 polynucleotides and identifying the region or regions of optimal sequence complementarity
  • SSPE 0 15M NaCl, lOmM NaH 2 P0 4 and 1 25mM EDTA, pH 74
  • SSC 0 15M NaCl and 15mM sodium citrate
  • T m melting temperature
  • each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide /expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
  • Ovary (CHO) cells human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays 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, those described in Gyuris et al, 1993, Cell 75: 791-803 and in Rossi et al, 1997, Proc. Natl. Acad. Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) 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. Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
  • 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.
  • 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.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al, Proc. Natl. Acad. Sci. U.S.A.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SOD severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, 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
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.
  • Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MR /lpr pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function preferably a B lymphocyte antigen function
  • 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.
  • the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of ⁇ e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • 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.
  • 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.
  • 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.
  • 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 transplant
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • 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, 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.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • 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.
  • 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).
  • FSH follicle stimulating hormone
  • 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.
  • 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 in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
  • the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule.
  • the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes.
  • Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth.
  • reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
  • Fragments of proteins of the present invention with cadherin activity can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • a protein of the invention may exhibit other anti- tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulm 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.
  • 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.
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • the addition of other known growth factors, such as IGF I (insulin like growth factor I) may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • CTCTCTGGAT TCAATCAAGG CTGATGGGAC CTCTCTGGAC TTCAGCACTT ACCGCAGTAG 180
  • AGTCTTCACT CACCAAGGAA AAGTGGCTCT GTATGGCAAG CTGGTGCAGT CAGCTCAGAA 660 TGAGAGGGAG AAACTTCAAA TAAAGATAGA TGAGATGGAT AAAATACTTA AGAAGATCGA 720
  • AACTGAAGAG GAGGAGCTTC AAAGAAATCT CTTAGAACTG GAGGTACCAA AAGAGCAGAC 900
  • CTCTAGCTCC GCAGCATTTG CAAAGTTTGA AATAACTTTG TTTCTCTCAG CCTATTATCC 1440 ATCTGTACCA TTACCTTCCA CCATTCAGAA TCACGTTGGG AACACTAGCC AAGATGATAT 1500
  • Lys lie Lys Ser Cys Phe Thr Lys Met Thr Lys Val Phe Thr His Gin 130 135 140
  • Lys lie Asp Asn Cys Leu Thr Glu Met Glu Thr Glu Thr Lys Asn Leu 180 185 190
  • AGCCATCAAC AACTCAGTGG GAAGTCCTTC TTCCATTAAG CTGGAGAACA AAGGGGACCT 480
  • GACTGTCCGC CAGCTTCTGG GGGAGCTCTA CGTGGACAAA GAGTATTTGG AGAAGCTCCT 660
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 4 :
  • Cys Lys Gly lie Leu Gin Lys Ala Glu Thr Leu Tyr Thr Met Gly Asp 85 90 95
  • Asp Leu lie Lys Gly Thr Met Lys Gly Gly Leu Thr Val Glu Asp Leu 210 215 220 lie Met Thr Gly lie Asn Tyr Leu Asp Thr His Ser Asn Phe Trp Arg 225 230 235 240
  • Gin Gin Lys Pro lie Tyr Ala Arg Glu Arg Asp Arg Lys Leu Met Gin 245 250 255
  • Glu Lys lie Pro Leu Ala Lys Thr Thr Leu Glu Lys Thr Trp Leu Phe 385 390 395 400
  • GTGTCCCCTC CTCCCTCTGC CCCCAGTGTT TCTTCTGATT TTTTTTTCCC CTTTCCCTCC 600 CTCCCTCTCC GCATTCTTCC CTTGGTTCAG CACAGGTAAA ACGGTTCCCC TCCCTCCCTG 660 CCTTCATGGA TCACCAGCTC ACGTCATGTT GCCTTCTCTT TTCTTTGTGT GTGTGTTTAT 720
  • GGAGCAGTCC AGCTTATGGG CCTGCTTCGA ACTTTAGTTG ACCCAGAGAA CATGCTAGCC 120
  • GAGCTCTCCA GCTTGGACCA
  • CACATTGCCC TTTTCTCAGG GAAGGAAATG GAAACAAAAA 1500
  • CAGTGGAATC ACGCATGCCG GAAGGGAAGT GGAGAAGGTT TTCAACGGAC TTAGCAACAT 240 GGGGAGCCAC ACCGGCAAGG AGTTGGACAA AGGCGTCCAG GGGCTCAACC ACGGCATGGA 300
  • AACCGCCGCC AGCACCATGG CCAGCACCAT TTCCGCCTAC AAGGAGAAGA TGAAGGAGCT 120 GTCGGTGCTG TCGCTCATCT GCTCCTGCTT CTACACACAG CCGCACCCCA ATACCGTCTA 180 CCAGTACGGG GACATGGAGG TGAAGCAGCT GGACAAGCGG GCCTCAGGCC AGAGCTTCGA 240
  • CTTCCAGAGC CACACCCAGG GACCACGGGA GGATCCTGAC CCCTGCAGGG CTCAGGGGTC 1980
  • TGTGTGTACC TTATGTCATA GCTTCTGGTG TTGTTCCTTT ACTAGGTGTT ACTGCGGAAA 2580 TGCAAAACTT AGTCCATCGG CGGATTTATC CATTTTTACT GATGGTCGTG GTATTGATGG 2640 CAATTTTGTC CTTCCAAGTC CGCCAGTTTA AGCGCCTTTA TGAACATATT AAAAATGACA 2700

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,XXX (converted to a provisional application from non-provisional application Ser. No. 08/878,715), filed June 19, 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 185 to nucleotide 1600;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1403 to nucleotide 1600; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:l from nucleotide 1 to nucleotide 850;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468; (h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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 185 to nucleotide 1600; the nucleotide sequence of SEQ ID NO:l from nucleotide 1403 to nucleotide 1600; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 850; the nucleotide sequence of the full-length protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:2, or 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 231 to amino acid 240 of SEQ ID NO:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO.l.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222; (c) fragments of the amino acid sequence of SEQ ID NO:2 comprising eight consecutive amino acids of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:2, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2having biological activity, the fragment comprising the amino acid sequence from amino acid 231 to amino acid 240 of SEQ ID NO:2.
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 47 to nucleotide 2065; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 1086 to nucleotide 1848;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dx290_l deposited under accession number ATCC 98468; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dx290_l deposited under accession number ATCC 98468; (g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 47 to nucleotide 2065; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1086 to nucleotide 1848; the nucleotide sequence of the full-length protein coding sequence of clone dx290_l deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone dx290_l deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468. 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 312 to amino acid 600. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or 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 331 to amino acid 340 of SEQ ID NO:4.
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 312 to amino acid 600;
(c) fragments of the amino acid sequence of SEQ ID NO:4 comprising eight consecutive amino acids of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468; 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 312 to amino acid 600. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4having biological activity, the fragment comprising the amino acid sequence from amino acid 331 to amino acid 340 of SEQ ID NO:4.
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 107 to nucleotide 724;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 218 to nucleotide 724;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 536 to nucleotide 866;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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 107 to nucleotide 724; the nucleotide sequence of SEQ ID NO:5 from nucleotide 218 to nucleotide 724; the nucleotide sequence of SEQ ID NO:5 from nucleotide 536 to nucleotide 866; the nucleotide sequence of the full-length protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468. 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 6 to amino acid 92. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:6, or 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 97 to amino acid 106 of SEQ ID NO:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:5.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6; (b) the amino acid sequence of SEQ ID NO:6 from amino acid 6 to amino acid 92;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising eight consecutive amino acids of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468; 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 6 to amino acid 92. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6having biological activity, the fragment comprising the amino acid sequence from amino acid 97 to amino acid 106 of SEQ ID NO:6.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleotide 31 to nucleotide 1230;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 289 to nucleotide 1230;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 344 to nucleotide 1119;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone er471_7 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone er471_7 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:7 from nucleotide 31 to nucleotide 1230; the nucleotide sequence of SEQ ID NO:7 from nucleotide 289 to nucleotide 1230; the nucleotide sequence of SEQ ID NO:7 from nucleotide 344 to nucleotide 1119; the nucleotide sequence of the full-length protein coding sequence of clone er471_7 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone er471_7 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid
363. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:8, or 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 195 to amino acid 204 of SEQ ID NO:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid 363;
(c) fragments of the amino acid sequence of SEQ ID NO:8 comprising eight consecutive amino acids of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid 363. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8having biological activity, the fragment comprising the amino acid sequence from amino acid 195 to amino acid 204 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 62 to nucleotide 322;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 571 to nucleotide 878;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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 62 to nucleotide 322; the nucleotide sequence of SEQ ID NO:9 from nucleotide 571 to nucleotide 878; the nucleotide sequence of the full-length protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or 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 38 to amino acid 47 of SEQ
ID NO:10.
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 eight consecutive amino acids of SEQ ID NO:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or 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 38 to amino acid 47 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 43 to nucleotide 1671;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 112 to nucleotide 1671;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 224 to nucleotide 679;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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 43 to nucleotide 1671; the nucleotide sequence of SEQ ID NO:ll from nucleotide 112 to nucleotide 1671; the nucleotide sequence of SEQ ID NO:ll from nucleotide 224 to nucleotide 679; the nucleotide sequence of the full-length protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468. 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 62 to amino acid 212. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:12, or 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 266 to amino acid 275 of SEQ ID NO:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NOill.
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 62 to amino acid 212;
(c) fragments of the amino acid sequence of SEQ ID NO:12 comprising eight consecutive amino acids of SEQ ID NO:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468; 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 62 to amino acid 212. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:12, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12having biological activity, the fragment comprising the amino acid sequence from amino acid 266 to amino acid 275 of SEQ ID NO:12.
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 17 to nucleotide 523;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 77 to nucleotide 523;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 392;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:14;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:13 from nucleotide 17 to nucleotide 523; the nucleotide sequence of SEQ ID NO:13 from nucleotide 77 to nucleotide 523; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 392; the nucleotide sequence of the full-length protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468. 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
125. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 14, or 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 79 to amino acid 88 of SEQ ID NO:14.
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 125;
(c) fragments of the amino acid sequence of SEQ ID NO:14 comprising eight consecutive amino acids of SEQ ID NO:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468; 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 125. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 14, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14having biological activity, the fragment comprising the amino acid sequence from amino acid 79 to amino acid 88 of SEQ ID NO:14.
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 2 to nucleotide 991;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 62 to nucleotide 991;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 2 to nucleotide 504;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:16;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:15 from nucleotide 2 to nucleotide 991; the nucleotide sequence of SEQ ID NO:15 from nucleotide 62 to nucleotide 991; the nucleotide sequence of SEQ ID NO:15 from nucleotide 2 to nucleotide 504; the nucleotide sequence of the full-length protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468. 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
167. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:16, or 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 160 to amino acid 169 of SEQ ID NO:16.
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 167;
(c) fragments of the amino acid sequence of SEQ ID NO:16 comprising eight consecutive amino acids of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468; 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 167. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO: 16, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16having biological activity, the fragment comprising the amino acid sequence from amino acid 160 to amino acid 169 of SEQ ID NO:16. 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 77 to nucleotide 616;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 164 to nucleotide 616;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 1 to nucleotide 415; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468; (g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468; (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 eight consecutive amino acids 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 that hybridizes 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 77 to nucleotide 616; the nucleotide sequence of SEQ ID NO:17 from nucleotide 164 to nucleotide 616; the nucleotide sequence of SEQ ID NO:17 from nucleotide 1 to nucleotide 415; the nucleotide sequence of the full-length protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468. 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 113. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:18, or 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 85 to amino acid 94 of SEQ ID NO:18. 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 113;
(c) fragments of the amino acid sequence of SEQ ID NO:18 comprising eight consecutive amino acids of SEQ ID NO:18; and (d) the amino acid sequence encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468; 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 113. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:18, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18having biological activity, the fragment comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:18.
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 564 to nucleotide 2813;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 705 to nucleotide 2813; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 793 to nucleotide 1628;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468; (h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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 564 to nucleotide 2813; the nucleotide sequence of SEQ ID NO:19 from nucleotide 705 to nucleotide 2813; the nucleotide sequence of SEQ ID NO:19 from nucleotide 793 to nucleotide 1628; the nucleotide sequence of the full-length protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468; or the nucleotide sequence of a mature protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468. 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 78 to amino acid
355. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or 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 370 to amino acid 379 of SEQ ID NO:20.
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 78 to amino acid 355;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising eight consecutive amino acids of SEQ ID NO:20; and (d) the amino acid sequence encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468; 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 78 to amino acid 355. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20having biological activity, the fragment comprising the amino acid sequence from amino acid 370 to amino acid 379 of SEQ ID NO:20.
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.
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 forms) 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 "dol5 4"
A polynucleotide of the present invention has been identified as clone "dol5_4"_ dol5_4 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. dol5_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dol5_4 protein").
The nucleotide sequence of dol5_4 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 dol5_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Amino acids 394 to 406 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 407, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone dol5_4 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for dol5_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. dol5_4 demonstrated at least some similarity with sequences identified as AA 113909 (zm80f 12.r 1 Stratagene neuroepithelium (#937231 ) Homo sapiens cDNA clone 531983 5'), AA189888 (mu55h06.rl Soares mouse lymph node NbMLN Mus musculus cDNA clone 643355 5'), and U52052 (Human S6 A-8 mRNA expressed in chromosome 6-suppressed melanoma cells). Based upon sequence similarity, dol5_4 proteins and each similar protein or peptide may share at least some activity.
Clone "dx290 1"
A polynucleotide of the present invention has been identified as clone "dx290_l". dx290_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. dx290_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dx290_l protein").
The nucleotide sequence of dx290_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dx290_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone dx290_l should be approximately 2300 bp. The nucleotide sequence disclosed herein for dx290_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. dx290_l demonstrated at least some similarity with the sequence identified as AA064383 (ml47h02.rl Stratagene mouse testis (#937308) Mus musculus cDNA clone 515187 5'). Based upon sequence similarity, dx290_l proteins and each similar protein or peptide may share at least some activity.
Clone "ek390 4" A polynucleotide of the present invention has been identified as clone "ek390_4". ek390_4 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. ek390_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ek390_4 protein").
The nucleotide sequence of ek390_4 as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ek390_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino acids 25 to 37 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 38, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ek390_4 should be approximately 1000 bp. The nucleotide sequence disclosed herein for ek390_4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ek390_4 demonstrated at least some similarity with sequences identified as AA075783 (zm89h02.rl Stratagene ovarian cancer (#937219) Homo sapiens cDNA clone 545139 5'), AA427538 (zw32g04.rl Soares ovary tumor NbHOT Homo sapiens cDNA clone 771030 5'), AA427539 (zw32g04.sl Soares ovary tumor NbHOT Homo sapiens cDNA clone 771030 3'), AA453353 (zx47a06.rl Soares testis NHT Homo sapiens cDNA clone 795346 5'), C20637 (HUMGS0004639, Human Gene Signature, 3'-directed cDNA sequence), R74326 (yl01c07.sl Homo sapiens cDNA clone 156972 3'), R74420 (yl01c07.rl Homo sapiens cDNA clone 156972 5'), T22914 (Human gene signature), U41197 (Human [TTTCj lO short tandem repeat polymorphism UM65,
D17S 1340), and X58237 (Human mRNA for anti-lectin antibody epitope (clone p36/8-6)). Based upon sequence similarity, ek390_4 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the ek390_4 protein sequence centered around amino acid 160 of SEQ ID NO:6. The nucleotide sequence of ek390_4 indicates that it may contain GGGA repeat sequences.
Clone "er471 7"
A polynucleotide of the present invention has been identified as clone "er471_7". er471_7 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. er471_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "er471_7 protein").
The nucleotide sequence of er471_7 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 er471_7 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8. Amino acids 74 to 86 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 87, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone er471_7 should be approximately 2250 bp.
The nucleotide sequence disclosed herein for er471_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. er471_7 demonstrated at least some similarity with sequences identified as AA039137 (mi98h06.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone 474683 5'), AA066962 (mm38g05.rl Stratagene mouse melanoma
(#937312) Mus musculus cDNA clone 523832 5'), AA189170 (zq47h05.sl Stratagene hNT neuron (#937233) Homo sapiens cDNA clone 632889 3'), AA609188 (afl2cl0.sl Soares testis NHT Homo sapiens cDNA clone 1031442 3'), and W07704 (zb02e02.rl Soares fetal lung NbHL19W Homo sapiens cDNA clone 300890 5' similar to SW:YN66_YEAST P40164 HYPOTHETICAL 98.1 KD PROTEIN IN SPX19-GCR2
INTERGENIC REGION). The predicted amino acid sequence disclosed herein for er471_7 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted er471_7 protein demonstrated at least some similarity to sequences identified as AFO 16448 (Cosmid F41E6 [Caenorhabditis elegans]) and L08407 (collagen type XVII [Mus musculus]). Based upon sequence similarity, er471_7 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts three potential transmembrane domains within the er471 7 protein sequence, centered around amino acids 40, 80, and 110 of SEQ ID NO:8, respectively.
Clone "fs40 3"
A polynucleotide of the present invention has been identified as clone "fs40_3". fs40_3 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. fs40_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fs40_3 protein").
The nucleotide sequence of fs40_3 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 fs40_3 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 fs40_3 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for fs40_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. fs40_3 demonstrated at least some similarity with sequences identified as AA411142 (zt37g01.rl Soares ovary tumor NbHOT Homo sapiens cDNA clone 724560 5'), AA412527 (zul2a03.sl Soares testis NHT Homo sapiens cDNA clone 731596 3'), AA565855 (nj32d09.sl NCI_CGAP_AA1 Homo sapiens cDNA clone IMAGE:994193), HI 7042 (ym39fl2.sl Homo sapiens cDNA clone 50584 3'), and T33280 (EST57284 Homo sapiens cDNA 3' end similar to None). Based upon sequence similarity, fs40_3 proteins and each similar protein or peptide may share at least some activity. The
TopPredll computer program predicts a potential transmembrane domain within the fs40_3 protein sequence at the C-terminus of SEQ ID NO:10. Clone "ga63 6"
A polynucleotide of the present invention has been identified as clone "ga63_6". ga63_6 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. ga63_6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ga63_6 protein").
The nucleotide sequence of ga63_6 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 ga63_6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ga63_6 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for ga63_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ga63_6 demonstrated at least some similarity with sequences identified as AA405433 (zul3hl0.rl Soares testis NHT Homo sapiens cDNA clone
731779 5'similar to TR G474970 G474970 SP32 PRECURSOR), AA406076 (zu67c02.sl Soares testis NHT Homo sapiens cDNA clone 743042 3' similar to TR:G475021 G475021 SP32 PRECURSOR), AA424694 (zul3hl0.sl Soares testis NHT Homo sapiens cDNA clone 731779 3' similar to TR G475021 G475021 SP32 PRECURSOR; contains element TARl repetitive element), D16200 (Pig mRNA for sρ32, partial sequence), D16203 (Guinea pig mRNA for sp32, complete eds), and D 17573 (Mouse mRNA for proacrosin-binding protein (sp32), complete eds). The predicted amino acid sequence disclosed herein for ga63_6 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted ga63_6 protein demonstrated at least some similarity to sequences identified as D 16200 (sp32 precursor
[Sus scrofa]), and D17574 (alternative splicing product for proacrosin-binding protein (sp32) [Mus musculus]). The sp32 protein is found in the acrosomal vescicle of sperm, which is involved in egg-sperm fusion in fertilization. This protein is initially synthesized as a 61 -kDa precursor protein with a putative signal peptide at the amino terminus. The carboxyl-terminal half of the precursor molecule corresponds to the mature sp32 protein. Thus, sp32 is produced by post-translational modification of the precursor. The binding of sp32 to proacrosin may be involved in packaging the acrosin zymogen into the acrosomal matrix. (Baba et al., 1994, 7. Biol. Chem. 269 (13): 10133-10140, which is incorporated by reference herein). Based upon sequence similarity, ga63_6 proteins and each similar protein or peptide may share at least some activity.
Clone "gm335 4" A polynucleotide of the present invention has been identified as clone "gm335_4"_ gm335_4 was isolated from a human adult uterus 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. gm335_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gm335_4 protein").
The nucleotide sequence of gm335_4 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 gm335_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14. 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 gm335_4 should be approximately 800 bp. The nucleotide sequence disclosed herein for gm335_4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gm335_4 demonstrated at least some similarity with sequences identified as AA055367 (zf20b05.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 377457 5'), AC002389 (Human DNA from chromosome 19 specific cosmid R28461, genomic sequence, complete sequence), W08522 (mb46hl0.rl Soares mouse p3NMF19.5
Mus musculus cDNA clone 332515 5'), and X93916 (S.scrofa mRNA (clone VIB11 ; expressed sequence tag)). Based upon sequence similarity, gm335_4 proteins and each similar protein or peptide may share at least some activity. Clone "hv370 9"
A polynucleotide of the present invention has been identified as clone "hy370_9". hy370_9 was isolated from a human adult trachea 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. hy370_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hy370_9 protein").
The nucleotide sequence of hy370_9 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 hy370_9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16. 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 hy370_9 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for hy370_9 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hy370_9 demonstrated at least some similarity with the sequence identified as AA763313 (vv89h07.rl Stratagene mouse skin (#937313) Mus musculus cDNA clone 1229629 5'). Based upon sequence similarity, hy370_9 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 hy370_9 protein sequence centered around amino acid 140 of SEQ ID NO:16.
Clone "ie47 4"
A polynucleotide of the present invention has been identified as clone "ie47_4". ie47_4 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. ie47_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ie47_4 protein"). The nucleotide sequence of ie47_4 as presently determined is reported in SEQ ID NO: 17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ie47_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18. Amino acids 17 to 29 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 30, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ie47_4 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for ie47_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ie47_4 demonstrated at least some similarity with sequences identified as AA071953 (mfl7h08.rl Life Tech mouse brain Mus musculus cDNA clone 405375 5' similar to TR G304421 G304421 SILENCER ELEMENT), AA207250 (zq82d05.sl Stratagene hNT neuron (#937233) Homo sapiens cDNA clone 648105 3' similar to TR G304421 G304421 SILENCER ELEMENT), L14938 (Chicken SCG10 protein mRNA, complete eds), L20260 (Mouse SCG10 gene sequence), R49053 (yg58c05.sl Homo sapiens cDNA clone 37017 3'), S82024 (SCG10 neuron-specific growth-associated protein/stathmin homolog [human, embryo, mRNA]), T25428 (Human gene signature HUMGS07594, T25428 standard; cDNA to mRNA), W54204 (md04al2.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone 367390 5' similar to SW:SCGB_XENLA Q09002 SCG10 PROTEIN HOMOLOG A), X71433 (X. laevis SCG10 mRNA), and Z99916 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 221G9; HTGS phase 1). The predicted amino acid sequence disclosed herein for ie47_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted ie47_4 protein demonstrated at least some similarity to sequences identified as L14938 (SCG10 protein [Gallus gallus]) and S82024 (SCG 10 neuron-specific growth-associated protein/stathmin homolog [human, embryo, Peptide] [Homo sapiens]). SCG10 protein is considered to be a membrane-bound protein present in neural growth cones and developing neurons (Maucuer et al, 1993, /. Biol. Chem. 268: 16420-16429; Stein et al, 1988, Neuron 1:463-476; which are incorporated by reference herein). Based upon sequence similarity, ie47_4 proteins and each similar protein or peptide may share at least some activity. Clone "s!95 10"
A polynucleotide of the present invention has been identified as clone "sl95_10". sl95_10 was isolated from a human adult neural tissue 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. sl95_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "sl95_10 protein").
The nucleotide sequence of sl95_10 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 sl95_10 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 35 to 47 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 48, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone sl95_10 should be approximately 3500 bp.
The nucleotide sequence disclosed herein for sl95_10 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. sl95_10 demonstrated at least some similarity with sequences identified as AA 113800 (zn65b05.sl Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone 563025 3' similar to TR:G600018 G600018 SSM4P), AA114062 (zn65b05.rl Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone 563025 5'), AA280316 (ztl0f06.sl Soares NbHTGBC Homo sapiens cDNA clone 712739 3'), AF009301 (Homo sapiens TEB4 protein mRNA, complete eds), N70344 (zaόOflO.sl Homo sapiens cDNA clone 296971 3'), R60474 (yhl3g07.rl Homo sapiens cDNA clone 43058 5'), and T26266
(standard; cDNA to mRNA; 148 BP, Human gene signature HUMGS08505). The predicted amino acid sequence disclosed herein for sl95_10 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted sl95_10 protein demonstrated at least some similarity to sequences identified as AF009301 (TEB4 protein [Homo sapiens]), X76715 (SSM4 gene product
[Saccharomyces cerevisiae]), Z46861 (Ssm4p [Saccharomyces cerevisiae]), and Z47047 (Ssm4p [Saccharomyces cerevisiae]). Based upon sequence similarity, sl95_10 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts eleven additional potential transmembrane domains within the sl95_10 protein sequence, centered around amino acids 130, 170, 210, 260, 320, 470, 520, 560, 600, 650, and 690 of SEQ ID NO:20, respectively. The nucleotide sequence of sl95_10 indicates that it may contain a simple GAA repeat region.
Deposit of Clones
Clones dol5_4, dx290_l, ek390_4, er471_7, fs40_3, ga63_6, gm335_4, hy370_9. ie47_4/ and sl95_10 were deposited on June 19, 1997 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an original deposit under the Budapest Treaty and were given the accession number ATCC 98468, 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), and the term of the deposit will comply with 37 C.F.R. § 1.806. 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 Figures 1A and IB, respectively. 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 an oligonucleotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence dol5_4 SEQ ID NO:21 dx290_l SEQ ID NO:22 ek390_4 SEQ ID NO:23 er471_7 SEQ ID NO:24 fs40_3 SEQ ID NO:25 ga63_6 SEQ ID O:26 gm335_4 SEQ ID NO:27 hy370_9 SEQ ID NO:28 ie47_4 SEQ ID NO:29 sl95_10 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-O-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any; (b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each
A or T and 4 degrees for each G or C). The oligonucleotide should preferably be labeled with g-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 Nad/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 immunoglobulm. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulm 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(s) 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(s) of the mature form(s) 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 BI, 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 homologues 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. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Species homologues 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 homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canis familiar is, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al, 1993, Nature
Genetics 3:103-112; Johansson et al, 1995, Genomics 25: 682-690; Lyons et al, 1997, Nature Genetics 15: 47-56; O'Brien et al, 1997, Trends in Genetics 13(10): 393-399; Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein).
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize 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 imgf000042_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 polynucleotides and identifying the region or regions of optimal sequence complementarity
+ SSPE (lxSSPE is 0 15M NaCl, lOmM NaH2P04 and 1 25mM EDTA, pH 74) 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 + 166(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 pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention. USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays 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, those described in Gyuris et al, 1993, Cell 75: 791-803 and in Rossi et al, 1997, Proc. Natl. Acad. Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation /Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J.
Immunol. 152: 1756-1761, 1994. Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al, Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, 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 receptoπligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MR /lpr pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells {e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of {e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen {e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W 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 immunoglobulm 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, HJ. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers. A protein of the present invention, which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention. Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity. A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/ 16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α 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 in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin /Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities. The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti- tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and /or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulm 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-α and TGF-β), and insulin-like growth factor (IGF). The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Treacy, Maurice
Spaulding, Vikki Agostino, Michael J. Howes, Steven H. Fechtel, Kim
(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: 1748 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
GTTTAGTGAT ACGACACAAG ATCGGGAGAT TTTTGATCAC CATACTGAAG AGGATATAGA 60 TAAAAGTGCT AACAGTGTAT TGATAAAAAA CCTGAGCAGG ACCCCATCTA GTTGCAGCAG 120
CTCTCTGGAT TCAATCAAGG CTGATGGGAC CTCTCTGGAC TTCAGCACTT ACCGCAGTAG 180
TCAAATGGAA TCACAGTTTC TCAGAGATAC TATTTGTGAA GAGAGCTTGA GGGAGAAACT 240
CCAAGATGGG AGAATAACAA TAAGGGAGTT CTTTATACTT CTCCAGGTCC ACATCTTGAT 300
ACAGAAACCC CGACAGAGCA ATCTCCCAGG CAATTTTACT GTAAACACAC CACCTACTCC 360 AGAAGACCTG ATGTTAAGTC AATATGTTTA CCGACCCAAG ATACAGATTT ATAGAGAAGA 420
TTGTGAGGCT CGTCGCCAAA AGATTGAAGA ATTAAAGCTT TCTGCATCGA ACCAAGATAA 480
GCTGTTGGTT GATATAAATA AGAACCTGTG GGAAAAAATG AGACACTGCT CTGACAAAGA 540
GCTGAAGGCC TTTGGAATTT ATCTTAACAA AATAAAGTCA TGTTTTACCA AGATGACTAA 600
AGTCTTCACT CACCAAGGAA AAGTGGCTCT GTATGGCAAG CTGGTGCAGT CAGCTCAGAA 660 TGAGAGGGAG AAACTTCAAA TAAAGATAGA TGAGATGGAT AAAATACTTA AGAAGATCGA 720
TAACTGCCTC ACTGAGATGG AAACAGAAAC TAAGAATTTG GAGGATGAAG AGAAAAACAA 780
TCCTGTGGAA GAATGGGATT CTGAAATGAG AGCTGCAGAA AAAGAATTGG AACAGCTGAA 840
AACTGAAGAG GAGGAGCTTC AAAGAAATCT CTTAGAACTG GAGGTACCAA AAGAGCAGAC 900
CCTTGCTCAA ATAGACTTTA TGCAAAAACA AAGAAATAGA ACTGAAGAGC TACTGGATCA 960 GTTGAGCTTG TCTGAGTGGG ATGTCGTTGA GTGGAGTGAT GATCAAGCTG TATTCACCTT 1020
TGTTTATGAC ACGATACAAC TCACCATCAC CTTTGAAGAG TCAGTTGTTG GTTTCCCTTT 1080
CCTGGACAAG CGTTATAGGA AGATTGTTGA TGTCAATTTT CAATCTCTGT TAGATGAGGA 1140
TCAAGCTCCT CCTTCCTCCC TTTTAGTTCA TAAGCTTATT TTCCAGTACG TTGAAGAAAA 1200
GGAATCCTGG AAGAAGACAT GTACAACCCA GCATCAGTTA CCCAAGATGC TTGAAGAATT 1260 CTCACTGGTA GTGCACCATT GCAGACTCCT TGGAGAGGAG ATTGAGTATT TAAAGAGATG 1320
GGGACCAAAT TATAACCTAA TGAACATAGA TATTAATAAT AATGAATTGA GACTTTTATT 1380
CTCTAGCTCC GCAGCATTTG CAAAGTTTGA AATAACTTTG TTTCTCTCAG CCTATTATCC 1440 ATCTGTACCA TTACCTTCCA CCATTCAGAA TCACGTTGGG AACACTAGCC AAGATGATAT 1500
TGCTACCATT CTATCTAAAG TGCCACTGGA GAACAACTAC CTGAAGAATG TAGTCAAGCA 1560
AATTTACCAA GATCTGTTTC AGGACTGCCA TTTCTACCAC TAGACCCTTG GACCACCATT 1620
GGAACAACCA AGCAGAATGT ACTTGATATT ATTTCAGGGT CCCATTGCTG TTCAGCCTTT 1680
GTTTTTACGT CATTACAAGC TGAGTAAAAT TCCTTCTGAT GATGTTATAA AAAAAAAAAA 1740
AAAAAAAA 1748 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Glu Ser Gin Phe Leu Arg Asp Thr lie Cys Glu Glu Ser Leu Arg 1 5 10 15
Glu Lys Leu Gin Asp Gly Arg lie Thr lie Arg Glu Phe Phe lie Leu 20 25 30
Leu Gin Val His lie Leu lie Gin Lys Pro Arg Gin Ser Asn Leu Pro 35 40 45
Gly Asn Phe Thr Val Asn Thr Pro Pro Thr Pro Glu Asp Leu Met Leu 50 55 60 Ser Gin Tyr Val Tyr Arg Pro Lys lie Gin lie Tyr Arg Glu Asp Cys 65 70 75 80
Glu Ala Arg Arg Gin Lys lie Glu Glu Leu Lys Leu Ser Ala Ser Asn 85 90 95
Gin Asp Lys Leu Leu Val Asp lie Asn Lys Asn Leu Trp Glu Lys Met 100 105 110
Arg His Cys Ser Asp Lys Glu Leu Lys Ala Phe Gly lie Tyr Leu Asn 115 120 125
Lys lie Lys Ser Cys Phe Thr Lys Met Thr Lys Val Phe Thr His Gin 130 135 140
Gly Lys Val Ala Leu Tyr Gly Lys Leu Val Gin Ser Ala Gin Asn Glu 145 150 155 160
Arg Glu Lys Leu Gin lie Lys lie Asp Glu Met Asp Lys lie Leu Lys 165 170 175
Lys lie Asp Asn Cys Leu Thr Glu Met Glu Thr Glu Thr Lys Asn Leu 180 185 190
Glu Asp Glu Glu Lys Asn Asn Pro Val Glu Glu Trp Asp Ser Glu Met 195 200 205
Arg Ala Ala Glu Lys Glu Leu Glu Gin Leu Lys Thr Glu Glu Glu Glu 210 215 220 Leu Gin Arg Asn Leu Leu Glu Leu Glu Val Pro Lys Glu Gin Thr Leu 225 230 235 240
Ala Gin lie Asp Phe Met Gin Lys Gin Arg Asn Arg Thr Glu Glu Leu 245 250 255
Leu Asp Gin Leu Ser Leu Ser Glu Trp Asp Val Val Glu Trp Ser Asp 260 265 270
Asp Gin Ala Val Phe Thr Phe Val Tyr Asp Thr lie Gin Leu Thr lie 275 280 285
Thr Phe Glu Glu Ser Val Val Gly Phe Pro Phe Leu Asp Lys Arg Tyr 290 295 300 Arg Lys lie Val Asp Val Asn Phe Gin Ser Leu Leu Asp Glu Asp Gin 305 310 315 320
Ala Pro Pro Ser Ser Leu Leu Val His Lys Leu lie Phe Gin Tyr Val 325 330 335
Glu Glu Lys Glu Ser Trp Lys Lys Thr Cys Thr Thr Gin His Gin Leu 340 345 350
Pro Lys Met Leu Glu Glu Phe Ser Leu Val Val His His Cys Arg Leu 355 360 365
Leu Gly Glu Glu lie Glu Tyr Leu Lys Arg Trp Gly Pro Asn Tyr Asn 370 375 380 Leu Met Asn lie Asp ie Asn Asn Asn Glu Leu Arg Leu Leu Phe Ser 385 390 395 400
Ser Ser Ala Ala Phe Ala Lys Phe Glu lie Thr Leu Phe Leu Ser Ala 405 410 415
Tyr Tyr Pro Ser Val Pro Leu Pro Ser Thr lie Gin Asn His Val Gly 420 425 430
Asn Thr Ser Gin Asp Asp lie Ala Thr lie Leu Ser Lys Val Pro Leu 435 440 445 Glu Asn Asn Tyr Leu Lys Asn Val Val Lys Gin lie Tyr Gin Asp Leu 450 455 460
Phe Gin Asp Cys His Phe Tyr His 465 470
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2298 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
CTTTTCTTTG ATTGTCTCTG CTTTAGCGTC TCTAAATCCG GTCACCATGT CGGACCCCGA 60
AGGCGAGACC TTGCGAAGCA CCTTTCCCTC TTATATGGCC GAAGGCGAGC GGCTCTACCT 120
GTGCGGGGAA TTTTCTAAAG CCGCGCAGAG CTTCAGCAAC GCTCTTTACC TTCAGGATGG 180
AGACAAGAAC TGCCTGGTTG CTCGCTCAAA GTGCTTCCTG AAGATGGGAG ACTTGGAGAG 240 ATCCCTGAAG GATGCTGARG CTTCGCTCCA GAGTGACCCA GCTTTCTGTA AGGGGATTTT 300
GCAAAAGGCT GAGACACTGT ACACCATGGG AGACTTTGAG TTTGCCTTGG TATTCTATCA 360
TCGARGCTAC AAGCTGARGC CTGATCGGGA ATTCARARTT GGCATTCAGA AAGCCCAGGA 420
AGCCATCAAC AACTCAGTGG GAAGTCCTTC TTCCATTAAG CTGGAGAACA AAGGGGACCT 480
CTCCTTCTTA AGCAAGCAGG CTGAGAATAT AAAAGCCCAG CAGAAGCCTC AGCCCATGAA 540 ACACCTCTTA CACCCCACCA AGGGAGAGCC CAAGTGGAAG GCCTCGCTCA AGAGTGAGAA 600
GACTGTCCGC CAGCTTCTGG GGGAGCTCTA CGTGGACAAA GAGTATTTGG AGAAGCTCCT 660
ATTGGATGAA GACCTGATCA AAGGCACCAT GAAGGGCGGC CTGACTGTGG AGGACCTCAT 720
CATGACGGGC ATCAACTACC TGGATACTCA CAGCAACTTC TGGAGGCAGC AGAAGCCGAT 780
CTACGCCAGG GAGCGGGACC GGAAGCTGAT GCAAGAGAAA TGGCTGCGGG ACCACAAACG 840 CCGTCCCTCA CAGACAGCCC ATTACATCCT CAAGAGCCTG GAGGACATTG ATATGTTGCT 900
CACAAGTGGC AGTGCTGAAG GGAGTCTTCA GAAAGCTGAG AAAGTGCTGA AGAAGGTACT 960
GGAATGGAAC AAGGAAGAGG TACCCAACAA GGATGAACTG GTTGGAAACT TGTATAGCTG 1020 CATAGGGAAT GCCCAGATTG AGCTGGGGCA GATGGAGGCA GCCCTGCAGA GCCACAGAAA 1080
GGACYTGGAG ATCGCCAAGG AATATGACCT TCCTGATGCA AAATCGAGAG CCCTTGACAA 1140 CATTGGCAGA GTTTTTGCCA GAGTTGGGAA ATTCCAGCAA GCCATTGACA CGTGGGAAGA 1200
AAAGATCCCT CTGGCAAAAA CCACCCTGGA GAAGACCTGG CTGTTCCACG AGATCGGCCG 1260
CTGCTACTTG GAGCTGGACC AGGCCTGGCA GGCCCAGAAT TATGGCGAGA AGTCCCAGCA 1320
GTGTGCCGAG GAGGAAGGGG ACATTGAGTG GCAACTGAAT GCCAGTGTTC TGGTGGCCCA 1380
GGCACAAGTG AAGCTGAGAG ACTTCGAGTC AGCCGTGAAC AATTTTGAGA AGGCCCTGGA 1440 GAGAGCAAAG CTTGTGCATA ACAACGAGGC GCAGCAGGCC ATCATCAGTG CCTTGGACGA 1500
TGCCAACAAG GGTATCATCA GAGAACTGAG GAAAACCAAC TACGTGGAGA ATCTCAAAGA 1560
AAAAAGCGAG GGAGAAGCTT CACTGTATGA AGATAGAATA ATAACAAGAG AGAAGGACAT 1620
GAGGAGAGTG AGAGATGAGC CCGAGAAGGT GGTGAAGCAG TGGGACCATA GTGAGGATGA 1680
GAAAGAGACA GATGAGGACG ATGAGGCTTT TGGGGAAGCT CTGCAGAGCC CAGCAAGCGG 1740 AAAGCAGAGT GTGGAAGCAG GAAAAGCCAG AAGCGATTTG GGAGCAGTTG CCAAGGGCCT 1800
GTCAGGAGAA TTAGGCACAA GATCAGGAGA AACAGGCAGG AAGCTACTAG AAGCTGGCAG 1860
AAGAGAGTCA AGAGAAATTT ATAGGAGGCC TTCGGGAGAA TTAGAGCAAA GACTCTCAGG 1920
AGAATTCAGC AGACAGGAAC CAGAAGAACT AAAGAAACTT TCAGAAGTGG GCAGAAGAGA 1980
SCCAGAAGAA YTGGGAAAAA CACAATTTGG AGAAATAGGA GAAACGAAAA AAACAGGAAA 2040 TGAGATGGAA AAGGAATATG AATGAAGCCA TCGGTAGAGA TGAGGATCAG GAAGCTGGTG 2100
TTCAGAGGGA TCATGGGATT TTATTAAACT GGATTTTCAA GCGATTTGTC TGTTATAGGA 2160
AAAATGAGGG TTTTACTTYT GCTGCTTTCC ATCACTATTT TGCCATTAAA TAGGTGTCTT 2220
TCACTCTTGC MAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2280
AAAAAAAAAA AAAAAAAA 2298 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 672 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Ser Asp Pro Glu Gly Glu Thr Leu Arg Ser Thr Phe Pro Ser Tyr 1 5 10 15
Met Ala Glu Gly Glu Arg Leu Tyr Leu Cys Gly Glu Phe Ser Lys Ala 20 25 30 Ala Gin Ser Phe Ser Asn Ala Leu Tyr Leu Gin Asp Gly Asp Lys Asn 35 40 45
Cys Leu Val Ala Arg Ser Lys Cys Phe Leu Lys Met Gly Asp Leu Glu 50 55 60
Arg Ser Leu Lys Asp Ala Glu Ala Ser Leu Gin Ser Asp Pro Ala Phe 65 70 75 80
Cys Lys Gly lie Leu Gin Lys Ala Glu Thr Leu Tyr Thr Met Gly Asp 85 90 95
Phe Glu Phe Ala Leu Val Phe Tyr His Arg Xaa Tyr Lys Leu Xaa Pro 100 105 110
Asp Arg Glu Phe Xaa Xaa Gly lie Gin Lys Ala Gin Glu Ala lie Asn 115 120 125
Asn Ser Val Gly Ser Pro Ser Ser lie Lys Leu Glu Asn Lys Gly Asp 130 135 140
Leu Ser Phe Leu Ser Lys Gin Ala Glu Asn lie Lys Ala Gin Gin Lys 145 150 155 160
Pro Gin Pro Met Lys His Leu Leu His Pro Thr Lys Gly Glu Pro Lys 165 170 175
Trp Lys Ala Ser Leu Lys Ser Glu Lys Thr Val Arg Gin Leu Leu Gly 180 185 190 Glu Leu Tyr Val Asp Lys Glu Tyr Leu Glu Lys Leu Leu Leu Asp Glu 195 200 205
Asp Leu lie Lys Gly Thr Met Lys Gly Gly Leu Thr Val Glu Asp Leu 210 215 220 lie Met Thr Gly lie Asn Tyr Leu Asp Thr His Ser Asn Phe Trp Arg 225 230 235 240
Gin Gin Lys Pro lie Tyr Ala Arg Glu Arg Asp Arg Lys Leu Met Gin 245 250 255
Glu Lys Trp Leu Arg Asp His Lys Arg Arg Pro Ser Gin Thr Ala His 260 265 270 Tyr lie Leu Lys Ser Leu Glu Asp lie Asp Met Leu Leu Thr Ser Gly 275 280 285
Ser Ala Glu Gly Ser Leu Gin Lys Ala Glu Lys Val Leu Lys Lys Val 290 295 300
Leu Glu Trp Asn Lys Glu Glu Val Pro Asn Lys Asp Glu Leu Val Gly 305 310 315 320
Asn Leu Tyr Ser Cys lie Gly Asn Ala Gin lie Glu Leu Gly Gin Met 325 330 335
Glu Ala Ala Leu Gin Ser His Arg Lys Asp Leu Glu lie Ala Lys Glu 340 345 350 Tyr Asp Leu Pro Asp Ala Lys Ser Arg Ala Leu Asp Asn lie Gly Arg 355 360 365
Val Phe Ala Arg Val Gly Lys Phe Gin Gin Ala lie Asp Thr Trp Glu 370 375 380
Glu Lys lie Pro Leu Ala Lys Thr Thr Leu Glu Lys Thr Trp Leu Phe 385 390 395 400
His Glu lie Gly Arg Cys Tyr Leu Glu Leu Asp Gin Ala Trp Gin Ala 405 410 415
Gin Asn Tyr Gly Glu Lys Ser Gin Gin Cys Ala Glu Glu Glu Gly Asp 420 425 430 He Glu Trp Gin Leu Asn Ala Ser Val Leu Val Ala Gin Ala Gin Val 435 440 445
Lys Leu Arg Asp Phe Glu Ser Ala Val Asn Asn Phe Glu Lys Ala Leu 450 455 460
Glu Arg Ala Lys Leu Val His Asn Asn Glu Ala Gin Gin Ala He He 465 470 475 480
Ser Ala Leu Asp Asp Ala Asn Lys Gly He He Arg Glu Leu Arg Lys 485 490 495
Thr Asn Tyr Val Glu Asn Leu Lys Glu Lys Ser Glu Gly Glu Ala Ser 500 505 510 Leu Tyr Glu Asp Arg He He Thr Arg Glu Lys Asp Met Arg Arg Val 515 520 525
Arg Asp Glu Pro Glu Lys Val Val Lys Gin Trp Asp His Ser Glu Asp 530 535 540
Glu Lys Glu Thr Asp Glu Asp Asp Glu Ala Phe Gly Glu Ala Leu Gin 545 550 555 560
Ser Pro Ala Ser Gly Lys Gin Ser Val Glu Ala Gly Lys Ala Arg Ser 565 570 575 Asp Leu Gly Ala Val Ala Lys Gly Leu Ser Gly Glu Leu Gly Thr Arg 580 585 590
Ser Gly Glu Thr Gly Arg Lys Leu Leu Glu Ala Gly Arg Arg Glu Ser 595 600 605
Arg Glu He Tyr Arg Arg Pro Ser Gly Glu Leu Glu Gin Arg Leu Ser 610 615 620 Gly Glu Phe Ser Arg Gin Glu Pro Glu Glu Leu Lys Lys Leu Ser Glu 625 630 635 640
Val Gly Arg Arg Xaa Pro Glu Glu Leu Gly Lys Thr Gin Phe Gly Glu 645 650 655
He Gly Glu Thr Lys Lys Thr Gly Asn Glu Met Glu Lys Glu Tyr Glu 660 665 670
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1010 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGAAGAGCCA CCATCCCTGC CCCCGTTTTC CCACCGGGGA GTCTGTACAG AGATTTTTCT 60
ACGTTTTTAT TTTTTGCCTC AGAGGGATGG GATTGGGGAG GAGGGGATGG GCAGCGGAGG 120
GTTGGGGGCA TGGTCTGCAG GCTCATCTGT GTCCGCCTTT CACTCCACTA ATGCTGTCTC 180
AGTGTTTTCT CTCTCTCTCT TTCGAGCTTG CACTCCGGTA CCCGACCCGG CGCCCTGGCC 240
CATCCCATGC CGGGGGGCCA GTGGAAAGAA GACAGGCCGT CCAGCCCGTG CCCGCCTGCG 300 GCGGGGGCAC CCAGCAAGCC CGCCCACCGC CCGCTGCCTC ACCTGCTTCG CCACAGACTC 360
TTGTTCCCAG CCCCTTGGGG CCTCCGTGTT TGGGGTGGGG GAGCTGCTTA GAGACTGTGC 420
CCGTCCTCGG CCCCCCACCC TGAAGTGCCA GCACCACCAG CACCAGATCT TCCGCCGCCA 480
CACCGCATTG AGGACACGCC GGCCGGGCCG CTTCGTCTCA AGTTGTATAA AGTTGTCTCC 540
GTGTCCCCTC CTCCCTCTGC CCCCAGTGTT TCTTCTGATT TTTTTTTCCC CTTTCCCTCC 600 CTCCCTCTCC GCATTCTTCC CTTGGTTCAG CACAGGTAAA ACGGTTCCCC TCCCTCCCTG 660 CCTTCATGGA TCACCAGCTC ACGTCATGTT GCCTTCTCTT TTCTTTGTGT GTGTGTTTAT 720
TTAAGTTATT TTTCTTCCTC CTCTCCCTTT TCTTTTTGGC CCTCCCTCCC TCCCTCTTCT 780
GCCATGTAAC TGGAGGATGT GCTATGAGTT TGCAAACAGC TGGACTGTCA GGCTGCTTTT 840
TTTTCCAGAT GTTCTTCTTC TGCTTCCCCT TCCCCTCCTC TCCCCTCCTT TTCCTTCCTT 900
CCTTCCTTTC CTTGGAGCAC TGAGCACCAT TTGGAAGCTT GAGAGAAACC AAAATTAAAG 960
AGAGAAAGAG AGAGCGTGCA CGCTCCTGCT TTGTCAAAAA AAAAAAAAAA 1010 (2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Gly Ser Gly Gly Leu Gly Ala Trp Ser Ala Gly Ser Ser Val Ser 1 5 10 15
Ala Phe His Ser Thr Asn Ala Val Ser Val Phe Ser Leu Ser Leu Phe 20 25 30
Arg Ala Cys Thr Pro Val Pro Asp Pro Ala Pro Trp Pro He Pro Cys 35 40 45
Arg Gly Ala Ser Gly Lys Lys Thr Gly Arg Pro Ala Arg Ala Arg Leu 50 55 60
Arg Arg Gly His Pro Ala Ser Pro Pro Thr Ala Arg Cys Leu Thr Cys 65 70 75 80
Phe Ala Thr Asp Ser Cys Ser Gin Pro Leu Gly Ala Ser Val Phe Gly 85 90 95
Val Gly Glu Leu Leu Arg Asp Cys Ala Arg Pro Arg Pro Pro Thr Leu 100 105 110
Lys Cys Gin His His Gin His Gin He Phe Arg Arg His Thr Ala Leu 115 120 125
Arg Thr Arg Arg Pro Gly Arg Phe Val Ser Ser Cys He Lys Leu Ser 130 135 140 Pro Cys Pro Leu Leu Pro Leu Pro Pro Val Phe Leu Leu He Phe Phe 145 150 155 160
Ser Pro Phe Pro Pro Ser Leu Ser Ala Phe Phe Pro Trp Phe Ser Thr 165 170 175
Gly Lys Thr Val Pro Leu Pro Pro Cys Leu His Gly Ser Pro Ala His 180 185 190
Val Met Leu Pro Ser Leu Phe Phe Val Cys Val Phe He 195 200 205
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2409 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
ATTTYGCTCA TCAACCTCAT TATAGAACAT ATGATTTGTG ATACAGATCC TGAACTTGGA 60
GGAGCAGTCC AGCTTATGGG CCTGCTTCGA ACTTTAGTTG ACCCAGAGAA CATGCTAGCC 120
ACTGCCMATA AAACASAAAA GACTGAATTT CTGGGTTTCT TCTACAAGCA CTGTATGCAT 180
GTTCTCWCTG CTCCTTTACT AGCAAATACA ACAGAAGACA AACCTAGTAA AGATGATTTT 240 CAGACTGCCC AACTATTGGC ACTTGTATTG GAATTGTTAA CATTTTGTGT GGAGCACCAT 300
ACCTACCACA TAAAGAACTA CATTATTAAT AAGGATATCC TCCGGAGAGT GCTAGTTCTT 360
ATGGCCTCGA AGCATGCTTT CTTGGCATTA TGTGCCCTTC GTTTTAAAAG AAAGATTATT 420
GGATTAAAAG ATGAGTTTTA CAACCGCTAC ATAATGAAAA GTTTTTTGTT TGAACCAGTA 480
GTGAAAGCAT TTCTCAACAA TGGATCCCGC TACAATCTGA TGAACTCTGC CATAATAGAG 540 ATGTTTGAAT TTATTAGAGT GGAAGATATA AAATCATTAA CTGCTCATGT AATTGAAAAT 600
TACTGGAAAG CACTGGAAGA TGTAGATTAT GTACAGACAT TTAAAGGATT AAAACTGAGA 660
TTTGAACAAC AAAGAGAAAG GCAAGATAAT CCCAAACTTG ACAGTATGCG TTCCATTTTG 720
AGGAATCACA GATATCGAAG AGATGCCAGA ACACTAGAAG ATGAAGAAGA GATGTGGTTT 780
AACACAGATG AAGATGACAT GGAAGATGGA GAAGCTGTAG TGTCTCCATC TGACAAAACT 840 AAAAATGATG ATGATATTAT GGATCCAATA AGTAAATTCA TGGAAAGGAA GAAATTAAAA 900 GAAAGTGAGG AAAAGGAAGT GCTTCTGAAA ACAAACCTTT CTGGACGGCA GAGCCCAAGT 960
TTCAAGCTTT CCCTGTCCAG TGGAACGAAG ACTAACCTCA CCAGCCAGTC ATCTACAACA 1020 AATCTGCCTG GTTCTCCGGG ATCACCTGGA TCCCCAGGAT CTCCAGGCTC TCCTGGATCC 1080
GTACCTAAAA ATACATCTCA GACGGCAGCT ATTACTACAA AGGGAGGCCT CGTGGGTCTG 1140
GTAGATTATC CTGATGATGA TGAAGATGAT GATGAGGATG AAGATAAGGA AGATACGTTA 1200
CCATTGTCAA AGAAAGCAAA ATTTGATTCA TAATAATGGC AACGGCCTAG GATCAGTACC 1260
TGTTGAAAAA AACTGGTTCT CCACCCCTCC CCCATACAAA ATCCACAAAA AAGCGCAGTG 1320 GTCTCTTGTG AATGACTGAC ACAGATCAGC CTCTTACACT TGACTTCTGC TCATCAAGTG 1380
CCAATTCAAT GGAGCAGGAG GAGGGGATAT CATATATTTA GGGGAAAGAC TTAAGCCTTT 1440
GAGCTCTCCA GCTTGGACCA CACATTGCCC TTTTCTCAGG GAAGGAAATG GAAACAAAAA 1500
GCCAACAGGG CAGGGGTTTT GTAAGTGGAA CTCTGGATTG ACTGGTCAGT TGCTACAATC 1560
AGAATATGCT TTCTTGGACC ATGTTTGAGA CTCAGAAGAA TGGCCTTTCT GCCATAATTC 1620 TTCACTAGTC AAGAATGCCA GCAGTTTCTT TGTATAAAGA GACCTGCCTT TAAAATCATA 1680
CATTCTGAAC ATTTTAGTCA AGCTACAACA GGTTTGGAAA ACCTCTGTGG GGGAGGGGCG 1740
AGTATAAAGT TTTCCTCTTT TTTAACTGTT CCCTTTGCCC TTCAAACTGC AGATATTTTT 1800
TTTTTTAAGT GGGGACTTCT CCCTACTTGA TTAAAGATTG AGTGGAATTC TAGATGTGGT 1860
CATTTGTGTC ATAATTTTTT TGTTTTATTT TGTTTTTGAT TTTTTTTTTC CTCCCCTGAG 1920 TGTATGCTTA GTTGTTGAGT ATATATATTT GGGACCATTA AAACTTTTTT TGATGTAATA 1980
TAACCTAACG TTGTGCTGGT ACCTGTTTTA CCATGTGTAA TTTTTGTTCT ACATCACAGT 2040
TCTTAATTTG TTTAGAGTTT TATGAAAGAT GGTATAGTTT TTATTGACAA AAGCAAAGTA 2100
ATCTTACAAC TATGTGCATA CAAAAGCAAT ACTATTTTGT GACTAAATAT TTTATATTAA 2160
AATTTACATC AGCAACTGTC TTGAGAATTC AGGGAAATAG AATGGAATTT AAAACTTCAA 2220 CAGTTTTGTT AAATCTAGAA ACATGAAATT RGTATTCCAA AGAGATTCTG AAATTTCTTT 2280
TCTKGGGGAA ATGACGGTAC ATTAAATCAA AATTGRGGAT GGATGATTTA AAAACATTTG 2340
ACTTTTTAAT AATAAAAAGA AAAGTGAAGA GTAAGAGAAA TTGTAAAAAA AAAAAAAAAA 2400
AAAAAAAAA 2409
(2) INFORMATION FOR SEQ ID NO : 8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 400 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met He Cys Asp Thr Asp Pro Glu Leu Gly Gly Ala Val Gin Leu Met 1 5 10 15
Gly Leu Leu Arg Thr Leu Val Asp Pro Glu Asn Met Leu Ala Thr Ala 20 25 30
Xaa Lys Thr Xaa Lys Thr Glu Phe Leu Gly Phe Phe Tyr Lys His Cys 35 40 45
Met His Val Leu Xaa Ala Pro Leu Leu Ala Asn Thr Thr Glu Asp Lys 50 55 60
Pro Ser Lys Asp Asp Phe Gin Thr Ala Gin Leu Leu Ala Leu Val Leu 65 70 75 80
Glu Leu Leu Thr Phe Cys Val Glu His His Thr Tyr His He Lys Asn 85 90 95
Tyr He He Asn Lys Asp He Leu Arg Arg Val Leu Val Leu Met Ala 100 105 110
Ser Lys His Ala Phe Leu Ala Leu Cys Ala Leu Arg Phe Lys Arg Lys 115 120 125
He He Gly Leu Lys Asp Glu Phe Tyr Asn Arg Tyr He Met Lys Ser 130 135 140 Phe Leu Phe Glu Pro Val Val Lys Ala Phe Leu Asn Asn Gly Ser Arg 145 150 155 160
Tyr Asn Leu Met Asn Ser Ala He He Glu Met Phe Glu Phe He Arg 165 170 175
Val Glu Asp He Lys Ser Leu Thr Ala His Val He Glu Asn Tyr Trp 180 185 190
Lys Ala Leu Glu Asp Val Asp Tyr Val Gin Thr Phe Lys Gly Leu Lys 195 200 205
Leu Arg Phe Glu Gin Gin Arg Glu Arg Gin Asp Asn Pro Lys Leu Asp 210 215 220 Ser Met Arg Ser He Leu Arg Asn His Arg Tyr Arg Arg Asp Ala Arg 225 23 0 235 240
Thr Leu Glu Asp Glu Glu Glu Met Trp Phe Asn Thr Asp Glu Asp Asp 245 250 255
Met Glu Asp Gly Glu Ala Val Val Ser Pro Ser Asp Lys Thr Lys Asn 260 265 270
Asp Asp Asp He Met Asp Pro He Ser Lys Phe Met Glu Arg Lys Lys 275 280 285
Leu Lys Glu Ser Glu Glu Lys Glu Val Leu Leu Lys Thr Asn Leu Ser 290 295 300 Gly Arg Gin Ser Pro Ser Phe Lys Leu Ser Leu Ser Ser Gly Thr Lys 305 310 315 320
Thr Asn Leu Thr Ser Gin Ser Ser Thr Thr Asn Leu Pro Gly Ser Pro 325 330 335
Gly Ser Pro Gly Ser Pro Gly Ser Pro Gly Ser Pro Gly Ser Val Pro 340 345 350
Lys Asn Thr Ser Gin Thr Ala Ala He Thr Thr Lys Gly Gly Leu Val 355 360 365
Gly Leu Val Asp Tyr Pro Asp Asp Asp Glu Asp Asp Asp Glu Asp Glu 370 375 380 Asp Lys Glu Asp Thr Leu Pro Leu Ser Lys Lys Ala Lys Phe Asp Ser 385 390 395 400
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 951 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
GCCAGGCAGG GTGTGGGGGC AGCTGTGCCA ATCTACCTCA CAGGCCCACC CCCTGCCGGG 60
CATGCCGTGG GATCATGGGC AGGGAAGGCT CTGGGGGTCG GAGACACCGC TGCTTAGCAC 120
CCCCAGCCAG AACACCCTGA GGGTCTCGGG GCTCTGGAGA GAGTGGGGCG GGAGGAAGAA 180 TTGGCACCTT CCTAGGGAAG GAGACGAGCG CTTCGCCTTG ATTCTCCGAG AAGCCTCCGA 240 GAAGTGCTTT AAGTGTGTTT GCATGCSCCA GGCGGTGGGC AGCGGGGGCC TGTCCARCCC 300
TCTCCCGCCA TCCTTCCCCA AGTGACGTCC ACTGCCTTGT CACCAGCGAC CTGCCTGTCA 360
TGCCCACCCC CTGAGGAAGC ATGGGGACCC TAACACCCTG GTGCCCTGCA CCAGACAGGC 420
CGTGGTCAGG CCCAGGCCAC CGGCCGGGTT CTGCCACARC TTCCCACGTG CTTGCTGACA 480
TGCSTGTGCC TGTGTGTGGT GTCTGTTGCT GTGTCGTGAA ACTGTGACCA TCACTCAGTC 540
CAAACAAGTG AGTGGCCCTS GAGGCCACAG TTATGCAACT TTCAGTGTGT GTCATAACGA 600
CGTCACTGCT TTTTAAACTC GATAACTCTT TATTTTAGTA AAATGCCCAG GAGTCCTGGA 660 AGCTACGCGG ACTTGCAGAG GTTTTATTTT TTGGCCTTAG AATCTGCAGA AATTAGGAGG 720
CACCGAGCCC AGCGCAGCAG CCTCGGACCC GGATTGCGTT TGCCTTAGCG GATATGTTTA 780
TACAGATGAA TATAAAATGT TTTTTTCTTT GGGCTTTTTG CTTCTTTTTT CCCCCCCTTC 840
TCACCTTCCC TTCTCCCTGA CCCCACCCCC CAAAAAAGCT ACTTCTTCAT TCCGTGGTAC 900
GATTATTTTT TTTAACTAAA GGAAGATAAA ATTCTAAAAA AAAAAAAAAA A 951 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Pro Trp Asp His Gly Gin Gly Arg Leu Trp Gly Ser Glu Thr Pro 1 5 10 15
Leu Leu Ser Thr Pro Ser Gin Asn Thr Leu Arg Val Ser Gly Leu Trp 20 25 30
Arg Glu Trp Gly Gly Arg Lys Asn Trp His Leu Pro Arg Glu Gly Asp 35 40 45
Glu Arg Phe Ala Leu He Leu Arg Glu Ala Ser Glu Lys Cys Phe Lys 50 55 60
Cys Val Cys Met Xaa Gin Ala Val Gly Ser Gly Gly Leu Ser Xaa Pro 65 70 75 80 Leu Pro Pro Ser Phe Pro Lys 85 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1899 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GGCCGCTTGT GTCCACGGGA CGCGGGCGGA TCTTCTCCGG CCATGAGGAA GCCAGCCGCT 60 GGCTTCCTTC CCTCACTCCT GAAGGTGCTG CTCCTGCCTC TGGCACCTGC CGCAGCCCAG 120
GATTCGACTC AGGCCTCCAC TCCAGGCAGC CCTCTCTCTC CTACCGAATA CGAACGCTTC 180
TTCGCACTGC TGACTCCAAC CTGGAAGGCA GAGACTACCT GCCGTCTCCG TGCAACCCAC 240
GGCTGCCGGA ATCCCACACT CGTCCAGCTG GACCAATATG AAAACCACGG CTTAGTGCCC 300
GATGGTGCTG TCTGCTCCAA CCTCCCTTAT GCCTCCTGGT TTGAGTCTTT CTGCCAGTTC 360 ACTCACTACC GTTGCTCCAA CCACGTCTAC TATGCCAAGA GAGTCCTGTG TTCCCAGCCA 420
GTCTCTATTC TCTCACCTAA CACTCTCAAG GAGATAGAAG CTTCAGCTGA AGTCTCACCC 480
ACCACGATGA CCTCCCCCAT CTCACCCCAC TTCACAGTGA CAGAACGCCA GACCTTCCAG 540
CCCTGGCCTG AGAGGCTCAG CAACAACGTG GAAGAGCTCC TACAATCCTC CTTGTCCCTG 600
GGAGGCCAGG AGCAAGCGCC AGAGCACAAG CAGGAGCAAG GAGTGGAGCA CAGGCAGGAG 660 CCGACACAAG AACACAAGCA GGAAGAGGGG CAGAAACAGG AAGAGCAAGA AGAGGAACAG 720
GAAGAGGAGG GAAAGCAGGA AGAAGGACAG GGGACTAAGG AGGGACGGGA GGCTGTGTCT 780
CAGCTGCAGA CAGACTCAGA GCCCAAGTTT CACTCTGAAT CTCTATCTTC TAACCCTTCC 840
TCTTTTGCTC CCCGGGTACG AGAAGTAGAG TCTACTCCTA TGATAATGGA GAACATCCAG 900
GAGCTCATTC GATCAGCCCA GGAAATAGAT GAAATGAATG AAATATATGA TGAGAACTCC 960 TACTGGAGAA ACCAAAACCC TGGCAGCCTC CTGCAGCTGC CCCACACAGA GGCCTTGCTG 1020
GTGCTGTGCT ATTCGATCGT GGAGAATACC TGCATCATAA CCCCCACAGC CAAGGCCTGG 1080
AAGTACATGG AGGAGGAGAT CCTTGGTTTC GGGAAGTCGG TCTGTGACAG CCTTGGGCGG 1140 CGACACATGT CTACCTGTGC CCTCTGTGAC TTCTGCTCCT TGAAGCTGGA GCAGTGCCAC 1200
TCAGAGGCCA GCCTGCAGCG GCAACAATGC GACACCTCCC ACAAGACTCC CTTTGTCAGC 1260 CCCTTGCTTG CCTCCCAGAG CCTGTCCATC GGCAACCAGG TAGGGTCCCC AGAATCAGGC 1320
CGCTTTTACG GGCTGGATTT GTACGGTGGG CTCCACATGG ACTTCTGGTG TGCCCGGCTT 1380
GCCACGAAAG GCTGTGAAGA TGTCCGAGTC TCTGGGTGGC TCCAGACTGA GTTCCTTAGC 1440
TTCCAGGATG GGGATTTCCC TACCAAGATT TGTGACACAG ACTATATCCA GTACCCAAAC 1500
TACTGTTCCT TCAAAAGCCA GCAGTGTCTG ATGAGAAACC GCAATCGGAA GGTGTCCCGC 1560 ATGAGATGTC TGCAGAATGA GACTTACAGT GCGCTGAGCC TGGCAAAAGT GAGGACGTTG 1620
TGCTTTCGAT GGAGCCAGGA GTTCAGCACC TTGACTCTAG GCCAGTTCGG ATGAGCTKGS 1680
GTTTATTTTG CCCACACCCC AGCCCAACCT GCCCASGTTC TCTATTGTTT TGAGACCCCA 1740
TTGCTTTCAG GCTGCCCCTT CTGGGTCTGT TACTCGGCCC CTAMTCACAT TTCCTTGGGT 1800
TGGAGCAACA GTCCCAGAGA GGGCCACGGT GGGAGCTGCG CCCTCCTTAA AAGATGACTT 1860 TACATAAAAT GTTGATCTTC AAAAAAAAAA AAAAAAAAA 1899
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 543 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Arg Lys Pro Ala Ala Gly Phe Leu Pro Ser Leu Leu Lys Val Leu
1 5 10 15 Leu Leu Pro Leu Ala Pro Ala Ala Ala Gin Asp Ser Thr Gin Ala Ser
20 25 30
Thr Pro Gly Ser Pro Leu Ser Pro Thr Glu Tyr Glu Arg Phe Phe Ala 35 40 45
Leu Leu Thr Pro Thr Trp Lys Ala Glu Thr Thr Cys Arg Leu Arg Ala 50 55 60
Thr His Gly Cys Arg Asn Pro Thr Leu Val Gin Leu Asp Gin Tyr Glu 65 70 75 80 Asn His Gly Leu Val Pro Asp Gly Ala Val Cys Ser Asn Leu Pro Tyr 85 90 95
Ala Ser Trp Phe Glu Ser Phe Cys Gin Phe Thr His Tyr Arg Cys Ser 100 105 110
Asn His Val Tyr Tyr Ala Lys Arg Val Leu Cys Ser Gin Pro Val Ser 115 120 125 He Leu Ser Pro Asn Thr Leu Lys Glu He Glu Ala Ser Ala Glu Val
130 135 140
Ser Pro Thr Thr Met Thr Ser Pro He Ser Pro His Phe Thr Val Thr 145 150 155 160
Glu Arg Gin Thr Phe Gin Pro Trp Pro Glu Arg Leu Ser Asn Asn Val 165 170 175
Glu Glu Leu Leu Gin Ser Ser Leu Ser Leu Gly Gly Gin Glu Gin Ala 180 185 190
Pro Glu His Lys Gin Glu Gin Gly Val Glu His Arg Gin Glu Pro Thr 195 200 205
Gin Glu His Lys Gin Glu Glu Gly Gin Lys Gin Glu Glu Gin Glu Glu 210 215 220
Glu Gin Glu Glu Glu Gly Lys Gin Glu Glu Gly Gin Gly Thr Lys Glu 225 230 235 240
Gly Arg Glu Ala Val Ser Gin Leu Gin Thr Asp Ser Glu Pro Lys Phe 245 250 255
His Ser Glu Ser Leu Ser Ser Asn Pro Ser Ser Phe Ala Pro Arg Val 260 265 270
Arg Glu Val Glu Ser Thr Pro Met He Met Glu Asn He Gin Glu Leu 275 280 285 He Arg Ser Ala Gin Glu He Asp Glu Met Asn Glu He Tyr Asp Glu 290 295 300
Asn Ser Tyr Trp Arg Asn Gin Asn Pro Gly Ser Leu Leu Gin Leu Pro 305 310 315 320
His Thr Glu Ala Leu Leu Val Leu Cys Tyr Ser He Val Glu Asn Thr 325 330 335
Cys He He Thr Pro Thr Ala Lys Ala Trp Lys Tyr Met Glu Glu Glu 340 345 350
He Leu Gly Phe Gly Lys Ser Val Cys Asp Ser Leu Gly Arg Arg His 355 360 365 Met Ser Thr Cys Ala Leu Cys Asp Phe Cys Ser Leu Lys Leu Glu Gin 370 375 380
Cys His Ser Glu Ala Ser Leu Gin Arg Gin Gin Cys Asp Thr Ser His 385 390 395 400
Lys Thr Pro Phe Val Ser Pro Leu Leu Ala Ser Gin Ser Leu Ser He 405 410 415
Gly Asn Gin Val Gly Ser Pro Glu Ser Gly Arg Phe Tyr Gly Leu Asp 420 425 430
Leu Tyr Gly Gly Leu His Met Asp Phe Trp Cys Ala Arg Leu Ala Thr 435 440 445 Lys Gly Cys Glu Asp Val Arg Val Ser Gly Trp Leu Gin Thr Glu Phe 450 455 460
Leu Ser Phe Gin Asp Gly Asp Phe Pro Thr Lys He Cys Asp Thr Asp 465 470 475 480
Tyr He Gin Tyr Pro Asn Tyr Cys Ser Phe Lys Ser Gin Gin Cys Leu 485 490 495
Met Arg Asn Arg Asn Arg Lys Val Ser Arg Met Arg Cys Leu Gin Asn 500 505 510
Glu Thr Tyr Ser Ala Leu Ser Leu Ala Lys Val Arg Thr Leu Cys Phe 515 520 525 Arg Trp Ser Gin Glu Phe Ser Thr Leu Thr Leu Gly Gin Phe Gly 530 535 540
(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 722 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CGACCTTCCC AGCAATATGC ATCTTGCACG TCTGGTCGGC TCCTGCTCCC TCCTTCTGCT 60 ACTGGGGGCC CTGTCTGGAT GGGCGGCCAG CGATGACCCC ATTGAGAAGG TCATTGAAGG 120
GATCAACCGA GGGCTGAGCA ATGCAGAGAG AGAGGTGGGC AAGGCCCTGG ATGGCATCAA 180
CAGTGGAATC ACGCATGCCG GAAGGGAAGT GGAGAAGGTT TTCAACGGAC TTAGCAACAT 240 GGGGAGCCAC ACCGGCAAGG AGTTGGACAA AGGCGTCCAG GGGCTCAACC ACGGCATGGA 300
CAAGGTTGCC CATGAGATCA ACCATGGTAT TGGACAAGCA GGAAAGGAAG CAGAGAAGCT 360 TGGCCATGGG GTCAACAACG CTGCTGGACA GGGCAACCAT CAAAGCGGAT CTTCCAGCCA 420
TCAAGGAGGG GCCACAACCA CGCCGTTAGC CTCTGGGGCC TCGGTCAACA CGCCTTTCAT 480
CAACCTTCCC GCCCTGTGGA GGAGCGTCGC CAACATCATG CCCTAAACTG GCATCCGGCC 540
TTGCTGGGAG AATAATGTCG CCGTTGTCAC ATCAGCTGAC ATGACCTGGA GGGGTTGGGG 600
GTGGGGGACA GGTTTCTGAA ATCCCTGAAG GGGGTTGTAC TGGGATTTGT GAATAAACTT 660 GATACACTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 720
AA 722
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met His Leu Ala Arg Leu Val Gly Ser Cys Ser Leu Leu Leu Leu Leu 1 5 10 15
Gly Ala Leu Ser Gly Trp Ala Ala Ser Asp Asp Pro He Glu Lys Val 20 25 30 He Glu Gly He Asn Arg Gly Leu Ser Asn Ala Glu Arg Glu Val Gly 35 40 45
Lys Ala Leu Asp Gly He Asn Ser Gly He Thr His Ala Gly Arg Glu 50 55 60
Val Glu Lys Val Phe Asn Gly Leu Ser Asn Met Gly Ser His Thr Gly 65 70 75 80
Lys Glu Leu Asp Lys Gly Val Gin Gly Leu Asn His Gly Met Asp Lys 85 90 95
Val Ala His Glu He Asn His Gly He Gly Gin Ala Gly Lys Glu Ala 100 105 110 Glu Lys Leu Gly His Gly Val Asn Asn Ala Ala Gly Gin Gly Asn His 115 120 125
Gin Ser Gly Ser Ser Ser His Gin Gly Gly Ala Thr Thr Thr Pro Leu 130 135 140
Ala Ser Gly Ala Ser Val Asn Thr Pro Phe He Asn Leu Pro Ala Leu 145 150 155 160
Trp Arg Ser Val Ala Asn He Met Pro 165
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AATGGCTTTT CTTCCTTCCT GGGTTTGTGT ACTAGTTGGT TCCTTTTCTG CTTCCTTAGC 60
AGGGACTTCC AATCTCTCAG AGACAGAGCC CCCTCTGTGG AAGGAGAGTC CTGGTCAGCT 120
CAGTGACTAC AGGGTGGAGA ACAGCATGTA CATTATTAAT CCCTGGGTAT ACCTTGAGAG 180
AATGGGGATG TATAAAATCA TATTGAATCA GACAGCCAGG TATTTTGCAA AATTTGCACC 240 AGATAATGAA CAGAATATTT TATGGGGGTT GCCTCTGCAG TATGGCTGGC AATATAGGAC 300
AGGCAGATTA GCTGATCCAA CCCGAAGGAC AAACTGTGGC TATGAATCTG GAGATCATAT 360
GTGCATCTCT GTGGACAGTT GGTGGGCTGA TTTGAATTAT TTTCTGTCTT CATTACCCTT 420
TCTTGCTGCG GTTGATTCTG GTGTAATGGG GATATCATCA GACCAAGTCA GGCTTTTGCC 480
CCCACCCAAG AATGAGAGGA AGTTTTGTTA TGATGTTTCT AGCTGTCGTT CATCCTTCCC 540 TGAGACAATG AACAAGTGGA ACACCTTTTA CCAGTATTTG CAGTCACCTT TTAGTAAGTT 600
TGATGATCTG TTGAAGTACT TATGGGCTGC ACACACTTCA ACCTTGGCAG ATAATATCAA 660
AAGTTTTGAA GACAGATATG ATTATTATTC TAAAGCAGAA GCGCATTTTG AGAGAAGTTG 720
GGTACTGGCT GTGGATCATT TAGCTGCAGT CCTCTTTCCT ACAACCTTGA TTAGATCATA 780
TAAGTTCCAG AAGGGCATGC CACCACGAAT TCTTCTTAAT ACTGATGTAG CCCCTTTCAT 840 CAGTGACTTT ACTGCTTTTC AGAATGTAGT CCTGGTTCTT CTAAATATGC TTGACAATGT 900 GGATAAATCT ATAGGTTATC TTTGTACAGA AAAATCTAAT GTATATAGAG ATCATTCGGA 960
ATCTAGCTCT AGAAGTTATG GAAATAACTC CTGAAACATT TAACTTCAAA CTTCAGGAAA 1020
TGATTAATGA ATTAAAAATG AAAAACTCGA ACTTGACAAT CAGTAATTTC AAAAAATTAA 1080
TGTCATCATG ACCATGTAGT TTATTCTTTC TGATATTTTT GATTTATGCT TATTTGTTAA 1140
GATCTTGTAC ATGTATTAAA AACTTAAATT AAATGCATTC AAGTTAAAAA AAAAAAAAAA 1200
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1240 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 330 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Ala Phe Leu Pro Ser Trp Val Cys Val Leu Val Gly Ser Phe Ser 1 5 10 15
Ala Ser Leu Ala Gly Thr Ser Asn Leu Ser Glu Thr Glu Pro Pro Leu 20 25 30
Trp Lys Glu Ser Pro Gly Gin Leu Ser Asp Tyr Arg Val Glu Asn Ser 35 40 45
Met Tyr He He Asn Pro Trp Val Tyr Leu Glu Arg Met Gly Met Tyr 50 55 60 Lys He He Leu Asn Gin Thr Ala Arg Tyr Phe Ala Lys Phe Ala Pro 65 70 75 80
Asp Asn Glu Gin Asn He Leu Trp Gly Leu Pro Leu Gin Tyr Gly Trp 85 90 95
Gin Tyr Arg Thr Gly Arg Leu Ala Asp Pro Thr Arg Arg Thr Asn Cys 100 105 110
Gly Tyr Glu Ser Gly Asp His Met Cys He Ser Val Asp Ser Trp Trp 115 120 125
Ala Asp Leu Asn Tyr Phe Leu Ser Ser Leu Pro Phe Leu Ala Ala Val
130 135 140 Asp Ser Gly Val Met Gly He Ser Ser Asp Gin Val Arg Leu Leu Pro 145 150 155 160
Pro Pro Lys Asn Glu Arg Lys Phe Cys Tyr Asp Val Ser Ser Cys Arg 165 170 175
Ser Ser Phe Pro Glu Thr Met Asn Lys Trp Asn Thr Phe Tyr Gin Tyr 180 185 190
Leu Gin Ser Pro Phe Ser Lys Phe Asp Asp Leu Leu Lys Tyr Leu Trp 195 200 205
Ala Ala His Thr Ser Thr Leu Ala Asp Asn He Lys Ser Phe Glu Asp 210 215 220 Arg Tyr Asp Tyr Tyr Ser Lys Ala Glu Ala His Phe Glu Arg Ser Trp 225 230 235 240
Val Leu Ala Val Asp His Leu Ala Ala Val Leu Phe Pro Thr Thr Leu 245 250 255
He Arg Ser Tyr Lys Phe Gin Lys Gly Met Pro Pro Arg He Leu Leu 260 265 270
Asn Thr Asp Val Ala Pro Phe He Ser Asp Phe Thr Ala Phe Gin Asn 275 280 285
Val Val Leu Val Leu Leu Asn Met Leu Asp Asn Val Asp Lys Ser He 290 295 300 Gly Tyr Leu Cys Thr Glu Lys Ser Asn Val Tyr Arg Asp His Ser Glu 305 310 315 320
Ser Ser Ser Arg Ser Tyr Gly Asn Asn Ser 325 330
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2261 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCAGCACCAG CCGTCTGCAG CTCCGGCCGC CACTTGCGCC TCTCCAGCCT CCGCAGGCCC 60
AACCGCCGCC AGCACCATGG CCAGCACCAT TTCCGCCTAC AAGGAGAAGA TGAAGGAGCT 120 GTCGGTGCTG TCGCTCATCT GCTCCTGCTT CTACACACAG CCGCACCCCA ATACCGTCTA 180 CCAGTACGGG GACATGGAGG TGAAGCAGCT GGACAAGCGG GCCTCAGGCC AGAGCTTCGA 240
GGTCATCCTC AAGTCCCCTT CTGACCTGTC CCCAGAGAGC CCTATGCTCT CCTCCCCACC 300 CAAGAAGAAG GACACCTCCC TGGAGGAGCT GCAAAAGCGG CTGGAGGCAG CCGAGGAGCG 360
GAGGAAGACG CAGGAGGCGC AGGTGCTGAA GCAGCTGGCG GAGCGGCGCG AGCACGAGCG 420
CGAGGTGCTG CACAAGGCGC TGGAGGAGAA TAACAACTTC AGCCGCCAGG CGGAGGAGAA 480
GCTCAACTAC AAGATGGAGC TCAGCAAGGA GATCCGCGAG GCACACCTGG CCGCACTGCG 540
CGAGCGGCTG CGCGAGAAGG AGCTGCACGC GGCCGAGGTG CGCAGGAACA AGGAGCAGCG 600 AGAAGAGATG TCGGGCTAAG GGCCCGGGAC GGGCGGCGCC CATCCTGCGA CAGAACACGT 660
TCGGGTTTTG GTTTTGTTTC GTTCACCTCT GTCTAGATGC AACTTTTGTT CCTCCTCCCC 720
CACCCCAGCC CCCAGCTTCA TGCTTCTCTT CCGCACTCAG CCGCCCTGCC CTGTCCTCGT 780
GGTGAGTCGC TGACCACGGC TTCCCCTGCA GGAGCCGCCG GGCGTGAGAC GCGGTCCCTC 840
GGTGCAGACA CCAGGCCGGG CGCGGCTGGG TCCCCCGGGG GCCCTGTGAG AGAGGTGGCG 900 GTGACCGTGG TAAACCCAGG GCGGTGGCGT GGGATCGCGG GTCCTTACGC TGGGCTGTCT 960
GGTCAGCACG TGCAGGTCAG GGCAGGTCCT CTGAGCCGGC GCCCCTGGCC AGCAGGCGAG 1020
GCTACAGTAC CTGCTGTCTT TCCAGGGGGA AGGGGCTCCC CATGAGGGAG GGGCGACGGG 1080
GGAGGGGGGT GATGGTGCCT GGGAGCCTGC GTGTGCAGCC GGTGCTTGTT GAACTGGCAG 1140
GCGGGTGGGT GGGGGCTGCA GCTTTCCTTA ATGTGGTTGC ACAGGGGTCC TCTGAGACCA 1200 CCTGGCGTGA GGTGGACACC CTGGGCCTTC CTGGAAGCCT GCAGTTGGGG GCCTGCCCTG 1260
AGTCTGCTGG GGAGTGGGCA TTCTCTGCCA GGGACCCATG AGCAGGCTGC ATGGTCTAGA 1320
GGTTGTGGGC AGCATGGACA GTCCCCCACT CAGAAGTGCA AGAGTTCCAA AGAGCCTCTG 1380
GCCCAGGCCC CTCCCCACCA GGGCTTTGCA GATGTCCTTG AAAGACCCAC CCTAGAGCCC 1440
TTTGGAGTGC TGGCCCCTCC TGTGCCCTCT GCCCTGGTGG AAGCGGCAGC CACAAGTCCT 1500 CCTCAGGGAG CCCCAAGGGG GATTTTGTGG GACCGCTGCC CACAGATCCA GGTGTTGGAA 1560
GGGCAGCGGG TAAGGTTCCC AAGCCAGCCC CAACACCCTT CCCACTTGGC ACCCAGAGGG 1620
GGCTGTGGGT GGAGGCCTGA CTCCAGGCCT CTCCTGCCCA CACCCTCTGG GCTGAGTTCC 1680
TTCTTTCCCT TGGACGCCCA GTGCTGGCCT TGGAGGACGG TCAGCTGGAG GATGGCGGTG 1740
GGGGAGGCTG TCTTTGTACC ACTGCAGCAT CCCCCACTTC TCCACGGAAG CCCCATCCCA 1800 AAGCTGCTGC CTGGCCCCTT GCTGTAAAGT GTGAAGGGGG CGGCTGAGTT CTCTTAGGAC 1860 CCAGAGCCAG GGCCCTCAAC TTCCATCCTG CGGGAGGCCT TGGCCGGGCA CTGCCAGTGT 1920
CTTCCAGAGC CACACCCAGG GACCACGGGA GGATCCTGAC CCCTGCAGGG CTCAGGGGTC 1980
AGCAGGGACC CACTGCCCCA TCTCCCTCTC CCCACCAAGA CAGCCCCAGA AGGAGCAGCC 2040
AGCTGGGATG GGAACCCAAG GCTGTCCACA TCTGGCTTTT GTGGGACTCA GAAAGGGAAG 2100
CAGAACTGAG GGCTGGGATA TTCCTCATGG TGGCAGCGCT CATAGCGAAA GCCTACTGTA 2160
ATATGCACCC ATCTCATCCA CGTAGTAAAG TGAACTTAAA AATTCAATCA AATGAACAAT 2220
TAAATAAACA CCTGTGTGTT TAAGAAAAAA AAAAAAAAAA A 2261 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Met Ala Ser Thr He Ser Ala Tyr Lys Glu Lys Met Lys Glu Leu Ser 1 5 10 15
Val Leu Ser Leu He Cys Ser Cys Phe Tyr Thr Gin Pro His Pro Asn 20 25 30
Thr Val Tyr Gin Tyr Gly Asp Met Glu Val Lys Gin Leu Asp Lys Arg 35 40 45
Ala Ser Gly Gin Ser Phe Glu Val He Leu Lys Ser Pro Ser Asp Leu 50 55 60
Ser Pro Glu Ser Pro Met Leu Ser Ser Pro Pro Lys Lys Lys Asp Thr 65 70 75 80
Ser Leu Glu Glu Leu Gin Lys Arg Leu Glu Ala Ala Glu Glu Arg Arg 85 90 95
Lys Thr Gin Glu Ala Gin Val Leu Lys Gin Leu Ala Glu Arg Arg Glu 100 105 110
His Glu Arg Glu Val Leu His Lys Ala Leu Glu Glu Asn Asn Asn Phe 115 120 125
Ser Arg Gin Ala Glu Glu Lys Leu Asn Tyr Lys Met Glu Leu Ser Lys 130 135 140 Glu He Arg Glu Ala His Leu Ala Ala Leu Arg Glu Arg Leu Arg Glu 145 150 155 160
Lys Glu Leu His Ala Ala Glu Val Arg Arg Asn Lys Glu Gin Arg Glu 165 170 175
Glu Met Ser Gly 180 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3109 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGCCAAAGAG GCCTAGGAGC CTCGTGGCTG CGTCACCGCC GCCCCCCCAG ACAAGATGGA 60
CACCGCGGAG GAAGACATAT GTAGAGTGTG TCGGTCAGAA GGAACACCTG AGAAACCGCT 120
TTATCATCCT TGTGTATGTA CTGGCAGTAT TAAGTTTATC CATCAAGAAT GCTTAGTTCA 180
ATGGCTGAAA CACAGTCGAA AAGAATACTG TGAATTATGC AAGCACAGAT TTGCTTTTAC 240
ACCAATTTAT TCTCCAGATA TGCCTTCACG GCTTCCAATT CAAGACATAT TTGCTGGACT 300 GGTTACAAGT ATTGGCACTG CAATACGATA TTGGTTTCAT TATACACTTG TGGCCTTTGC 360
ATGGTTGGGA GTTGTTCCTC TTACAGCATG CCGCATCTAC AAGTGCTTGT TTACTGGCTC 420
CCGTGAGCTC ACTACTGACG CTGCCCATTA GATATGCTGT CAACCGGAAA ATTTGTTGGC 480
AGATTGTTTG CAGGGTTGTT TTGTGGTGAC GTGCACACTG TGTGCATTCA TCAGCCTGGT 540
GTGGTTGAGA GAGCAGATAG TCCATGGGGG AGCACCAATT TGGTTGGAGC ATGCTGCCCC 600 ACCGTTCAAT GCTGCGGGGC ATCACCAAAA TGAGGCTCCA GCAGGAGGAA ATGGTGCAGA 660
AAATGTTGCT GCTGATCAGC CTGCTAACCC ACCAGCTGAG AACGCAGTGG TGGGGGAAAA 720
CCCTGATGCC CAGGATGACC AGGCAGAAGA GGAGGAGGAG GACAATGAGG AGGAAGATGA 780
CGCTGGTGTG GAGGATGGCG GCAGATGCTA ATAACGGAGC CCAGGATGAC ATGAATTGGA 840
ATGCTTTAGA ATGGGACCGA GCTGCTGAAG AGCTTACATG GGAAAGAATG CTAGGACTTG 900 ATGGATCACT AGTTTTTCTG GAACATGTCT TCTGGGTGGT ATCTTTAAAT ACACTGTTCA 960 TTCTTGTTTT TGCATTTTGC CCTTACCATA TTGGTCATTT CTCCCTTGTT GGTTTGGGAT 1020
TTGAAGAACA CGTCCAAGCA TCTCATTTTG AAGGCCTAAT CACAACCATA GTTGGGTATA 1080 TACTTTTAGC AATAACACTG ATAATTTGTC ATGGCTTGGC AACTCTTGTG AAATTTCATA 1140
GATCTCGTCG CTTACTGGGA GTCTGCTATA TTGTTGTTAA GGTCTCTTTG TTAGTGGTGG 1200
TAGAAATTGG AGTATTCCCT CTCATTTGTG GTTGGTGGCT GGATATCTGT TCCTTGGAAA 1260
TGTTTGATGC TACTCTGAAA GATCGAGAAC TGAGCTTTCA GTCGGCTCCA GGTACTACCA 1320
TGTTTCTGCA TTGGCTAGTG GGAATGGTAT ATGTCTTCTA CTTTGCCTCC TTCATTCTAT 1380 TACTGAGAGA GGTACTTCGA CCTGGTGTCC TGTGGTTTCT AAGGAATTTG AATGATCCAG 1440
ATTTCAATCC AGTACAGGAA ATGATCCATT TGCCAATATA TAGGCATCTC CGAAGATTTA 1500
TTTTGTCAGT GATTGTCTTT GGCTCCATTG TCCTCCTGAT GCTTTGGCTT CCTATACGTA 1560
TAATTAAGAG TGTGCTGCCT AATTTTCTTC CATACAATGT CATGCTCTAC AGTGATGCTC 1620
CAGTGAGTGA ACTGTCCCTC GAGCTGCTTC TGCTTCAGGT TGTCTTGCCA GCATTACTCG 1680 AACAGGGACA CACGAGGCAG TGGCTGAAGG GGCTGGTGCG AGCGTGGACT GTGACCGCCG 1740
GATACTTGCT GGATCTTCAT TCTTATTTAT TGGGAGACCA GGAAGAAAAT GAAAACAGTG 1800
CAAATCAACA AGTTAACAAT AATCAGCATG CTCGAAATAA CAACGCTATT CCTGTGGTGG 1860
GAGAAGGCCT TCATGCAGCC CACCAAGCCA TACTCCAGCA GGGAGGGCCT GTTGGCTTTC 1920
AGCCTTACCG CCGACCTTTA AATTTTCCAC TCAGGATATT TCTGTTGATT GTCTTCATGT 1980 GTATAACATT ACTGATTGCC AGCCTCATCT GCCTTACTTT ACCAGTATTT GCTGGCCGTT 2040
GGTTAATGTC GTTTTGGACG GGGACTGCCA AAATCCATGA GCTCTACACA GCTGCTTGTG 2100
GTCTCTATGT TTGCTGGCTA ACCATAAGGG CTGTGACGGT GATGGTGGCA TGGATGCCTC 2160
AGGGACGCAG AGTGATCTTC CAGAAGGTTA AAGAGTGGTC TCTCATGATC ATGAAGACTT 2220
TGATAGTTGC GGTGCTGTTG GCTGGAGTTG TCCCTCTCCT TCTGGGGCTC CTGTTTGAGC 2280 TGGTCATTGT GGCTCCCCTG AGGGTTCCCT TGGATCAGAC TCCTCTTTTT TATCCATGGC 2340
AGGACTGGGC ACTTGGAGTC CTGCATGCCA AAATCATTGC AGCTATAACA TTGATGGGTC 2400
CTCAGTGGTG GTTGAAAACT GTAATTGAAC AGGTTTACGC AAATGGCATC CGGAACATTG 2460
ACCTTCACTA TATTGTTCGT AAACTGGCAG CTCCCGTGAT CTCTGTGCTG TTGCTTTCCC 2520
TGTGTGTACC TTATGTCATA GCTTCTGGTG TTGTTCCTTT ACTAGGTGTT ACTGCGGAAA 2580 TGCAAAACTT AGTCCATCGG CGGATTTATC CATTTTTACT GATGGTCGTG GTATTGATGG 2640 CAATTTTGTC CTTCCAAGTC CGCCAGTTTA AGCGCCTTTA TGAACATATT AAAAATGACA 2700
AGTACCTTGK GGGTCAASGA CTCGGTGAAC TACGAACGGA AATCTGGGCA AACAAGGCTC 2760 ATCTCCACCA CCTCCACAGT CATCCCAAGA ATAAAGTAGT TGTCTCAACA ACTTGACCTT 2820
CCCCTTTACA TGTCCTTTTT TGTGGACTTC TCTCTTKGGA GATTTTTCCC AGTGATCTCT 2880
CAGCGTKGTT TTTAAGTTAA AKGTATTKGA CTTGTGTTCT CAGCATTCAG AGAGCAGCGG 2940
TGTAAGATTC TGCTGTTCTC CCTGGATCTT CTGACATKAC TGCTGTCTGA GATTTGTATA 3000
TGKGTAAATA CAAGTTCCTT GATACCCTAA AACCTTGGAT TAAACAGAAT GTGCATKGTA 3060 CATCTTTAAA CAAAATGKAT ATTAATTTAT TAAAAAAAAA AAAAAAAAA 3109
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 750 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Gly Glu His Gin Phe Gly Trp Ser Met Leu Pro His Arg Ser Met 1 5 10 15 Leu Arg Gly He Thr Lys Met Arg Leu Gin Gin Glu Glu Met Val Gin
20 25 30
Lys Met Leu Leu Leu He Ser Leu Leu Thr His Gin Leu Arg Thr Gin 35 40 45
Trp Trp Gly Lys Thr Leu Met Pro Arg Met Thr Arg Gin Lys Arg Arg 50 55 60
Arg Arg Thr Met Arg Arg Lys Met Thr Leu Val Trp Arg Met Ala Ala 65 70 75 80
Asp Ala Asn Asn Gly Ala Gin Asp Asp Met Asn Trp Asn Ala Leu Glu 85 90 95
Trp Asp Arg Ala Ala Glu Glu Leu Thr Trp Glu Arg Met Leu Gly Leu 100 105 110
Asp Gly Ser Leu Val Phe Leu Glu His Val Phe Trp Val Val Ser Leu 115 120 125 Asn Thr Leu Phe He Leu Val Phe Ala Phe Cys Pro Tyr His He Gly 130 135 140
His Phe Ser Leu Val Gly Leu Gly Phe Glu Glu His Val Gin Ala Ser 145 150 155 160
His Phe Glu Gly Leu He Thr Thr He Val Gly Tyr He Leu Leu Ala 165 170 175 He Thr Leu He He Cys His Gly Leu Ala Thr Leu Val Lys Phe His
180 185 190
Arg Ser Arg Arg Leu Leu Gly Val Cys Tyr He Val Val Lys Val Ser 195 200 205
Leu Leu Val Val Val Glu He Gly Val Phe Pro Leu He Cys Gly Trp 210 215 220
Trp Leu Asp He Cys Ser Leu Glu Met Phe Asp Ala Thr Leu Lys Asp 225 230 235 240
Arg Glu Leu Ser Phe Gin Ser Ala Pro Gly Thr Thr Met Phe Leu His 245 250 255
Trp Leu Val Gly Met Val Tyr Val Phe Tyr Phe Ala Ser Phe He Leu 260 265 270
Leu Leu Arg Glu Val Leu Arg Pro Gly Val Leu Trp Phe Leu Arg Asn 275 280 285
Leu Asn Asp Pro Asp Phe Asn Pro Val Gin Glu Met He His Leu Pro 290 295 300
He Tyr Arg His Leu Arg Arg Phe He Leu Ser Val He Val Phe Gly 305 310 315 320
Ser He Val Leu Leu Met Leu Trp Leu Pro He Arg He He Lys Ser 325 330 335 Val Leu Pro Asn Phe Leu Pro Tyr Asn Val Met Leu Tyr Ser Asp Ala
340 345 350
Pro Val Ser Glu Leu Ser Leu Glu Leu Leu Leu Leu Gin Val Val Leu 355 360 365
Pro Ala Leu Leu Glu Gin Gly His Thr Arg Gin Trp Leu Lys Gly Leu 370 375 380
Val Arg Ala Trp Thr Val Thr Ala Gly Tyr Leu Leu Asp Leu His Ser 385 390 395 400
Tyr Leu Leu Gly Asp Gin Glu Glu Asn Glu Asn Ser Ala Asn Gin Gin 405 410 415 Val Asn Asn Asn Gin His Ala Arg Asn Asn Asn Ala He Pro Val Val 420 425 430
Gly Glu Gly Leu His Ala Ala His Gin Ala He Leu Gin Gin Gly Gly 435 440 445
Pro Val Gly Phe Gin Pro Tyr Arg Arg Pro Leu Asn Phe Pro Leu Arg 450 455 460
He Phe Leu Leu He Val Phe Met Cys He Thr Leu Leu He Ala Ser 465 470 475 480
Leu He Cys Leu Thr Leu Pro Val Phe Ala Gly Arg Trp Leu Met Ser 485 490 495 Phe Trp Thr Gly Thr Ala Lys He His Glu Leu Tyr Thr Ala Ala Cys
500 505 510
Gly Leu Tyr Val Cys Trp Leu Thr He Arg Ala Val Thr Val Met Val 515 520 525
Ala Trp Met Pro Gin Gly Arg Arg Val He Phe Gin Lys Val Lys Glu 530 535 540
Trp Ser Leu Met He Met Lys Thr Leu He Val Ala Val Leu Leu Ala 545 550 555 560
Gly Val Val Pro Leu Leu Leu Gly Leu Leu Phe Glu Leu Val He Val 565 570 575
Ala Pro Leu Arg Val Pro Leu Asp Gin Thr Pro Leu Phe Tyr Pro Trp 580 585 590
Gin Asp Trp Ala Leu Gly Val Leu His Ala Lys He He Ala Ala He 595 600 605
Thr Leu Met Gly Pro Gin Trp Trp Leu Lys Thr Val He Glu Gin Val 610 615 620
Tyr Ala Asn Gly He Arg Asn He Asp Leu His Tyr He Val Arg Lys 625 630 635 640
Leu Ala Ala Pro Val He Ser Val Leu Leu Leu Ser Leu Cys Val Pro 645 650 655 Tyr Val He Ala Ser Gly Val Val Pro Leu Leu Gly Val Thr Ala Glu
660 665 670
Met Gin Asn Leu Val His Arg Arg He Tyr Pro Phe Leu Leu Met Val 675 680 685
Val Val Leu Met Ala He Leu Ser Phe Gin Val Arg Gin Phe Lys Arg 690 695 700
Leu Tyr Glu His He Lys Asn Asp Lys Tyr Leu Xaa Gly Gin Xaa Leu 705 710 715 720 Gly Glu Leu Arg Thr Glu He Trp Ala Asn Lys Ala His Leu His His 725 730 735
Leu His Ser His Pro Lys Asn Lys Val Val Val Ser Thr Thr 740 745 750
(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:
TNTTTGAAGT TTCTCCCTCT CATTCTGAG 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 = "oligonucleotide1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GNTTCTCCAC GTAGTTGGTT TTCCTCAGT 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: CNACATGACG TGAGCTGGTG ATCCATGAA 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: ANTTGGGCTC TGCCGTCCAG AAAGGTTTG 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: GNAGCTACGC GGACTTGCAG AGGTTTTAT 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: TNGGTGAGAG AATAGAGACT GGCTGGGAA 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: ANGAGCCGAC CAGACGTGCA AGATGCATA 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:
ANCTGACCAG GACTCTCCTT CCACAGAGG 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: TNTAGGCGGA AATGGTGCTG GCCATGGTG 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:
ANATATCCAG CCACCAACCA CAAATGAGA 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 185 to nucleotide 1600;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1403 to nucleotide 1600;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 850;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dol5_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes 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. 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 1 to amino acid 222;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising eight consecutive amino acids of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone dol5_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
8. The protein of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
9. The protein of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 222.
10. A composition comprising the protein of claim 7 and a pharmaceutically acceptable carrier.
11. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:l.
12. 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 47 to nucleotide 2065;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1086 to nucleotide 1848;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dx290_l deposited under accession number ATCC 98468;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dx290_l deposited under accession number ATCC 98468;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
13. 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 312 to amino acid 600; (c) fragments of the amino acid sequence of SEQ ID NO:4 comprising eight consecutive amino acids of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone dx290_l deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
14. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
15. 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 107 to nucleotide 724;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 218 to nucleotide 724;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 536 to nucleotide 866;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ek390_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
16. 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 6 to amino acid 92;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising eight consecutive amino acids of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone ek390_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
17. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:5.
18. 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 31 to nucleotide 1230;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 289 to nucleotide 1230;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 344 to nucleotide 1119;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone er471_7 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468; (g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone er471_7 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
19. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino acid 111 to amino acid 363;
(c) fragments of the amino acid sequence of SEQ ID NO:8 comprising eight consecutive amino acids of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone er471_7 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
20. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
21. 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 62 to nucleotide 322;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 571 to nucleotide 878;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fs40_3 deposited under accession number ATCC 98468;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
22. 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 eight consecutive amino acids of SEQ ID NO:10; and
(c) the amino acid sequence encoded by the cDNA insert of clone fs40_3 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
23. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
24. 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 43 to nucleotide 1671;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 112 to nucleotide 1671;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 224 to nucleotide 679;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ga63_6 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
25. 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 62 to amino acid 212;
(c) fragments of the amino acid sequence of SEQ ID NO:12 comprising eight consecutive amino acids of SEQ ID NO:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone ga63_6 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
26. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:ll.
27. 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 17 to nucleotide 523;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 from nucleotide 77 to nucleotide 523;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 392;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gm335_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:14;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
28. 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 125;
(c) fragments of the amino acid sequence of SEQ ID NO: 14 comprising eight consecutive amino acids of SEQ ID NO:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone gm335_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
29. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:13.
30. 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 2 to nucleotide 991;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 62 to nucleotide 991;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 2 to nucleotide 504; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hy370_9 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:16;
(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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
31. 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 167;
(c) fragments of the amino acid sequence of SEQ ID NO: 16 comprising eight consecutive amino acids of SEQ ID NO:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone hy370_9 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
32. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:15.
33. 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 77 to nucleotide 616;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 164 to nucleotide 616;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 1 to nucleotide 415;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ie47_4 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
34. 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 113;
(c) fragments of the amino acid sequence of SEQ ID NO:18 comprising eight consecutive amino acids of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone ie47_4 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
35. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:17.
36. 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 564 to nucleotide 2813;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 705 to nucleotide 2813;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 793 to nucleotide 1628;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone sl95_10 deposited under accession number ATCC 98468;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468;
(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 eight consecutive amino acids 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 that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
37. 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 78 to amino acid 355;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising eight consecutive amino acids of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone sl95_10 deposited under accession number ATCC 98468; the protein being substantially free from other mammalian proteins.
38. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:19.
PCT/US1998/012516 1997-06-19 1998-06-18 Secreted proteins and polynucleotides encoding them WO1998057976A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP50468199A JP2002513294A (en) 1997-06-19 1998-06-18 Secreted proteins and polynucleotides encoding them
CA002294569A CA2294569A1 (en) 1997-06-19 1998-06-18 Secreted proteins and polynucleotides encoding them
EP98930279A EP1003770A4 (en) 1997-06-19 1998-06-18 Secreted proteins and polynucleotides encoding them
AU79704/98A AU7970498A (en) 1997-06-19 1998-06-18 Secreted proteins and polynucleotides encoding them

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US87871597A 1997-06-19 1997-06-19
US9858898A 1998-06-17 1998-06-17
US09/098,588 1998-06-17
US08/878,715 1998-06-17

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JP (1) JP2002513294A (en)
AU (1) AU7970498A (en)
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WO2001040271A2 (en) * 1999-12-01 2001-06-07 Ludwig Institute For Cancer Research Cancer associated antigens and uses therefor

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JP5504562B2 (en) * 2006-12-20 2014-05-28 東レ株式会社 Cancer detection method

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US5708157A (en) * 1996-07-26 1998-01-13 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
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US5770209A (en) * 1991-08-30 1998-06-23 University Of South Florida Acceleration of wound healing using connective tissue growth factor
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US5792628A (en) * 1997-03-14 1998-08-11 Bowman; Michael Secreted protein, BA3.1, and polynucleotides encoding same

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WO2001040271A2 (en) * 1999-12-01 2001-06-07 Ludwig Institute For Cancer Research Cancer associated antigens and uses therefor
WO2001040271A3 (en) * 1999-12-01 2002-04-18 Ludwig Inst Cancer Res Cancer associated antigens and uses therefor

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EP1003770A4 (en) 2002-10-30
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JP2002513294A (en) 2002-05-08
EP1003770A1 (en) 2000-05-31

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