WO1998021332A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998021332A2
WO1998021332A2 PCT/US1997/020740 US9720740W WO9821332A2 WO 1998021332 A2 WO1998021332 A2 WO 1998021332A2 US 9720740 W US9720740 W US 9720740W WO 9821332 A2 WO9821332 A2 WO 9821332A2
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Prior art keywords
seq
sequence
protein
polynucleotide
clone
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PCT/US1997/020740
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French (fr)
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WO1998021332A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
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Genetics Institute, Inc.
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Priority to CA002270873A priority Critical patent/CA2270873A1/en
Priority to JP52282098A priority patent/JP2002514058A/en
Priority to EP97947497A priority patent/EP0942974A2/en
Priority to AU52562/98A priority patent/AU5256298A/en
Publication of WO1998021332A2 publication Critical patent/WO1998021332A2/en
Publication of WO1998021332A3 publication Critical patent/WO1998021332A3/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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • 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
    • 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
    • A61P7/06—Antianaemics

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:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261
  • the present invention provides a composition comp ⁇ sing a protein, wherem said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of
  • a polynucleotide encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO:6 having biological activity (h) a polynucleotide encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO:6 having biological activity; (1) a polynucleotide which is an allelic variant of a polynucleotide of
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261
  • the present invention provides a polynucleotide encoding a protein comprising the ammo acid sequence of SEQ ID NO 6 from amino acid 1 to ammo acid 160.
  • the present invention provides a composition comprising a protem, wherein said protein comprises an ammo acid sequence selected from the group consisting of.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of.
  • a polvnucleohde capable of hvb ⁇ dizmg under stringent condihons to any one of the polynucleotides specified in (a)-( ⁇ )
  • such polynucleohde comprises the nucleohde sequence of SEQ ID NO: 1
  • the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261
  • the present invention provides a polynucleohde encoding a protem comprising the ammo acid sequence of SEQ ID NO 8 from ammo acid 87 to amino acid 164.
  • the present invention provides a composition comprising a protem, wherein said protem comprises an ammo acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of
  • a polynucleohde comprising the nucleohde sequence of the full- length protem codmg sequence of clone AX8_1 deposited under accession number ATCC 98261, (f) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261;
  • a polynucleohde encoding a protein comprising the amino acid sequence of SEQ ID NO:10, (]) a polynucleohde encodmg a protein comp ⁇ smg a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity;
  • such polynucleotide comprises the nucleohde sequence of SEQ ID NO 9 from nucleohde 242 to nucleotide 1060; the nucleohde sequence of SEQ ID NO 9 from nucleotide 596 to nucleotide 1060; the nucleohde sequence of SEQ ID NO:9 from nucleohde 10 to nucleohde 373; the nucleohde sequence of the full-length protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261, or the nucleotide sequence of the mature protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261
  • the present invention provides a polynucleotide encoding a prote
  • the present invention provides a composition comprising a protem, wherem said protein comprises an ammo acid sequence selected from the group consishng of: (a) the ammo acid sequence of SEQ ID NO.10;
  • the present invenhon provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-( ⁇ ).
  • such polynucleohde comprises the nucleotide sequence of SEQ ID NO: 11 from nucleohde 773 to nucleotide 928; the nucleohde sequence of SEQ ID NO.11 from nucleohde 815 to nucleohde 928; the nucleohde sequence of the full-length protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261, or the nucleohde sequence of the mature protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261
  • the present invention provides a composition comprising a protem, wherem said protein comp ⁇ ses an ammo acid sequence selected from the group consisting of
  • the present invenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of
  • such polynucleohde comprises the nucleohde sequence of SEQ ID NO 14 from nucleohde 174 to nucleohde 440; the nucleohde sequence of SEQ ID NO:14 from nucleotide 1 to nucleohde 313; the nucleotide sequence of the full-length protein codmg sequence of clone BD339_1 deposited under accession number ATCC 98261; or the nucleohde sequence of the mature protem coding sequence of clone BD339_1 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert ot clone BD339_1 deposited under accession number ATCC 98261
  • the present invention provides a polynucleohde encoding a protein comprising the amino acid sequence of SEQ ID NO.15 from amino acid 1 to amino acid
  • the present invention provides a composition comprising a protem, wherein said protem comprises an ammo acid sequence selected from the group consisting of:
  • protem (d) the amino acid sequence encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261, the protem being substantially free from other mammalian proteins
  • protem comprises the ammo acid sequence of SEQ ID NO 15 or the ammo acid sequence of SEQ ID NO 15 from ammo acid 1 to ammo acid 46
  • the present mvenhon provides a composition comprising an isolated polvnucleohde selected from the group consisting of
  • a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-( ⁇ )
  • such polynucleohde comprises the nucleotide sequence of SEQ ID NO:16 from nucleohde 509 to nucleohde 619, the nucleohde sequence of SEQ ID NO:16 from nucleohde 1 to nucleohde 580; the nucleotide sequence of the full-length protein codmg sequence of clone BD427_1 deposited under accession number ATCC 98261; or the nucleohde sequence of the mature protein coding sequence of clone BD427_1 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261.
  • the present invention provides a composition comprising a protem, wherem said protem comprises an ammo acid sequence selected from the group consisting of:
  • amino acid sequence encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261 the protein being substantially free from other mammalian proteins
  • protein comprises the ammo acid sequence of SEQ ID NO 17 or the amino acid sequence of SEQ ID NO.17 from amino acid 1 to amino acid 24
  • the present invenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of.
  • a polynucleohde comprising the nucleohde sequence of the full- length protein coding sequence of clone BL229_22 deposited under accession number ATCC 98261, (d) a polynucleohde encoding the full-length protein encoded by the cDNA msert of clone BL229_22 deposited under accession number ATCC 98261,
  • such polynucleohde comprises the nucleotide sequence of SEQ ID NO 18 from nucleotide 300 to nucleohde 360, the nucleohde sequence of the full-length protem coding sequence of clone BL229_22 deposited under accession number ATCC 98261, or the nucleotide sequence of the mature protem coding sequence of clone BL229_22 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protem encoded bv the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 18 from nucleotide 300 to nucleohde 360, the nucleohde sequence of the full-length protem coding sequence of clone BL229_22 deposited under accession number ATCC 98261, or the nucleotide sequence of the
  • the present invention provides a composition comprising a protem, wherein said protein comprises an ammo acid sequence selected from the group consisting of (a) the ammo acid sequence of SEQ ID NO 19,
  • protem the amino acid sequence encoded by the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins.
  • protem comprises the amino acid sequence of SEQ ID NO:19
  • the present mvenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of- (a) a polynucleohde comprising the nucleotide sequence of SEQ ID NO: 1
  • such polynucleohde comprises the nucleohde sequence of SEQ ID NO:21 from nucleohde 604 to nucleotide 771; the nucleohde sequence of SEQ ID NO:21 from nucleohde 1 to nucleohde 684; the nucleohde sequence of the full-length protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261; or the nucleotide sequence of the mature protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261.
  • the polynucleohde encodes the full-length or mature protein encoded bv the cDNA insert of clone BV123_16 deposited under accession number ATCC 98261.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 27.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:22 or the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 27.
  • the present invenhon provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261;
  • a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261;
  • such polynucleohde comprises the nucleotide sequence of SEQ ID NO.23 from nucleotide 43 to nucleo de 297; the nucleotide sequence of SEQ ID NO:23 from nucleotide 94 to nucleotide 297, the nucleohde sequence of SEQ ID NO.23 from nucleohde 1 to nucleohde 379; the nucleohde sequence of the full-length protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261, or the nucleohde sequence of the mature protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261
  • the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261.
  • the present invention provides a composition comprising a protem, wherem said protein comprises an ammo acid sequence selected from the group consisting of.
  • the polynucleohde is operably linked to an expression control sequence
  • the invention also provides a host cell, including bacterial, yeast, msect and mammalian cells, transformed with such polynucleotide compositions. Processes are also provided for producing a protem, which comprise:
  • protem produced according to such methods is also provided by the present invenhon
  • Preferred embodiments include those in which the protein produced by such process is a mature form of the protem
  • 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 invenhon.
  • Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian sub j ect a therapeutically effechve amount of a composition comprising a protem of the present invention and a pharmaceutically acceptable carrier
  • Figures 1A and IB are schemahc representahons of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
  • nucleotide and amino acid sequences are reported below for each clone and protem disclosed in the present applicahon.
  • the nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted ammo acid sequence (both full-length and mature) can then be determined from such nucleohde sequence.
  • the ammo 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 protem and determining its sequence. For each disclosed protein applicants have identified what they have determmed to be the read g 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 ammo acid sequence
  • “Secreted' proteins include without limitation proteins secreted wholly (e.g , soluble protems) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted” proteins also include without limitahon proteins which are transported across the membrane of the endoplasmic rehculum
  • a polynucleotide of the present invention has been identified as clone "AJ20_2 ' AJ20_2 was isolated from a human adult testes cDNA library using methods which are selechve 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 protem AJ20_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AJ20_2 protein").
  • nucleohde sequence of the 5 portion of AJ20_2 as presently determined is reported m SEQ ID NO.l What applicants presently believe is the proper reading frame for the codmg region is indicated m SEQ ID NO.2.
  • the predicted amino acid sequence of the AJ20_2 protem corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO.2
  • Ammo acids 8 to 20 are a predicted leader /signal sequence, with the predicted mature ammo acid sequence beginning at amino acid 21, or are a transmembrane domam. Additional nucleotide sequence from the 3' portion of AJ20_2, including the polyA tail, is reported in SEQ ID NO:3.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone AJ20_2 should be approximately 850 bp
  • nucleotide sequence disclosed herein for AJ20_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. Clone 'AR440 1"
  • AR440_1 A polynucleohde of the present invenhon has been identified as clone "AR440_1 AR440_1 was isolated from a human adult rehna cDNA library usmg methods which are selechve tor 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 ammo acid sequence of the encoded protein.
  • AR440_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "AR440_1 protem”)
  • AR440_1 should be approximately 1400 bp
  • nucleohde sequence disclosed herein for AR440_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols No hits were found in the database
  • nucleohde sequence of AR440_1 indicates that it may contain an Alu repetitive element
  • AS164_1 A polynucleohde of the present invention has been identified as clone "AS164_1' AS164_1 was isolated from a human fetal bram cDNA library usmg methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat No 5,536,637), or was identified as encodmg a secreted or transmembrane protem on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • AS164_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS164_1 protein”)
  • the nucleohde sequence of AS164_1 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 AS164_1 protein corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO:8
  • the EcoRI /Noil restriction fragment obtainable from the deposit containing clone AS164_1 should be approximately 1600 bp.
  • AS164_1 demonstrated at least some similarity with sequences identified as H24668 (yl40hl0.rl Homo sapiens cDNA clone 160771 5'), N29757
  • the predicted ammo acid sequence disclosed herein for AS164_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted AS164_1 protein demonstrated at least some similarity to sequences identified as A20359_l (ryanodine receptor gene product [Homo sapiens]) and U78866 (putahve argrnme-aspartate- ⁇ ch RNA binding protein [Arabidopsis tha ana]) Based upon sequence similarity, AS164_1 proteins and each similar protein or peptide may share at least some activity
  • the predicted AS164_1 protein sequence also contains repeated Asp- Arg RNA-bmdmg mohfs
  • a polynucleotide of the present invention has been identified as clone "AX8_1"
  • AX8_1 was isolated from a human adult testes cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protein.
  • AX8_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "AX8_1 protein")
  • AX8_1 The nucleohde sequence of AX8_1 as presently determmed is reported in SEQ ID NO:9 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the AX8_1 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 10.
  • Ammo acids 106 to 118 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at ammo acid 119, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone AX8_1 should be approximately 2300 bp
  • the TopPredll computer program predicts three potential transmembrane domains within the AX8_1 protein sequence, centered around ammo acids 111, 144, and 182 of SEQ ID NO.10
  • BD176_3 was isolated from a human fetal kidney cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U S Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem BD176_3 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BD176_3 protein”)
  • nucleohde sequence of the 5 portion of BD176_3 as presently determined is reported in SEQ ID NO 11 What applicants presently believe is the proper reading frame for the coding region is indicated m SEQ ID NO 12
  • the predicted ammo acid sequence of the BD176_3 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 12
  • Amino acids 2 to 14 are a predicted leader/signal sequence, with the predicted mature ammo acid sequence beginning at ammo acid 15, or are a transmembrane domain
  • Additional nucleohde sequence from the 3 porhon of BD176_3, including the polyA tail, is reported in SEQ ID NO.13
  • the EcoRI/Notl restriction fragment obtainable from the deposit containmg clone
  • BD176_3 should be approximately 1300 bp
  • BD176_3 The nucleotide sequence disclosed herein for BD176_3 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols. BD176_3 demonstrated at least some similarity with sequences identified as AA029679 (ze94gl0 rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 366690 5 ), D45913 (Mouse NLRR-1 mRNA for leucine- ⁇ ch-repeat protem, complete cds), R55610 (yg88h08.rl Homo sapiens cDNA clone 40606 5'), and T07640 (EST05530 Homo sapiens cDNA clone HFBEM16) The predicted amino acid sequence disclosed herein for BD176_3 was searched agamst the GenPept and GeneSeq ammo acid sequence databases using the BLASTX search protocol.
  • BD176_3 protem demonstrated at least some similarity to sequences identified as D45913 (leucine- ⁇ ch-repeat protein [Mus museums]) and M59472 (asparagine- ⁇ ch anhgen Pfa55-6 [Plasmodium fal ⁇ parum]). Based upon sequence similarity, BD176_3 proteins and each similar protein or peptide may share at least some achvity.
  • BD339_1 A polynucleohde of the present mvenhon has been idenhhed as clone "BD339_1".
  • BD339_1 was isolated from a human fetal kidney cDNA library usmg methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem.
  • BD339_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BD339_1 protem' )
  • the nucleohde sequence of BD339_1 as presently determined is reported in SEQ ID NO: 1
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone BD339_1 should be approximately 650 bp.
  • BD339_1 The nucleotide sequence disclosed herein for BD339_1 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols. BD339_1 demonstrated at least some similarity with sequences idenhhed as H82422 (yu80d08.sl Homo sapiens cDNA clone 240111 3), N62058 (EST53c05 Homo sapiens cDNA clone), U21730 Human 5'-nucleohdase (CD73)), W01979 (za30h09 rl
  • BD339_1 proteins and each similar protem or pephde may share at least some achvity.
  • the TopPredll computer program predicts three potential transmembrane domains withm the BD339_1 protem sequence,centered around ammo acids 14, 46, and 76 of SEQ ID NO:15. Clone "BD427 1"
  • BD427_1 A polynucleohde of the present invention has been idenhhed as clone "BD427_1".
  • BD427_1 was isolated from a human fetal kidney cDNA library using methods which are selechve 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 BD427_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BD427_1 protein”)
  • the EcoRI /Notl restriction fragment obtainable from the deposit containing clone BD427_1 should be approximately 1810 bp
  • the nucleotide sequence disclosed herein for BD427_1 was searched against the
  • GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols BD427_1 demonstrated at least some similarity with sequences idenhhed as AA027122 (zk04a03 rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 469516 5'), N24735 (yx56b02.sl Homo sapiens cDNA clone 265707 3'), and W84644 (zd91a06 rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 356818 5 ) Based upon sequence similarity, BD427_1 proteins and each similar protein or pephde may share at least some achvity.
  • BL229_22 was isolated from a human adult testes cDNA library using methods which are selechve 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 protem BL229_22 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BL229_22 protem").
  • nucleotide sequence of the 5' porhon of BL229_22 as presently determined is reported in SEQ ID NO: 18 What applicants presently believe is the proper readmg frame for the coding region is indicated in SEQ ID NO.19.
  • the predicted ammo acid sequence of the BL229_22 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:19 Additional nucleotide sequence from the 3' portion of BL229_22, including the polyA tail, is reported in SEQ ID NO:20.
  • the EcoRI/Notl restriction fragment obtamable from the deposit contammg clone BL229_22 should be approximately 870 bp.
  • nucleohde sequence disclosed herein for BL229_22 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols No hits were found in the database.
  • BV123_16 A polynucleohde of the present invenhon has been idenhhed as clone "BV123_16".
  • BV123_16 was isolated from a human adult bram cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U.S Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem BV123_16 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BV123_16 protein”)
  • nucleohde sequence of BV123_16 as presently determined is reported in SEQ ID NO.21 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the BV123_16 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO.22.
  • the EcoRI /Notl restriction fragment obtainable from the deposit contammg clone BV123_16 should be approximately 1080 bp
  • nucleohde sequence disclosed herein for BV123_16 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and
  • BV123_16 demonstrated at least some similarity with sequences identified as H29610 (ym61e03.sl Homo sapiens cDNA clone 52653 3'), H52374 (yq81bl2 rl Homo sapiens cDNA clone 202175 5'), H66213 (yul ⁇ hlO.sl Homo sapiens cDNA), L08092 (Homo sapiens dystrophin (DMD) gene, mtron 7, transposon- ke sequence), L35670 (Homo sapiens (subclone H8 10_g5 from PI 35 H5 C8) DNA sequence),
  • M62716 Human CSP-B gene flanking sequence
  • N46985 yy83a05.sl Homo sapiens cDNA clone 2801123'
  • R94603 yq38a04.sl Homo sapiens cDNA clone 198030 3'
  • U91321 Human chromosome 16pl3 BAC clone CIT987SK-363E6, complete sequence
  • Z82200 Human DNA sequence from clone J333E231 Based upon sequence similarity, BV123_16 proteins and each similar protem 01 peptide may share at least some activity.
  • CH377_1 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • CH377_1 is a full-length clone, mcluding the entire coding sequence of a secreted protein (also referred to herein as "CH377_1 protein")
  • nucleohde sequence of CH377_1 as presently determined is reported m SEQ ID NO 23 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the CH377_1 protem corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO 24
  • Amino acids 5 to 17 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18, or are a transmembrane domain
  • the EcoRI /Notl restriction fragment obtainable from the deposit contammg clone CH377_1 should be approximately 570 bp
  • the nucleohde sequence disclosed herein for CH377_1 was searched against the
  • CH377_1 demonstrated at least some similarity with sequences idenhhed as AA507382 (nh73b01.sl NCI_CGAP_Brl 1 Homo sapiens cDNA clone IMAGE 964105) and N70479 (za74fl2.sl Homo sapiens cDNA clone 298319 3') Based upon sequence similarity, CH377_1 proteins and each similar protein or peptide may share at least some activity
  • Clones AJ20_2, AR440_1, AS164_1, AX8_1, BD176_3, BD339_1, BD427_1, BL229_22, BV123_16, and CH377_1 were deposited on November 15, 1996 with the
  • Each clone has been transfected into separate bacterial cells (E coli) in this composite deposit. Each clone can be removed from the vector m which it was deposited by performing an EcoRI/Notl digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate fragment for such clone Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig.
  • the pED6dpc2 vector (“pED6") was derived from pED6dpcl by insertion of a new polylmker 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 polylmker, and insertion of the M13 origin of replication m the Clal site.
  • the deposited clone can become "flipped" (I e , in the reverse orientation) in the deposited isolate
  • the cDNA insert can still be isolated by digestion with EcoRI and Notl However, Notl will then produce the 5' site and EcoRI will produce the 3' site tor placement of the cDNA in proper orientation for expression in a suitable vector
  • the cDNA may also be expressed from the vectors in which they were deposited
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone This sequence can be derived from the sequences provided herein, or from a combination of those sequences The sequence of the oligonucleotide probe that was used to isolate each full-length clone is idenhhed 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- ,2 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 mcorporated into the probe should be quantitated by measurement m 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 moculate a sterile culture flask contammg 25 ml of sterile L-broth containing ampicilhn 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 Ahquots 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 ampicilhn at 100 ⁇ g/ml and agar at 1.5% in a 150 mm pet ⁇ dish when grown overnight at 37°C Other known methods of obtaining dishnct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/hter, 88.2 g Na citrate/liter, adjusted to pH 7 0 with NaOH) contammg 0 5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm hlter).
  • 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 invenhon 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 desc ⁇ bed 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 bmdmg sites.
  • fragments of the protein may be fused through "linker ' sequences to the Fc portion of an immunoglobulin
  • a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions
  • a protein - IgM fusion would generate a decavalent form of the protein of the invenhon
  • the present invention also provides both full-length and mature forms of the disclosed proteins.
  • the full-length form of the such protems is identified in the sequence listing by translation of the nucleohde sequence of each disclosed clone.
  • the mature form of such protein may be obtamed by expression of the disclosed full-length polynucleohde (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • sequence of the mature form of the protein may also be determinable from the ammo acid sequence of the full-length form
  • the present invention also provides genes correspondmg to the cDNA sequences disclosed herein "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which the cDNA sequences are derived and any conhguous regions of the genome necessary for the regulated expression of such genes, mcluding but not limited to codmg sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements
  • the corresponding genes can be isolated m accordance with known methods using the sequence mformahon disclosed herein Such methods include the preparahon of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials Where the protein of the present invenhon is membrane-bound (e g , is a receptor), the present invenhon also provides for soluble forms of such protem In such forms part or all of the mtracellular and transmembr
  • 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 protem, where sequence identity is determmed by comparmg the ammo 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 ammo acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins
  • Species homologs of the disclosed polynucleotides and protems are also provided by the present invention
  • a "species homologue" is a protein or polynucleotide
  • the present mvenhon also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preterably highly stringent condihons, 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 hvb ⁇ d length is that anticipated for the hybridized reg ⁇ on(s) of the hybridizing polynucleotides When hybridizing a polynucleohde to a target polvnucleohde of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleohde When polynucleotides of known sequence are hybridized, the hybrid length can be determined bv aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity
  • ⁇ SSPE (lxSSPE is 0 15M NaCl, lOmM NaH,P0 4 , and 1 25mM EDTA, pH 7 4) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
  • T 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 mav 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)
  • 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 hgated polynucleohde/expression control sequence
  • a number of types of cells may act as suitable host cells for expression of the protem 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 m vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • cell strains derived from m vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast 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 Eschenchia coli, Bacillus subtihs, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous protems If the protem is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylahon or glycosylation of the appropriate sites, in order to obtam the functional protem. Such covalent attachments may be accomplished using known chemical or enzymatic methods
  • the protem may also be produced by operably linking the isolated polynucleohde of the invention to suitable control sequences m one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences m 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.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed"
  • the protein of the invention mav be prepared by culturmg 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 protem may also include an affinity column containing agents which will bind to the protem, one or more column steps over such affinity resins as concanavahn A-agarose, hepa ⁇ n-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography usmg such resms as phenyl ether
  • fusion protein such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxm (TRX).
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxm
  • Kits for expression and purification of such fusion protems are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respechvely
  • the protem 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
  • the protein of the invention may also be expressed as a product of transgenic ammals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells contammg a nucleotide sequence encoding the protein.
  • the protem mav also be produced by known conventional chemical synthesis
  • protems of the present invention by synthetic means are known to those skilled m the art
  • the synthetically-constructed protem 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
  • they mav be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeuhc compounds and in immunological processes for the development of anhbodies
  • protems provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or dehberatelv engineered
  • modifications in the peptide or DNA sequences can be made by those skilled m the art using known techniques
  • Modifications of mterest in the protem sequences may include the alterahon, subs tuhon, replacement, insertion or deletion of a selected amino acid residue m the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another ammo acid to alter the conformation of the molecule.
  • polynucleotides and protems of the present mvenhon 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 mvenhon may be provided by administration or use of such protems or by administration or use of polynucleohdes encodmg such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA)
  • the polynucleohdes provided bv the present invenhon can be used by the research community for various purposes
  • the polynucleohdes can be used to express recombinant protem for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protem is preferentially expressed (either conshtuhvelv or at a particular stage of hssue differenhahon or development or m 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 m patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences, as a source of mformahon to derive PCR primers for genehc fingerprinting; as a probe to "subtract-out known sequences in the process of discovering other novel polynucleohdes; for selechng and making oligo
  • the polynucleohde encodes a protein which bmds or potentially binds to another protem (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyu ⁇ s et al, Cell 75:791-803 (1993)) to identify polynucleohdes encoding the other protem with which binding occurs or to identify inhibitors of the binding interaction
  • the protems 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 (mcluding the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids, as markers for hssues in which the corresponding protem is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements Such uses mclude without limitation use as a protem or ammo acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate
  • the protem or polynucleohde 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 m the form of powder, pills, solutions, suspensions or capsules
  • the protein or polynucleohde of the invention can be added to the medium in or on which the microorganism is cultured
  • a protem of the present invention may exhibit cytokine, cell proliferation (either inducmg or inhibiting) or cell differentiation (either inducmg or inhibiting) achvity or may induce production of other cytokmes m certain cell populations
  • cytokine cytokine
  • cell proliferation either inducmg or inhibiting
  • cell differentiation either inducmg or inhibiting
  • achvity may induce production of other cytokmes m certain cell populations
  • Many protem factors discovered to date, mcluding all known cytokmes have exhibited achvity 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, RB
  • the activity of a protem 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 m: Current Protocols m Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Marguhes, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al, J. Immunol. 137:3494-3500, 1986, Bertagnolli et al, J. Immunol.
  • Assays tor cytokine production and /or proliferation of spleen cells, lymph node cells or thvmocytes include, without limitation, those desc ⁇ bed m: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J E e.a Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoiehc and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukm 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; deV ⁇ es et al., J. Exp. Med. 173.1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl Acad. So. U S.A. 80:2931-2938, 1983,
  • a protem 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 protem mav be useful in the treatment of various immune deficiencies and disorders (mcluding severe combined immunodeficiency (SOD)), e.g., m 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
  • m 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
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, mcluding infections by HIV, hepatitis viruses, herpesvirus
  • Autoimmune disorders which may be treated using a protein of the present invenhon include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroidihs, insulin dependent diabetes mellihs, myasthema gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • Such a protein of the present mvenhon may also to be useful in the treatment of allergic reachons and condihons, such as asthma (particularly allergic asthma) or other respiratory problems
  • Other conditions, in which immune suppression is desired may also be treatable using a protem of the present invention
  • the proteins of the invention may also be possible to immune responses, m 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-anhgen-specific, process which requires continuous exposure of the T cells to the suppressive agent Tolerance, which involves inducing non-responsiveness or anergy m T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tole ⁇ zing agent has ceased Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen m the absence of the tole ⁇ zing 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 lymphokme synthesis by activated T cells, will be useful m situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) For example, blockage of T cell function should result m 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
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural l ⁇ gand(s) on immune cells such as a soluble, monome ⁇ c form of a peptide having B7-2 activity alone or m conjunction with a monome ⁇ c form of a peptide having an activity of another B lymphocyte anhgen (e g , B7-
  • B lymphocyte antigen function 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 an gen-blockmg 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 funchon of a combinahon 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 m humans
  • animal models that are predictive of efficacy m humans
  • appropriate systems which can be used include allogeneic cardiac grafts m rats and xenogeneic pancreahc islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion protems in vivo as described in Lenschow et al , Science 257789-792 (1992) and Turka et al , Proc Natl Acad Sci USA, 89 11102-11105 (1992)
  • 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 cytokmes and autoantibodies involved in the pathology of the diseases
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms
  • Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell achvahon and prevent production of autoantibodies or T cell-derived cytokmes which may be involved in the disease process
  • blocking reagents may induce antigen-specific tolerance of autoreachve T cells which could lead to long-term relief from the disease
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lprfl
  • anti-viral immune responses may be enhanced m an mfected patient by removmg 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 remtroducing the in vitro achvated T cells into the pahent.
  • 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 o ⁇ the protein on their surface, and reintroduce the transfected cells into the patient
  • the mfected cells would now be capable of delivering a costimulatorv 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
  • the tumor cell can be transfected to express a combination of pephdes.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector direchng the expression ot a peptide having B7-2-hke activity alone, or in conjunction with a peptide having B7-l-l ⁇ ke activity and /or B7-3-hke 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
  • rumor 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 protem and ⁇ 2 microglobulm protein or an MHC class II a chain protem and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II protems on the cell surface.
  • a gene encoding an anhsense construct which blocks expression of an MHC class II associated protein, such as the mva ⁇ ant 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 anhgens 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 achvity of a protem of the mvenhon may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitahon, those described in: Current Protocols m Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Marguhes, 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.
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Mahszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, protems expressed by dendritic cells that achvate naive T-cells) include, without limitation, those described in. Guery et al, J.
  • lymphocyte survival/apoptosis which will identify, among others, proteins that prevent apoptosis after superanhgen induction and protems that regulate lymphocyte homeostasis
  • Assays for lymphocyte survival/apoptosis include, without limitahon, those described m: 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 protems that influence early steps of T-cell commitment and development include, without limitation, those described in: Anhca 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 margmal biological achvity in support of colony forming cells or of factor-dependent cell lines mdicates involvement in regulating hematopoiesis, e.g.
  • m supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokmes 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 supporhng 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; m supporhng the growth and proliferation of megakarvocvtes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocvtopema, and generally for use m place of or complimentary to platelet transfusions, and /or in supporting the growth and proliferation of hematopoie c stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find
  • achvitv of a protein of the invention mav, among other means, be measured bv the following methods Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al Cellular Biology 15:141-151, 1995, Keller et al , Molecular and Cellular Biology 13.473-486, 1993, McClanahan et al , Blood
  • Assays for stem cell survival and differentiation include, without limitation, those described m. 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 proliferahve potential, McNTece, LK.
  • a protem of the present mvenhon 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 m 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 protem of the invenhon may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation ot artificial j oints
  • De novo bone formation induced by an osteogenic agent contributes to the repair of congemtal, trauma induced, or oncologic resection induced cramofacial defects, and also is useful in cosmetic plastic surgery
  • a protein of this invention mav also be used in the treatment of periodontal disease, and in other tooth repair processes
  • Such agents may prov ide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells
  • a protem ot the mvenhon may also be useful in the treatment of osteoporosis or osteoarth ⁇ tis, 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 mav be attributable to the protein of the present invention is tendon/ligament formation
  • a protein of the present invention which induces tendon/ gament-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, deformihes and other tendon or ligament defects in humans and other animals
  • the protein of the present invention mav also be useful for proliferation of neural cells and for regeneration of nerve and bram tissue, i e for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degenerahon death or trauma to neural cells or nerve tissue
  • a protein mav 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 svstem diseases, such as Alzheimer s, Parkinson s disease, Hunhngton s disease, amvotrophic lateral sclerosis, and Shy-Drager syndrome
  • diseases of the peripheral nervous system such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous svstem diseases, such as Alzheimer s, Parkinson s disease, Hunhngton s disease, amvotrophic lateral sclerosis, and Shy-Drager syndrome
  • Further conditions which mav be treated in accordance with the present invenhon m clude mechanical and fraumah
  • Protems of the invenhon may also be useful to promote better or faster closure of non-healing wounds, including without limitahon pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like
  • a protem 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, skm, endothelium), muscle (smooth, skeletal or cardiac) and vascular (mcludmg 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 hbrosis, reperfusion in j ury 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 achvity of a protein of the invention may, among other means, be measured by the following methods.
  • Assays for hssue generation activity include, without limitation, those described in. International Patent Publication No W095/ 16035 (bone, cartilage, tendon); International Patent Publication No W095/ 05846 (nerve, neuronal), International Patent Publication No. WO91/07491 (sk , endothelium )
  • 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 activms and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a protem of the present invention alone or m heterodimers with a member of the mhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • the protem of the mvenhon may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules m stimulating FSH release from cells of the anterior pituitary.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to mcrease the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protem of the invention may, among other means, be measured by the following methods- Assays for activin /inhibin achvity 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. So USA 83.3091-3095, 1986
  • a protem of the present invention may have chemotachc or chemokmetic activity (e.g , act as a chemokme) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells.
  • chemotachc or chemokmetic activity e.g , act as a chemokme
  • mammalian cells including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells.
  • Chemotactic and chemokmetic proteins can be used to mobilize or attract a desired cell population to a desired site of action
  • Chemotactic or chemokmetic 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 shmulate directed movement of cells Whether a particular protem has chemotactic activity for a populahon of cells can be readily determined by employing such protem or peptide in any known assay for cell chemotaxis.
  • the achvity of a protem of the invenhon may, among other means, be measured by the following methods
  • Assays for chemotachc achvity 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 m clude, without limitahon, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Marguhes, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokmes 6.12.1-6.12.28; Taub et al.
  • a protein of the invention may also exhibit hemostahc or thrombolyhc activity As a result such a protem is expected to be useful in treatment of various coagulation disorders (mcluding hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostahc events in treating wounds resulting from trauma, surgery or other causes
  • a protem of the invention mav 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 , stroke)
  • the achvity of a protein of the invenhon may, among other means, be measured by the following methods
  • Assav for hemostahc and thrombolvhc achvity m clude, without limitation, those described m Linet et al , J Chn Pharmacol 26 131-140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrev et al , Fibrmolvsis 5 71-79 (1991), Schaub, Prostaglandms 35 467-474, 1988
  • a protein of the present invention mav 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 selechns, mtegrms and their ligands) and receptor/ gand 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/hgand interaction
  • a protein of the present invention may themselves be useful as inhibitors of receptor/hgand interactions
  • the activity of a protem of the invention may, among other means, be measured by the following
  • Suitable assays for receptor-ligand activity include without limitation those described m Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, 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, Shtt et al., Cell 80:661-670, 1995.
  • Protems of the present invenhon may also exhibit anti-inflammatory achvity.
  • 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
  • Protems exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as sephc shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion in j ury, endotoxm lethality, arthritis, complement-mediated hyperacute re j ection, nephritis, cytokine or chemokme-mduced lung mjury, inflammatory bowel disease, Crohn s disease or resulting from
  • Cadhe ⁇ ns are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types Loss or alterahon of normal cadherm expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherm malfunction is also implicated in other human diseases, such as pemphigus vulga ⁇ s and pemphigus fo aceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
  • the cadhe ⁇ n superfamily includes well over forty members, each with a distinct pattern of expression All members of the superfamily have in common conserved extracellular repeats (cadherm domains), but structural differences are found in other parts of the molecule.
  • the cadherm domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion Only a few ammo acids in the first cadherm domain provide the basis for homophi c adhesion; modification of this recognition site can change the specificity of a cadherm so that mstead of recognizing only itself, the mutant molecule can now also bind to a different cadhe ⁇ n. In addition, some cadherms engage in heterophihc adhesion with other cadhenns E-cadhe ⁇ n, one member of the cadhe ⁇ n superfamily, is expressed m epithelial cell types.
  • Cancer cells have also been shown to express cadherms of a different tissue type than their origin, thus allowing these cells to invade and metastasize m a different tissue in the body.
  • Proteins of the present mvenhon with cadherm achvity, and polynucleohdes of the present invention encoding such proteins can be substituted in these cells for the inappropriately expressed cadherms, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize
  • proteins of the present invention with cadherm activity can be used to generate antibodies recognizing and binding to cadherms
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherms, prevenhng the cells from formmg a tumor elsewhere.
  • Such an anti-cadherm 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 cadherm expression there will be, and this decrease m cadherm expression can be detected by the use of a cadhe ⁇ n-bmding antibody.
  • Fragments of proteins of the present invention with cadherm activity preferably a polypeptide comprising a decapephde of the cadherm recognition site, and polynucleotides of the present invention encoding such protem 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.
  • 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 characterishcs, 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;
  • 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 ca ⁇ ier) 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 effechveness 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, TNF1, 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 mul timers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and /or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes B lymphocytes will respond to antigen through their surface immunoglobulin receptor T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins MHC and structurally related protems 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
  • TCR T cell receptor
  • the antigen components could also be supplied as purified MHC-pephde complexes alone or with co-stimulatory molecules that can directly signal T cells Alternatively antibodies able to bind surface lmmunolgobu n 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 invenhon may be in the form of a liposome in which protein of the present mvenhon is combined, m addition to other pharmaceutically acceptable carriers, with amphipathic agents such as hpids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers m aqueous solution
  • hpids for liposomal formulation include, without limitation, monoglyce ⁇ des, diglyce ⁇ des, sulfatides, lysolecithin, phosphohpids, saponin, bile acids, and the like
  • Preparation of such liposomal formulahons is withm 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, l e , treatment, healing, prevenhon or amelioration of the relevant medical condition, or an increase m rate of treatment, healmg, prevention or amelioration of such conditions
  • an individual active ingredient, administered alone the term refers to that mgredient alone
  • the term refers to combined amounts of the active ingredients that result m the therapeutic effect, whether administered in combination, se ⁇ ally or simultaneously
  • a therapeutically effective amount of protem 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 mvenhon either alone or in combmahon with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors When co-administered with one or more cytokines, lymph
  • 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, mtrape ⁇ toneal, 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 pharmaceuhcal composihon of the mvenhon may additionally contain a solid carrier such as a gelatin or an ad j uvant
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present mvenhon, and preferably from about 25 to 90% prote 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 saccha ⁇ de solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol
  • the pharmaceuhcal composihon contains from about 0 5 to
  • protem of the present invention When a therapeutically effective amount of protem of the present invention is admmistered bv intravenous, cutaneous or subcutaneous in j ection, protein of the present mvenhon will be m the form of a pyrogen-free, parenterally acceptable aqueous solution
  • a preferred pharmaceuhcal composihon for intravenous, cutaneous, or subcutaneous in j ection should contain, m addition to protem of the present invention, an isotomc vehicle such as Sodium Chloride Injection, Ringer s Injection, Dextrose In j ection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • an isotomc vehicle such as Sodium Chloride Injection, Ringer s Injection, Dextrose In j ection, 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 protem of the present invenhon in the pharmaceuhcal composihon of the present invenhon 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.
  • compositions used to practice the method of the present mvenhon should contain about 0 01 ⁇ g to about 100 mg (preferably about 0 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 durahon of each applicahon of the protem 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 durahon of intravenous therapy using the pharmaceutical composition of the present invenhon
  • 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 pephde immunogens additionally may contam a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH)
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies bmdmg to the protein of the invention may be useful diagnostic agents for the lmmunodetection of the protem.
  • Neutralizing monoclonal antibodies bmdmg to the protein may also be useful therapeutics for both condihons associated with the protein and also m the treatment of some forms of cancer where abnormal expression of the protem is involved.
  • 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 mjected 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 protem of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be admmistered simultaneously or sequentially with the composition in the methods of the invention
  • the composihon would include a matrix capable of the composition in the methods of the invention.
  • compositions may be biodegradable and chemically defined calcium sulfate, tncalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhyd ⁇ des
  • potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen
  • Further matrices are comprised of pure protems 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 tncalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium- alummate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty.
  • a sequestering agent such as carboxymethvl 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- methvlcellulose, and carboxymethylcellulose, the most preferred being catiomc salts of carboxvmethvlcellulose (CMC)
  • CMC carboxvmethvlcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polvmer and poly(v ⁇ nvl alcohol)
  • the amount of sequestermg 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 polvmer matrix and to provide appropriate handling of the composihon, yet not so much that the progenitor cells are prevented from infil
  • protems of the invention may be combined with other agents beneficial to the treatment of the bone and/or carhlage defect, wound, or tissue in queshon
  • agents include various growth factors such as epidermal growth factor
  • EGF platelet derived growth factor
  • TGF- ⁇ transforming growth factors
  • TGF- ⁇ TGF- ⁇
  • IGF insulin-like growth factor
  • compositions are also presently valuable for veterinary applications
  • the dosage regimen of a protem-contammg pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the protems, 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 hssue (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 m 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, histomorphomet ⁇ c determinations and tetracyclme labeling.
  • Polynucleohdes of the present mvenhon can also be used for gene therapy. Such polynucleohdes can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleohdes of the mvenhon may also be administered by other known methods for mfroduchon of nucleic acid into a cell or organism (mcludmg, 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 invenhon 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.
  • CTGTGTTCCA GCTGACAGAC GTGCTAATTA TCCTGTTTTC TGTGTCCAGA CAAAGACTAC 300
  • CTGCTCTAGG 430 (2) INFORMATION FOR SEQ ID NO : 2 :
  • CACGAAGCCC CAGCATTAT TGTGGATGAA TATAAGGCAC TACTGCAGTC TCAGTTAAG 480
  • Gin Glu Ala lie Ser Phe Gin Asp Arg Tyr Lys Glu Leu Gin Glu Lys 20 25 30
  • TGCAAGAGTC ACCTTCTGGA CTGCTGCCCC CATGACATCC TGGCTGGGAC GCGCATGGAT 240 TTAGGAGAAT GTACCAAAAT CCACGACTTG GCCCTCCGAG CAGATTATGA GATTGCAAGT 300
  • CTCCTTGCTA AAGCCGAACA GCTAGGGGCT GAAGGTAATG TGGATGAATC CCAGAAGATT 540
  • MOLECULE TYPE protein
  • Glu Glu lie Ser Ala Glu Val Ser Ala Lys Ala Gly Lys Val His Glu 115 120 125
  • Val Ala Arg lie lie Asn Gly lie lie lie lie Ser Val Lys Thr Arg Ser 210 215 220
  • Lys lie 245 250 255
  • Asn Asn Ala Val Leu Thr Thr Val Leu Asn Lys Met lie Asn Asp Arg 20 25 30 Met Gly Phe Pro Cys Ala Phe Thr Ser Ser Met Thr Leu Pro Glu Ala 35 40 45 lie Arg Arg Lys 50
  • Trp Val Lys Val lie Gin Lys Leu Tyr Thr lie Phe Ala Phe Phe Ser 35 40 45
  • GTCACTTTCA GATTTCAATT TGAGGTTAAG TATATAAAGC ACATCCCAAT TTTATATGCT 180 GCCTTGAGAA AATTACAGGA TGCACGGCAA TTTGTAGGAA TTTCAAATGG GATCATTTAA 240
  • TTCTCTAGTC AATATCTTTA GTGATYTTYT TTAATAAACA TGRAAGCAAA GRAAAAAAAA 480

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 the following applications: Ser. No. 08/749,745, filed November 15, 1996, and Ser No. 08/867,678, filed June 2, 1997
FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins
BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e g , cytokmes, such as lymphokines, interferons, CSFs and mterleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polvnucleotides "directlv" in the sense that they relv 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 vaπous 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 68 to nucleotide 430;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 128 to nucleotide 430; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone AJ20_2 deposited under accession number ATCC 98261;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ20_2 deposited under accession number ATCC 98261;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:l from nucleotide 68 to nucleotide 430; the nucleotide sequence of SEQ ID NO:l from nucleotide 128 to nucleotide 430; the nucleotide sequence of the full-length protein coding sequence of clone AJ20_2 deposited under accession number ATCC 98261; or the nucleotide sequence of the mature protein coding sequence of clone AJ20_2 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:l or SEQ ID NO:3.
In other embodiments, the present invention provides a composition compπsing a protein, wherem said protein comprises an amino acid sequence selected from the group consisting of:
(a) the ammo acid sequence of SEQ ID NO.2, (b) fragments of the ammo acid sequence of SEQ ID NO.2; and
(c) the ammo acid sequence encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence 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.5,
(b) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO:5 from nucleotide 289 to nucleotide 780,
(c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone AR440_1 deposited under accession number ATCC 98261;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261,
(e) a polynucleotide compπsmg the nucleotide sequence of the mature protein coding sequence of clone AR440_1 deposited under accession number ATCC 98261,
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(h) a polynucleotide encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO:6 having biological activity; (1) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protem of (g) or (h) above ; and (k) a polynucleohde capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:5 from nucleotide 289 to nucleotide 780; the nucleotide sequence of the full-length protein coding sequence of clone AR440_1 deposited under accession number ATCC 98261, or the nucleohde sequence of the mature protem coding sequence of clone AR440_1 deposited under accession number ATCC 98261. In other preferred embodiments, the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the ammo acid sequence of SEQ ID NO 6 from amino acid 1 to ammo acid 160.
Other embodiments provide the gene corresponding to the cDN A sequence of SEQ
ID NO 5 or SEQ ID NO-4
In other embodiments, the present invention provides a composition comprising a protem, wherein said protein comprises an ammo acid sequence selected from the group consisting of.
(a) the ammo acid sequence of SEQ ID NO.6;
(b) the amino acid sequence of SEQ ID NO.6 from ammo acid 1 to amino acid 160,
(c) fragments of the amino acid sequence of SEQ ID NO.6; and (d) the amino acid sequence encoded by the cDNA insert of clone
AR440_1 deposited under accession number ATCC 98261; 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 ammo acid 1 to amino acid 160. 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 polynucleohde comprising the nucleotide sequence of SEQ ID NO:7 from nucleohde 76 to nucleohde 1050;
(c) a polynucleohde comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 331 to nucleohde 567, (d) a polynucleotide comprising the nucleohde sequence of the full- length protein coding sequence of clone AS164_1 deposited under accession number ATCC 98261,
(e) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261; (f) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone AS164_1 deposited under accession number
ATCC 98261,
(g) a polynucleo de encoding the mature protein encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261, (h) a polynucleotide encoding a protem comprising the ammo acid sequence of SEQ ID NO'8,
(I) a polynucleohde encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO 8 having biological activity,
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (I) above , and
(1) a polvnucleohde capable of hvbπdizmg under stringent condihons to any one of the polynucleotides specified in (a)-(ι) Preferably, such polynucleohde comprises the nucleohde sequence of SEQ ID
NO.7 from nucleohde 76 to nucleohde 1050, the nucleohde sequence of SEQ ID NO.7 from nucleohde 331 to nucleohde 567; the nucleotide sequence of the full-length protein codmg sequence of clone AS164_1 deposited under accession number ATCC 98261; or the nucleohde sequence of the mature protein coding sequence of clone AS164_1 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261 In yet other preferred embodiments, the present invention provides a polynucleohde encoding a protem comprising the ammo acid sequence of SEQ ID NO 8 from ammo acid 87 to amino acid 164.
Other embodiments provide the gene coπespondmg to the cDNA sequence of SEQ ID NO.7 In other embodiments, the present invention provides a composition comprising a protem, wherein said protem comprises an ammo acid sequence selected from the group consisting of:
(a) the ammo acid sequence of SEQ ID NO:8;
(b) the ammo acid sequence of SEQ ID NO.8 from ammo acid 87 to amino acid 164,
(c) fragments of the amino acid sequence of SEQ ID NO.8; and
(d) the amino acid sequence encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261, the protem 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 ammo acid 87 to amino acid 164 in one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide comprising the nucleohde sequence of SEQ ID NO.9,
(b) a polynucleohde comprising the nucleohde sequence of SEQ ID NO.9 from nucleotide 242 to nucleotide 1060,
(c) a polynucleohde comprising the nucleohde sequence of SEQ ID NO:9 from nucleohde 596 to nucleotide 1060, (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO-9 from nucleotide 10 to nucleohde 373,
(e) a polynucleohde comprising the nucleohde sequence of the full- length protem codmg sequence of clone AX8_1 deposited under accession number ATCC 98261, (f) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261;
(g) a polynucleohde compπsmg the nucleohde sequence of the mature protem codmg sequence of clone AX8_1 deposited under accession number ATCC 98261; (h) a polynucleohde encoding the mature protem encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261;
(1) a polynucleohde encoding a protein comprising the amino acid sequence of SEQ ID NO:10, (]) a polynucleohde encodmg a protein compπsmg a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleohde of (a)-(h) above;
(1) a polynucleohde which encodes a species homologue of the protem of (I) or (j) above ; and
(m) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(j)
Preferably, such polynucleotide comprises the nucleohde sequence of SEQ ID NO 9 from nucleohde 242 to nucleotide 1060; the nucleohde sequence of SEQ ID NO 9 from nucleotide 596 to nucleotide 1060; the nucleohde sequence of SEQ ID NO:9 from nucleohde 10 to nucleohde 373; the nucleohde sequence of the full-length protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261, or the nucleotide sequence of the mature protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protem comprising the amino acid sequence of SEQ ID NO.10 from amino acid 1 to ammo acid 44. 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 protem, wherem said protein comprises an ammo acid sequence selected from the group consishng of: (a) the ammo acid sequence of SEQ ID NO.10;
(b) the ammo acid sequence of SEQ ID NO.10 from amino acid 1 to amino acid 44;
(c) fragments of the ammo acid sequence of SEQ ID NO: 10; and (d) the ammo acid sequence encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261, the protem being substantially free from other mammalian proteins. Preferably such protein compπses the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to ammo acid 44
In one embodiment, the present invenhon provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleohde comprising the nucleohde sequence of SEQ ID NO:ll; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO 11 from nucleohde 773 to nucleohde 928;
(c) a polynucleohde comprising the nucleotide sequence of SEQ ID NO 11 from nucleotide 815 to nucleohde 928,
(d) a polynucleohde comprising the nucleotide sequence of the full- length protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261,
(e) a polynucleotide encoding the full-length protem encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261;
(f) a polynucleohde compπsmg the nucleohde sequence of the mature protem coding sequence of clone BD176_3 deposited under accession number
ATCC 98261,
(g) a polynucleohde encoding the mature protein encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding a protem comprising the amino acid sequence of SEQ ID NO.12,
(l) a polynucleohde encodmg a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological achvity;
(j) a polynucleohde which is an allelic variant of a polynucleohde of
(a)-(g) above, (k) a polynucleohde which encodes a species homologue of the protem of (h) or (I) above ; and
(1) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(ι). Preferably, such polynucleohde comprises the nucleotide sequence of SEQ ID NO: 11 from nucleohde 773 to nucleotide 928; the nucleohde sequence of SEQ ID NO.11 from nucleohde 815 to nucleohde 928; the nucleohde sequence of the full-length protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261, or the nucleohde sequence of the mature protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO.ll or SEQ ID NO.13.
In other embodiments, the present invention provides a composition comprising a protem, wherem said protein compπses an ammo acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO:12, (b) fragments of the amino acid sequence of SEQ ID NO 12, and
(c) the ammo acid sequence encoded by the cDNA insert of clone
BD176_3 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins. Preferably such protem comprises the amino acid sequence of SEQ ID NO.12. In one embodiment, the present invenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of
(a) a polynucleotide comprising the nucleohde sequence of SEQ ID NO.14,
(b) a polynucleohde comprising the nucleo de sequence of SEQ ID NO:14 from nucleohde 174 to nucleohde 440;
(c) a polynucleohde comprising the nucleotide sequence of SEQ ID NO 14 from nucleohde 1 to nucleo de 313,
(d) a polynucleohde comprising the nucleohde sequence of the full- length protein coding sequence of clone BD339_1 deposited under accession number ATCC 98261;
(e) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261; (f) a polynucleohde compπsmg the nucleohde sequence of the mature protem coding sequence of clone BD339_1 deposited under accession number ATCC 98261,
(g) a polynucleohde encoding the mature protem encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261;
(h) a polynucleohde encoding a protein comprising the ammo acid sequence of SEQ ID NO.15,
(l) a polynucleohde encodmg a protein comprising a fragment of the amino acid sequence of SEQ ID NO.15 having biological activity; (j) a polynucleohde which is an allelic variant of a polynucleohde of
(a)-(g) above,
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (l) above , and
(1) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(ι)
Preferably, such polynucleohde comprises the nucleohde sequence of SEQ ID NO 14 from nucleohde 174 to nucleohde 440; the nucleohde sequence of SEQ ID NO:14 from nucleotide 1 to nucleohde 313; the nucleotide sequence of the full-length protein codmg sequence of clone BD339_1 deposited under accession number ATCC 98261; or the nucleohde sequence of the mature protem coding sequence of clone BD339_1 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert ot clone BD339_1 deposited under accession number ATCC 98261 In yet other preferred embodiments, the present invention provides a polynucleohde encoding a protein comprising the amino acid sequence of SEQ ID NO.15 from amino acid 1 to amino acid
46.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:14.
In other embodiments, the present invention provides a composition comprising a protem, wherein said protem comprises an ammo acid sequence selected from the group consisting of:
(a) the ammo acid sequence of SEQ ID NO:15;
(b) the amino acid sequence of SEQ ID NO.15 from ammo acid 1 to
Figure imgf000012_0001
(c) fragments of the ammo acid sequence of SEQ ID NO 15, and
(d) the amino acid sequence encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261, the protem being substantially free from other mammalian proteins Preferably such protem comprises the ammo acid sequence of SEQ ID NO 15 or the ammo acid sequence of SEQ ID NO 15 from ammo acid 1 to ammo acid 46
In one embodiment, the present mvenhon provides a composition comprising an isolated polvnucleohde selected from the group consisting of
(a) a polynucleotide comprising the nucleohde sequence of SEQ ID NO 16,
(b) a polvnucleohde comprising the nucleotide sequence of SEQ ID NO 16 from nucleotide 509 to nucleohde 619,
(c) a polvnucleohde comprising the nucleohde sequence of SEQ ID NO 16 from nucleo de 1 to nucleohde 580, (d) a polvnucleohde comprising the nucleohde sequence of the full- length protein codmg sequence of clone BD427_1 deposited under accession number ATCC 98261,
(e) a polynucleohde encoding the full-length protem encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261, (f) a polvnucleohde compπsmg the nucleohde sequence of the mature protem coding sequence of clone BD427_1 deposited under accession number ATCC 98261,
(g) a polynucleohde encodmg the mature protein encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261, (h) a polvnucleohde encoding a protein comprising the amino acid sequence of SEQ ID NO 17,
(I) a polynucleohde encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO 17 having biological activity,
()) a polynucleohde which is an allelic variant of a polynucleohde of (a)-(g) above,
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (l) above , and
(1) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(ι) Preferably, such polynucleohde comprises the nucleotide sequence of SEQ ID NO:16 from nucleohde 509 to nucleohde 619, the nucleohde sequence of SEQ ID NO:16 from nucleohde 1 to nucleohde 580; the nucleotide sequence of the full-length protein codmg sequence of clone BD427_1 deposited under accession number ATCC 98261; or the nucleohde sequence of the mature protein coding sequence of clone BD427_1 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protem encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261. In yet other preferred embodiments, the present invention provides a polynucleohde encoding a protem comprising the ammo acid sequence of SEQ ID NO:17 from amino acid 1 to amino acid 24
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 16.
In other embodiments, the present invention provides a composition comprising a protem, wherem said protem comprises an ammo acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO.17,
(b) the amino acid sequence of SEQ ID NO 17 from amino acid 1 to amino acid 24, (c) fragments of the amino acid sequence of SEQ ID NO.17; and
(d) the amino acid sequence encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins Preferably such protein comprises the ammo acid sequence of SEQ ID NO 17 or the amino acid sequence of SEQ ID NO.17 from amino acid 1 to amino acid 24
In one embodiment, the present invenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of.
(a) a polynucleohde comprising the nucleohde sequence of SEQ ID NO:18, (b) a polynucleohde comprising the nucleohde sequence of SEQ ID
NO: 18 from nucleohde 300 to nucleohde 360,
(c) a polynucleohde comprising the nucleohde sequence of the full- length protein coding sequence of clone BL229_22 deposited under accession number ATCC 98261, (d) a polynucleohde encoding the full-length protein encoded by the cDNA msert of clone BL229_22 deposited under accession number ATCC 98261,
(e) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone BL229_22 deposited under accession number ATCC 98261,
(f) a polynucleohde encoding the mature protein encoded by the cDNA msert of clone BL229_22 deposited under accession number ATCC 98261,
(g) a polynucleohde encoding a protem comprising the amino acid sequence of SEQ ID NO 19, (h) a polvnucleohde encoding a protem comprising a fragment of the ammo acid sequence of SEQ ID NO 19 having biological activity,
(l) a polvnucleohde which is an allelic variant of a polynucleohde of
(a)-(f) above,
()) a polvnucleohde which encodes a species homologue of the protem of (g) or (h) above , and
(k) a polynucleohde capable of hybridizing under stringent condihons to anv one of the polvnucleohdes specified in (a)-(h)
Preferably, such polynucleohde comprises the nucleotide sequence of SEQ ID NO 18 from nucleotide 300 to nucleohde 360, the nucleohde sequence of the full-length protem coding sequence of clone BL229_22 deposited under accession number ATCC 98261, or the nucleotide sequence of the mature protem coding sequence of clone BL229_22 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protem encoded bv the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO 18 or SEQ ID NO 20
In other embodiments, the present invention provides a composition comprising a protem, wherein said protein comprises an ammo acid sequence selected from the group consisting of (a) the ammo acid sequence of SEQ ID NO 19,
(b) fragments of the amino acid sequence of SEQ ID NO 19, and
(c) the amino acid sequence encoded by the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins. Preferably such protem comprises the amino acid sequence of SEQ ID NO:19
In one embodiment, the present mvenhon provides a composition comprising an isolated polynucleohde selected from the group consisting of- (a) a polynucleohde comprising the nucleotide sequence of SEQ ID
NO.21,
(b) a polynucleotide comprising the nucleohde sequence of SEQ ID NO:21 from nucleohde 604 to nucleohde 771;
(c) a polynucleohde comprising the nucleotide sequence of SEQ ID NO:21 from nucleohde 1 to nucleotide 684,
(d) a polynucleohde comprising the nucleotide sequence of the full- length protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261,
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BV123_16 deposited under accession number ATCC 98261,
(f) a polynucleohde comprising the nucleotide sequence of the mature protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261,
(g) a polynucleotide encoding the mature protein encoded by the cDNA msert of clone BV123_16 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO.22,
(I) a polynucleohde encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO.22 having biological activity, (j) a polynucleohde which is an allelic variant of a polynucleohde of
(a)-(g) above,
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (l) above ; and
(1) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(ι).
Preferably, such polynucleohde comprises the nucleohde sequence of SEQ ID NO:21 from nucleohde 604 to nucleotide 771; the nucleohde sequence of SEQ ID NO:21 from nucleohde 1 to nucleohde 684; the nucleohde sequence of the full-length protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261; or the nucleotide sequence of the mature protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261. In other preferred embodiments, the polynucleohde encodes the full-length or mature protein encoded bv the cDNA insert of clone BV123_16 deposited under accession number ATCC 98261. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 27.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:21. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:22;
(b) the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 27;
(c) fragments of the amino acid sequence of SEQ ID NO:22; and
(d) the amino acid sequence encoded by the cDNA insert of clone BV123_16 deposited under accession number ATCC 98261; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:22 or the amino acid sequence of SEQ ID NO:22 from amino acid 1 to amino acid 27.
In one embodiment, the present invenhon provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23 from nucleotide 43 to nucleotide 297;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23 from nucleohde 94 to nucleotide 297; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:23 from nucleotide 1 to nucleotide 379;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261; (f) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261;
(g) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261,
(h) a polynucleohde encoding the mature protein encoded by the cDNA msert of clone CH377_1 deposited under accession number ATCC 98261;
(I) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:24; (j) a polynucleohde encodmg a protein comprising a fragment of the ammo acid sequence of SEQ ID NO.24 having biological activity,
(k) a polynucleohde which is an allelic variant of a polynucleohde of (a)-(h) above;
(1) a polynucleohde which encodes a species homologue of the protem of (l) or (j) above , and
(m) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleohde comprises the nucleotide sequence of SEQ ID NO.23 from nucleotide 43 to nucleo de 297; the nucleotide sequence of SEQ ID NO:23 from nucleotide 94 to nucleotide 297, the nucleohde sequence of SEQ ID NO.23 from nucleohde 1 to nucleohde 379; the nucleohde sequence of the full-length protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261, or the nucleohde sequence of the mature protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261 In other preferred embodiments, the polynucleohde encodes the full-length or mature protein encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261.
Other embodiments provide the gene correspondmg to the cDNA sequence of SEQ ID NO.23.
In other embodiments, the present invention provides a composition comprising a protem, wherem said protein comprises an ammo acid sequence selected from the group consisting of.
(a) the ammo acid sequence of SEQ ID NO:24,
(b) fragments of the amino acid sequence of SEQ ID NO:24, and (c) the amino acid sequence encoded by the cDNA insert of clone
CH377_1 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins. Preferably such protem compπses the amino acid sequence of SEQ ID NO.24. In certain preferred embodiments, the polynucleohde is operably linked to an expression control sequence The invention also provides a host cell, including bacterial, yeast, msect and mammalian cells, transformed with such polynucleotide compositions. Processes are also provided for producing a protem, which comprise:
(a) growing a culture of the host cell transformed with such polynucleohde compositions in a suitable culture medium; and
(b) puπfving the protem from the culture.
The protem produced according to such methods is also provided by the present invenhon Preferred embodiments include those in which the protein produced by such process is a mature form of the protem 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 invenhon.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effechve amount of a composition comprising a protem of the present invention and a pharmaceutically acceptable carrier
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are schemahc representahons 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 protem disclosed in the present applicahon. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted ammo acid sequence (both full-length and mature) can then be determined from such nucleohde sequence. The ammo 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 protem and determining its sequence. For each disclosed protein applicants have identified what they have determmed to be the read g 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 ammo acid sequence "Secreted' proteins include without limitation proteins secreted wholly (e.g , soluble protems) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitahon proteins which are transported across the membrane of the endoplasmic rehculum
Clone 'AT20 2"
A polynucleotide of the present invention has been identified as clone "AJ20_2 ' AJ20_2 was isolated from a human adult testes cDNA library using methods which are selechve 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 protem AJ20_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AJ20_2 protein").
The nucleohde sequence of the 5 portion of AJ20_2 as presently determined is reported m SEQ ID NO.l What applicants presently believe is the proper reading frame for the codmg region is indicated m SEQ ID NO.2. The predicted amino acid sequence of the AJ20_2 protem corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO.2 Ammo acids 8 to 20 are a predicted leader /signal sequence, with the predicted mature ammo acid sequence beginning at amino acid 21, or are a transmembrane domam. Additional nucleotide sequence from the 3' portion of AJ20_2, including the polyA tail, is reported in SEQ ID NO:3.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone AJ20_2 should be approximately 850 bp
The nucleotide sequence disclosed herein for AJ20_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. Clone 'AR440 1"
A polynucleohde of the present invenhon has been identified as clone "AR440_1 AR440_1 was isolated from a human adult rehna cDNA library usmg methods which are selechve tor 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 ammo acid sequence of the encoded protein. AR440_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "AR440_1 protem")
The parhal nucleohde sequence of AR440_1, including its 3' end and any identified polyA tail, as presently determined is reported in SEQ ID NO 5 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO.6 The predicted amino acid sequence of the AR440_1 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO.6 Additional nucleohde sequence from the 5 porhon of AR440_1 is reported m SEQ ID NO 4 The EcoRI/Notl restriction fragment obtainable from the deposit containing clone
AR440_1 should be approximately 1400 bp
The nucleohde sequence disclosed herein for AR440_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols No hits were found in the database The nucleohde sequence of AR440_1 indicates that it may contain an Alu repetitive element
Clone 'AS164 1"
A polynucleohde of the present invention has been identified as clone "AS164_1' AS164_1 was isolated from a human fetal bram cDNA library usmg methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat No 5,536,637), or was identified as encodmg a secreted or transmembrane protem on the basis of computer analysis of the amino acid sequence of the encoded protein. AS164_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS164_1 protein")
The nucleohde sequence of AS164_1 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 AS164_1 protein corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO:8 The EcoRI /Noil restriction fragment obtainable from the deposit containing clone AS164_1 should be approximately 1600 bp.
The nucleotide sequence disclosed herein for AS164_1 was searched against the
GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols. AS164_1 demonstrated at least some similarity with sequences identified as H24668 (yl40hl0.rl Homo sapiens cDNA clone 160771 5'), N29757
(yw90hl0.sl Homo sapiens cDNA clone 259555 3'), T62184 (yb96d08.rl Homo sapiens cDNA clone 79023 5'), Z69706 (Human DNA sequence from cosmid COS12 from a conhg from the hp of the short arm of chromosome 16, spanning 2Mb of 16pl3.3. Contams ESTs, Flanking sequences of 3' alpha globm H), and Z69890 (Human DNA sequence from cosmid RJ14 from a conhg from the hp of the short arm of chromosome 16, spanning 2Mb of 16pl3 3. Contams ESTs and CpG island) The predicted ammo acid sequence disclosed herein for AS164_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol The predicted AS164_1 protein demonstrated at least some similarity to sequences identified as A20359_l (ryanodine receptor gene product [Homo sapiens]) and U78866 (putahve argrnme-aspartate-πch RNA binding protein [Arabidopsis tha ana]) Based upon sequence similarity, AS164_1 proteins and each similar protein or peptide may share at least some activity The predicted AS164_1 protein sequence also contains repeated Asp- Arg RNA-bmdmg mohfs
Clone "AX8 1 '
A polynucleotide of the present invention has been identified as clone "AX8_1" AX8_1 was isolated from a human adult testes cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protein. AX8_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "AX8_1 protein")
The nucleohde sequence of AX8_1 as presently determmed is reported in SEQ ID NO:9 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the AX8_1 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 10. Ammo acids 106 to 118 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at ammo acid 119, or are a transmembrane domain. The EcoRI/Notl restriction fragment obtainable from the deposit containing clone AX8_1 should be approximately 2300 bp
The nucleohde sequence disclosed herein for AX8_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database The TopPredll computer program predicts three potential transmembrane domains within the AX8_1 protein sequence, centered around ammo acids 111, 144, and 182 of SEQ ID NO.10
Clone BD176 3" A polvnucleohde of the present invenhon has been idenhhed as clone "BD176_3"
BD176_3 was isolated from a human fetal kidney cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U S Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem BD176_3 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BD176_3 protein")
The nucleohde sequence of the 5 portion of BD176_3 as presently determined is reported in SEQ ID NO 11 What applicants presently believe is the proper reading frame for the coding region is indicated m SEQ ID NO 12 The predicted ammo acid sequence of the BD176_3 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 12 Amino acids 2 to 14 are a predicted leader/signal sequence, with the predicted mature ammo acid sequence beginning at ammo acid 15, or are a transmembrane domain Additional nucleohde sequence from the 3 porhon of BD176_3, including the polyA tail, is reported in SEQ ID NO.13 The EcoRI/Notl restriction fragment obtainable from the deposit containmg clone
BD176_3 should be approximately 1300 bp
The nucleotide sequence disclosed herein for BD176_3 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols. BD176_3 demonstrated at least some similarity with sequences identified as AA029679 (ze94gl0 rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 366690 5 ), D45913 (Mouse NLRR-1 mRNA for leucine-πch-repeat protem, complete cds), R55610 (yg88h08.rl Homo sapiens cDNA clone 40606 5'), and T07640 (EST05530 Homo sapiens cDNA clone HFBEM16) The predicted amino acid sequence disclosed herein for BD176_3 was searched agamst the GenPept and GeneSeq ammo acid sequence databases using the BLASTX search protocol. The predicted BD176_3 protem demonstrated at least some similarity to sequences identified as D45913 (leucine-πch-repeat protein [Mus museums]) and M59472 (asparagine-πch anhgen Pfa55-6 [Plasmodium falαparum]). Based upon sequence similarity, BD176_3 proteins and each similar protein or peptide may share at least some achvity.
Clone "BD339 1"
A polynucleohde of the present mvenhon has been idenhhed as clone "BD339_1". BD339_1 was isolated from a human fetal kidney cDNA library usmg methods which are selechve for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem. BD339_1 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BD339_1 protem' ) The nucleohde sequence of BD339_1 as presently determined is reported in SEQ
ID NO:14 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the BD339_1 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO.15.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone BD339_1 should be approximately 650 bp.
The nucleotide sequence disclosed herein for BD339_1 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols. BD339_1 demonstrated at least some similarity with sequences idenhhed as H82422 (yu80d08.sl Homo sapiens cDNA clone 240111 3), N62058 (EST53c05 Homo sapiens cDNA clone), U21730 Human 5'-nucleohdase (CD73)), W01979 (za30h09 rl
Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone 294113 5'), and W02015 (za32bll.rl Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone 294237 5'). Based upon sequence similarity, BD339_1 proteins and each similar protem or pephde may share at least some achvity. The TopPredll computer program predicts three potential transmembrane domains withm the BD339_1 protem sequence,centered around ammo acids 14, 46, and 76 of SEQ ID NO:15. Clone "BD427 1"
A polynucleohde of the present invention has been idenhhed as clone "BD427_1". BD427_1 was isolated from a human fetal kidney cDNA library using methods which are selechve 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 BD427_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BD427_1 protein")
The nucleohde sequence of BD427_1 as presently determined is reported in SEQ ID NO.16 What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the BD427_1 protem corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO 17
The EcoRI /Notl restriction fragment obtainable from the deposit containing clone BD427_1 should be approximately 1810 bp The nucleotide sequence disclosed herein for BD427_1 was searched against the
GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols BD427_1 demonstrated at least some similarity with sequences idenhhed as AA027122 (zk04a03 rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 469516 5'), N24735 (yx56b02.sl Homo sapiens cDNA clone 265707 3'), and W84644 (zd91a06 rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 356818 5 ) Based upon sequence similarity, BD427_1 proteins and each similar protein or pephde may share at least some achvity.
Clone 'BL229 22" A polynucleohde of the present mvenhon has been idenhhed as clone "BL229_22"
BL229_22 was isolated from a human adult testes cDNA library using methods which are selechve 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 protem BL229_22 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BL229_22 protem").
The nucleotide sequence of the 5' porhon of BL229_22 as presently determined is reported in SEQ ID NO: 18 What applicants presently believe is the proper readmg frame for the coding region is indicated in SEQ ID NO.19. The predicted ammo acid sequence of the BL229_22 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:19 Additional nucleotide sequence from the 3' portion of BL229_22, including the polyA tail, is reported in SEQ ID NO:20.
The EcoRI/Notl restriction fragment obtamable from the deposit contammg clone BL229_22 should be approximately 870 bp.
The nucleohde sequence disclosed herein for BL229_22 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and FASTA search protocols No hits were found in the database.
Clone "BV123 16"
A polynucleohde of the present invenhon has been idenhhed as clone "BV123_16". BV123_16 was isolated from a human adult bram cDNA library using methods which are selechve for cDNAs encoding secreted proteins (see U.S Pat No 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the ammo acid sequence of the encoded protem BV123_16 is a full-length clone, including the entire coding sequence of a secreted protem (also referred to herein as "BV123_16 protein")
The nucleohde sequence of BV123_16 as presently determined is reported in SEQ ID NO.21 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the BV123_16 protem corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO.22.
The EcoRI /Notl restriction fragment obtainable from the deposit contammg clone BV123_16 should be approximately 1080 bp
The nucleohde sequence disclosed herein for BV123_16 was searched against the GenBank and GeneSeq nucleohde sequence databases using BLASTN/BLASTX and
FASTA search protocols. BV123_16 demonstrated at least some similarity with sequences identified as H29610 (ym61e03.sl Homo sapiens cDNA clone 52653 3'), H52374 (yq81bl2 rl Homo sapiens cDNA clone 202175 5'), H66213 (yulόhlO.sl Homo sapiens cDNA), L08092 (Homo sapiens dystrophin (DMD) gene, mtron 7, transposon- ke sequence), L35670 (Homo sapiens (subclone H8 10_g5 from PI 35 H5 C8) DNA sequence),
M62716 (Human CSP-B gene flanking sequence), N46985 (yy83a05.sl Homo sapiens cDNA clone 2801123'), R94603 (yq38a04.sl Homo sapiens cDNA clone 198030 3'), U91321 (Human chromosome 16pl3 BAC clone CIT987SK-363E6, complete sequence), and Z82200 (Human DNA sequence from clone J333E231) Based upon sequence similarity, BV123_16 proteins and each similar protem 01 peptide may share at least some activity.
Clone CH377 1" A polynucleohde of the present invenhon has been idenhhed as clone "CH377_1".
CH377_1 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protem on the basis of computer analysis of the amino acid sequence of the encoded protein. CH377_1 is a full-length clone, mcluding the entire coding sequence of a secreted protein (also referred to herein as "CH377_1 protein")
The nucleohde sequence of CH377_1 as presently determined is reported m SEQ ID NO 23 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the CH377_1 protem corresponding to the foregoing nucleohde sequence is reported in SEQ ID NO 24 Amino acids 5 to 17 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18, or are a transmembrane domain
The EcoRI /Notl restriction fragment obtainable from the deposit contammg clone CH377_1 should be approximately 570 bp The nucleohde sequence disclosed herein for CH377_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols CH377_1 demonstrated at least some similarity with sequences idenhhed as AA507382 (nh73b01.sl NCI_CGAP_Brl 1 Homo sapiens cDNA clone IMAGE 964105) and N70479 (za74fl2.sl Homo sapiens cDNA clone 298319 3') Based upon sequence similarity, CH377_1 proteins and each similar protein or peptide may share at least some activity
Deposit of Clones
Clones AJ20_2, AR440_1, AS164_1, AX8_1, BD176_3, BD339_1, BD427_1, BL229_22, BV123_16, and CH377_1 were deposited on November 15, 1996 with the
American Type Culture Collection as an oπgmal deposit under the Budapest Treaty and were given the accession number ATCC 98261, from which each clone comprising a parhcular polynucleohde is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granhng of the patent, except for the requirements specified in 37 C F.R § 1 808(b)
Each clone has been transfected into separate bacterial cells (E coli) in this composite deposit. Each clone can be removed from the vector m which it was deposited by performing an EcoRI/Notl digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate fragment for such clone Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1 The pED6dpc2 vector ("pED6") was derived from pED6dpcl by insertion of a new polylmker 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 polylmker, and insertion of the M13 origin of replication m the Clal site. In some instances, the deposited clone can become "flipped" (I e , in the reverse orientation) in the deposited isolate In such instances, the cDNA insert can still be isolated by digestion with EcoRI and Notl However, Notl will then produce the 5' site and EcoRI will produce the 3' site tor placement of the cDNA in proper orientation for expression in a suitable vector The cDNA may also be expressed from the vectors in which they were deposited
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone This sequence can be derived from the sequences provided herein, or from a combination of those sequences The sequence of the oligonucleotide probe that was used to isolate each full-length clone is idenhhed below, and should be most reliable in isolating the clone of interest
Clone Probe Sequence
AJ20_2 SEQ ID NO:25
AR440_1 SEQ ID NO:26
AS164_1 SEQ ID NO:27 A AXX88__11 SEQ ID NO:28
BD176_3 SEQ ID NO:29
BD339_1 SEQ ID NO:30
BD427_1 SEQ ID NO:31
BL229 22 SEQ ID NO.32 BV123_16 SEQ ID NO 33
CH377_1 SEQ ID NO.34
In the sequences listed above which include an N at position 2, that position is occupied in preferred probes /primers by a biotmylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1- dιmethoxytπtyloxv-2-(N-bιotιnyl-4-ammobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N- dnsopropyl)-phosphoramadιte) (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-,2P 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 mcorporated into the probe should be quantitated by measurement m 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 moculate a sterile culture flask contammg 25 ml of sterile L-broth containing ampicilhn 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 Ahquots 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 ampicilhn at 100 μg/ml and agar at 1.5% in a 150 mm petπ dish when grown overnight at 37°C Other known methods of obtaining dishnct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/hter, 88.2 g Na citrate/liter, adjusted to pH 7 0 with NaOH) contammg 0 5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm hlter). 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 invenhon 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 descπbed 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 bmdmg sites. For example, fragments of the protein may be fused through "linker ' sequences to the Fc portion of an immunoglobulin For a bivalent form ot the protem, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions For example, a protein - IgM fusion would generate a decavalent form of the protein of the invenhon
The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such protems is identified in the sequence listing by translation of the nucleohde sequence of each disclosed clone. The mature form of such protein may be obtamed by expression of the disclosed full-length polynucleohde (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
The sequence of the mature form of the protein may also be determinable from the ammo acid sequence of the full-length form
The present invention also provides genes correspondmg to the cDNA sequences disclosed herein "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which the cDNA sequences are derived and any conhguous regions of the genome necessary for the regulated expression of such genes, mcluding but not limited to codmg sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements The corresponding genes can be isolated m accordance with known methods using the sequence mformahon disclosed herein Such methods include the preparahon of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials Where the protein of the present invenhon is membrane-bound (e g , is a receptor), the present invenhon also provides for soluble forms of such protem In such forms part or all of the mtracellular and transmembrane domams of the protem are deleted such that the protem is fullv secreted from the cell in which it is expressed The mtracellular and transmembrane domains of protems of the invenhon can be idenhhed 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 protem, where sequence identity is determmed by comparmg the ammo 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 ammo acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins Species homologs of the disclosed polynucleotides and protems are also provided by the present invention As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
The present mvenhon also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preterably highly stringent condihons, 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 imgf000033_0001
J The hvbπd length is that anticipated for the hybridized regιon(s) of the hybridizing polynucleotides When hybridizing a polynucleohde to a target polvnucleohde of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleohde When polynucleotides of known sequence are hybridized, the hybrid length can be determined bv aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity
■ SSPE (lxSSPE is 0 15M NaCl, lOmM NaH,P04, and 1 25mM EDTA, pH 7 4) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
*T„ - TR The hybndizahon temperature for hybrids anticipated to be less than 50 base pairs m length should be 5-10 C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations For hybrids less than 18 base pairs in length, Tm("C) = 2(# of A + T bases) + 4(# of G + C bases) For hybrids between 18 and 49 base pairs in length, Tm( C) = 81 5 + 16 6(log10[Na+]) + 0 41(%G+C) - (600/N), where N is the number of bases in the hvbπd, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC = 0 165 M) Additional examples of stringency condihons for polynucleotide hybridization are provided m Sambrook, J , E.F. Fπtsch, and T Mamatis, 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.
Preterably, 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 mav 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 hgated polynucleohde/expression control sequence
A number of types of cells may act as suitable host cells for expression of the protem 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 m 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 Eschenchia coli, Bacillus subtihs, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous protems If the protem is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylahon or glycosylation of the appropriate sites, in order to obtam the functional protem. Such covalent attachments may be accomplished using known chemical or enzymatic methods
The protem may also be produced by operably linking the isolated polynucleohde of the invention to suitable control sequences m 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 mav be prepared by culturmg 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 protem may also include an affinity column containing agents which will bind to the protem, one or more column steps over such affinity resins as concanavahn A-agarose, hepaπn-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography usmg such resms as phenyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography Alternatively, the protein of the mvenhon 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 thioredoxm (TRX). Kits for expression and purification of such fusion protems are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respechvely The protem 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 protem Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protem The protein thus purified is substantially tree 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 ammals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells contammg a nucleotide sequence encoding the protein. The protem mav also be produced by known conventional chemical synthesis
Methods for constructing the protems of the present invention by synthetic means are known to those skilled m the art The synthetically-constructed protem 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 mav be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeuhc compounds and in immunological processes for the development of anhbodies
The protems provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or dehberatelv engineered For example, modifications in the peptide or DNA sequences can be made by those skilled m the art using known techniques Modifications of mterest in the protem sequences may include the alterahon, subs tuhon, replacement, insertion or deletion of a selected amino acid residue m the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another ammo acid to alter the conformation of the molecule. Techniques for such alteration, subshtuhon, replacement, inserhon or deletion are well known to those skilled in the art (see, e.g., U S Patent No 4,518,584). Preferably, such alterahon, subshtuhon, replacement, insertion or deletion retains the desired activity of the protein
Other fragments and deπvahves of the sequences of proteins which would be expected to retain protein achvity m whole or m part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled m the art given the disclosures herein. Such modifications are believed to be encompassed by the present mvenhon. USES AND BIOLOGICAL ACTIVITY
The polynucleotides and protems of the present mvenhon 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 mvenhon may be provided by administration or use of such protems or by administration or use of polynucleohdes encodmg such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA)
Research Uses and Utilities The polynucleohdes provided bv the present invenhon can be used by the research community for various purposes The polynucleohdes can be used to express recombinant protem for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protem is preferentially expressed (either conshtuhvelv or at a particular stage of hssue differenhahon or development or m 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 m patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences, as a source of mformahon to derive PCR primers for genehc fingerprinting; as a probe to "subtract-out known sequences in the process of discovering other novel polynucleohdes; for selechng and making oligomers for attachment to a gene chip" or other support, including for examination of expression patterns, to raise anh-protem antibodies using DNA immunization techniques, and as an anhgen to raise anh-DNA anhbodies or elicit another immune response. Where the polynucleohde encodes a protein which bmds or potentially binds to another protem (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuπs et al, Cell 75:791-803 (1993)) to identify polynucleohdes encoding the other protem with which binding occurs or to identify inhibitors of the binding interaction The protems 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 (mcluding the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids, as markers for hssues in which the corresponding protem is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or m a disease state); and, of course, to isolate correlative receptors or gands Where the protem binds or potentially binds to another protem (such as, for example, in a receptor-ligand interaction), the protem can be used to identify the other protein with which bmdmg occurs or to identify inhibitors of the bmdmg interaction Protems involved in these bmdmg 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 Fπtsch and T Mamatis eds , 1989, and "Methods in Enzvmology Guide to Molecular Clonmg 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 mclude without limitation use as a protem or ammo acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate In such cases the protem or polynucleohde 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 m the form of powder, pills, solutions, suspensions or capsules In the case ot microorganisms, the protein or polynucleohde of the invention can be added to the medium in or on which the microorganism is cultured
Cvtokine and Cell Proliferation/ Differentiation Activity
A protem of the present invention may exhibit cytokine, cell proliferation (either inducmg or inhibiting) or cell differentiation (either inducmg or inhibiting) achvity or may induce production of other cytokmes m certain cell populations Many protem factors discovered to date, mcluding all known cytokmes, have exhibited achvity 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 protem 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 m: Current Protocols m Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Marguhes, 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 tor cytokine production and /or proliferation of spleen cells, lymph node cells or thvmocytes include, without limitation, those descπbed m: 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 hematopoiehc and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukm 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; deVπes et al., J. Exp. Med. 173.1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl Acad. So. 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 Interleukm 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 Interleukm 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 m Immunology, Ed by J E Coligan, A.M. Kruisbeek, D H. Margu es, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocvte Function; Chapter 6, Cytokmes and their cellular receptors, Chapter 7, Immunologic studies m 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 Shmulahng or Suppressing Activity
A protem 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 protem mav be useful in the treatment of various immune deficiencies and disorders (mcluding severe combined immunodeficiency (SOD)), e.g., m 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, mcluding infections by HIV, hepatitis viruses, herpesviruses, mycobacteπa, Leishmania spp , malaria spp and various fungal mfechons such as candidiasis. Of course, in this regard, a protem of the present invenhon 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 invenhon include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroidihs, insulin dependent diabetes mellihs, myasthema gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present mvenhon may also to be useful in the treatment of allergic reachons and condihons, 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 protem of the present invention
Usmg the proteins of the invention it may also be possible to immune responses, m 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-anhgen-specific, process which requires continuous exposure of the T cells to the suppressive agent Tolerance, which involves inducing non-responsiveness or anergy m T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the toleπzing agent has ceased Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen m the absence of the toleπzing 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 lymphokme synthesis by activated T cells, will be useful m situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) For example, blockage of T cell function should result m 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 bv 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 lιgand(s) on immune cells (such as a soluble, monomeπc form of a peptide having B7-2 activity alone or m conjunction with a monomeπc form of a peptide having an activity of another B lymphocyte anhgen (e g , B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural lιgand(s) on the immune cells without transmitting the corresponding coshmulatory 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 an gen-blockmg 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 funchon of a combinahon 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 m humans Examples of appropriate systems which can be used include allogeneic cardiac grafts m rats and xenogeneic pancreahc islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion protems in vivo as described in Lenschow et al , Science 257789-792 (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 cytokmes and autoantibodies involved in the pathology of the diseases Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell achvahon and prevent production of autoantibodies or T cell-derived cytokmes which may be involved in the disease process Additionally, blocking reagents may induce antigen-specific tolerance of autoreachve T cells which could lead to long-term relief from the disease The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lprflpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mel tus in NOD mice and BB rats, and murme 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 funchon), as a means of up regulating immune responses, may also be useful in therapy Upregulahon of immune responses may be in the form of enhancing an exishng 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 m an mfected patient by removmg 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 remtroducing the in vitro achvated T cells into the pahent. 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 oϊ the protein on their surface, and reintroduce the transfected cells into the patient The mfected cells would now be capable of delivering a costimulatorv 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 pephdes. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector direchng the expression ot a peptide having B7-2-hke activity alone, or in conjunction with a peptide having B7-l-lιke activity and /or B7-3-hke 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 anhgen(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, rumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e g , a cytoplasmic-domain truncated portion) of an MHC class I α chain protem and β2 microglobulm protein or an MHC class II a chain protem and an MHC class II β chain protein to thereby express MHC class I or MHC class II protems on the cell surface. Expression of the appropriate class I or class II MHC in conjunchon with a pephde havmg 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 anhsense construct which blocks expression of an MHC class II associated protein, such as the mvaπant 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 anhgens 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 achvity of a protem of the mvenhon may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitahon, those described in: Current Protocols m Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Marguhes, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J Immunol. 128:1968-1974, 1982; Handa et al, J. Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Mahszewski, 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, protems 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. Marguhes, 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, protems expressed by dendritic cells that achvate 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 superanhgen induction and protems that regulate lymphocyte homeostasis) include, without limitahon, those described m: 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 protems that influence early steps of T-cell commitment and development include, without limitation, those described in: Anhca 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 margmal biological achvity in support of colony forming cells or of factor-dependent cell lines mdicates involvement in regulating hematopoiesis, e.g. m supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokmes, 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 supporhng 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; m supporhng the growth and proliferation of megakarvocvtes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocvtopema, and generally for use m place of or complimentary to platelet transfusions, and /or in supporting the growth and proliferation of hematopoie c 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 usuallv treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobmuπa), 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 achvitv of a protein of the invention mav, among other means, be measured bv 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, protems that regulate lvmpho-hematopoiesis) include, without limitation, those described m. 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 proliferahve potential, McNTece, LK. and Bπddell, 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 m the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994, Long term culture initiating cell assay, Sutherland, H J In Culture of Hematopoietic Cells R I Freshney, et al eds Vol pp 139-162, Wiley-Liss, Inc , New York, NY 1994
Tissue Growth Activity A protem of the present mvenhon 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 m 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 protem of the invenhon may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation ot artificial joints De novo bone formation induced by an osteogenic agent contributes to the repair of congemtal, trauma induced, or oncologic resection induced cramofacial defects, and also is useful in cosmetic plastic surgery
A protein of this invention mav also be used in the treatment of periodontal disease, and in other tooth repair processes Such agents may prov ide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells A protem ot the mvenhon may also be useful in the treatment of osteoporosis or osteoarthπtis, 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 mav be attributable to the protein of the present invention is tendon/ligament formation A protein of the present invention, which induces tendon/ gament-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, deformihes and other tendon or ligament defects in humans and other animals Such a preparation employing a tendon /hgament-like tissue inducing protem 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-hke 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 hssue 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 mclude an appropriate matrix and /or sequestermg agent as a carrier as is well known in the art
The protein of the present invention mav also be useful for proliferation of neural cells and for regeneration of nerve and bram tissue, i e for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degenerahon death or trauma to neural cells or nerve tissue More specifically, a protein mav 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 svstem diseases, such as Alzheimer s, Parkinson s disease, Hunhngton s disease, amvotrophic lateral sclerosis, and Shy-Drager syndrome Further conditions which mav be treated in accordance with the present invenhon mclude mechanical and fraumahc disorders, such as spmal 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 protem of the invention
Protems of the invenhon may also be useful to promote better or faster closure of non-healing wounds, including without limitahon pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like
It is expected that a protem 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, skm, endothelium), muscle (smooth, skeletal or cardiac) and vascular (mcludmg 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 hbrosis, 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 achvity of a protein of the invention may, among other means, be measured by the following methods.
Assays for hssue generation activity include, without limitation, those described in. International Patent Publication No W095/ 16035 (bone, cartilage, tendon); International Patent Publication No W095/ 05846 (nerve, neuronal), International Patent Publication No. WO91/07491 (sk , endothelium )
Assays for wound healing achvity mclude, without limitation, those described m 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 activms and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protem of the present invention, alone or m heterodimers with a member of the mhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protem of the mvenhon, as a homodimer or as a heterodimer with other protein subunits of the rnhibin- β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules m 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 mcrease the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protem of the invention may, among other means, be measured by the following methods- Assays for activin /inhibin achvity 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. So USA 83.3091-3095, 1986
Chemotactic /Chemokmetic Activity
A protem of the present invention may have chemotachc or chemokmetic activity (e.g , act as a chemokme) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells. Chemotactic and chemokmetic proteins can be used to mobilize or attract a desired cell population to a desired site of action Chemotactic or chemokmetic 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 shmulate directed movement of cells Whether a particular protem has chemotactic activity for a populahon of cells can be readily determined by employing such protem or peptide in any known assay for cell chemotaxis.
The achvity of a protem of the invenhon may, among other means, be measured by the following methods
Assays for chemotachc achvity (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 mclude, without limitahon, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Marguhes, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokmes 6.12.1-6.12.28; Taub et al. J. Chn. Invest. 95:1370-1376, 1995; Lmd 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. Hemostahc and Thrombolvhc Activity
A protein of the invention may also exhibit hemostahc or thrombolyhc activity As a result such a protem is expected to be useful in treatment of various coagulation disorders (mcluding hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostahc events in treating wounds resulting from trauma, surgery or other causes A protem of the invention mav 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 , stroke) The achvity of a protein of the invenhon may, among other means, be measured by the following methods
Assav for hemostahc and thrombolvhc achvity mclude, without limitation, those described m Linet et al , J Chn Pharmacol 26 131-140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrev et al , Fibrmolvsis 5 71-79 (1991), Schaub, Prostaglandms 35 467-474, 1988
Receptor/Ligand Activity
A protein of the present invention mav 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 selechns, mtegrms and their ligands) and receptor/ gand 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/hgand interaction A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/hgand interactions The activity of a protem of the invention may, among other means, be measured by the following methods
Suitable assays for receptor-ligand activity include without limitation those described m Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, 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, Shtt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity
Protems of the present invenhon may also exhibit anti-inflammatory achvity. 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 Protems exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as sephc shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxm lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokme-mduced lung mjury, inflammatory bowel disease, Crohn s disease or resulting from over production ot cytokmes such as TNF or IL-1 Proteins of the invenhon may also be useful to treat anaphylaxis and hypersensihvity to an antigenic substance or material.
Cadheπn /Tumor Invasion Suppressor Achvity
Cadheπns are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types Loss or alterahon of normal cadherm expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherm malfunction is also implicated in other human diseases, such as pemphigus vulgaπs and pemphigus fo aceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities. The cadheπn superfamily includes well over forty members, each with a distinct pattern of expression All members of the superfamily have in common conserved extracellular repeats (cadherm domains), but structural differences are found in other parts of the molecule. The cadherm domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion Only a few ammo acids in the first cadherm domain provide the basis for homophi c adhesion; modification of this recognition site can change the specificity of a cadherm so that mstead of recognizing only itself, the mutant molecule can now also bind to a different cadheπn. In addition, some cadherms engage in heterophihc adhesion with other cadhenns E-cadheπn, one member of the cadheπn superfamily, is expressed m epithelial cell types. Pathologically, if E-cadheπn expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes Transfection of cancer cell lines with polynucleotides expressing E-cadherm 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, reintroducmg E-cadheπn expression reverts carcinomas to a less advanced stage It is likely that other cadherms have the same invasion suppressor role in carcinomas derived from other hssue types Therefore, protems of the present invention with cadherm activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer Introducing such proteins or polynucleohdes mto cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherm expression
Cancer cells have also been shown to express cadherms of a different tissue type than their origin, thus allowing these cells to invade and metastasize m a different tissue in the body. Proteins of the present mvenhon with cadherm achvity, and polynucleohdes of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherms, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize
Additionally, proteins of the present invention with cadherm activity, and polynucleohdes of the present invention encoding such protems, can used to generate antibodies recognizing and binding to cadherms Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherms, prevenhng the cells from formmg a tumor elsewhere. Such an anti-cadherm 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 cadherm expression there will be, and this decrease m cadherm expression can be detected by the use of a cadheπn-bmding antibody.
Fragments of proteins of the present invention with cadherm activity, preferably a polypeptide comprising a decapephde of the cadherm recognition site, and polynucleotides of the present invention encoding such protem 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 characterishcs, 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 caπier) 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 effechveness 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, TNF1, 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 mul timers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and /or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes B lymphocytes will respond to antigen through their surface immunoglobulin receptor T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins MHC and structurally related protems 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-pephde complexes alone or with co-stimulatory molecules that can directly signal T cells Alternatively antibodies able to bind surface lmmunolgobu n 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 invenhon may be in the form of a liposome in which protein of the present mvenhon is combined, m addition to other pharmaceutically acceptable carriers, with amphipathic agents such as hpids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers m aqueous solution Suitable hpids for liposomal formulation mclude, without limitation, monoglyceπdes, diglyceπdes, sulfatides, lysolecithin, phosphohpids, saponin, bile acids, and the like Preparation of such liposomal formulahons is withm 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, l e , treatment, healing, prevenhon or amelioration of the relevant medical condition, or an increase m rate of treatment, healmg, prevention or amelioration of such conditions When applied to an individual active ingredient, administered alone, the term refers to that mgredient alone When applied to a combination, the term refers to combined amounts of the active ingredients that result m the therapeutic effect, whether administered in combination, seπally or simultaneously In practicing the method of treatment or use of the present invenhon, a therapeutically effective amount of protem 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 mvenhon either alone or in combmahon 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, protem of the present invention may be administered either simultaneously with the cytokιne(s), lymphokιne(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 protem of the present invention in combination with cytokιne(s), lymphokιne(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, mtrapeπtoneal, 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 pharmaceuhcal composihon of the mvenhon 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 mvenhon, and preferably from about 25 to 90% prote of the present invention When admmistered m 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 sacchaπde solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol When administered in liquid form, the pharmaceuhcal composihon contains from about 0 5 to 90% by weight of protem of the present invention, and preferably from about 1 to 50% protein of the present invenhon
When a therapeutically effective amount of protem of the present invention is admmistered bv intravenous, cutaneous or subcutaneous injection, protein of the present mvenhon will be m the form of a pyrogen-free, parenterally acceptable aqueous solution
The preparahon 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 pharmaceuhcal composihon for intravenous, cutaneous, or subcutaneous injection should contain, m addition to protem of the present invention, an isotomc 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 protem of the present invenhon in the pharmaceuhcal composihon of the present invenhon 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 mvenhon should contain about 0 01 μg to about 100 mg (preferably about 0 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 durahon of each applicahon of the protem 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 durahon of intravenous therapy using the pharmaceutical composition of the present invenhon
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 pephde immunogens additionally may contam 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. Merπfield, J. Amer.Chem.Soc. 85, 2149-2154 (1963), J.L. Krstenansky, et al , FEBS Lett
211, 10 (1987) Monoclonal antibodies bmdmg to the protein of the invention may be useful diagnostic agents for the lmmunodetection of the protem. Neutralizing monoclonal antibodies bmdmg to the protein may also be useful therapeutics for both condihons associated with the protein and also m the treatment of some forms of cancer where abnormal expression of the protem is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal anhbodies against the protein may be useful in detechng 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 mjected 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 protem of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be admmistered simultaneously or sequentially with the composition in the methods of the invention Preferably for bone and/or cartilage formation, the composihon would include a matrix capable of delivering the protein-containing composihon 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 biocompatibihty, biodegradabihty, 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, tncalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydπdes Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen Further matrices are comprised of pure protems 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 tncalciumphosphate. The bioceramics may be altered in composition, such as in calcium- alummate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty. Presently preferred is a 50 50 (mole weight) copolymer of lactic acid and glycohc 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 carboxymethvl 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- methvlcellulose, and carboxymethylcellulose, the most preferred being catiomc salts of carboxvmethvlcellulose (CMC) Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polvmer and poly(vιnvl alcohol) The amount of sequestermg 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 polvmer matrix and to provide appropriate handling of the composihon, 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, protems of the invention may be combined with other agents beneficial to the treatment of the bone and/or carhlage defect, wound, or tissue in queshon 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 protem-contammg pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the protems, 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 hssue (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 m 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, histomorphometπc determinations and tetracyclme labeling.
Polynucleohdes of the present mvenhon can also be used for gene therapy. Such polynucleohdes can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleohdes of the mvenhon may also be administered by other known methods for mfroduchon of nucleic acid into a cell or organism (mcludmg, 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 invenhon 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:
(1) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Merberg, David Treacy, Maurice Spauldmg, Vikki Agostmo, Michael J.
(11) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(in) NUMBER OF SEQUENCES: 34
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET. 87 CamoπdgePark 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: Patentln Release #1.0, Version #1.30
(vi ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vin) 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 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 430 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESΞ : double
(D) TOPOLOGY: linear in) MOLECULE TYPE: cDNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
TAAAGATCTG TGTTCAGAGT CATACTGAAY AGAGACTTCT GGACTCTATA GAACCCACTG 60
CCTCCTGATG AAGTCCCTAC TGTTCACCCT TGCAGTTTTT ATGCTCCTGG CCCAATTGGT 120
CTCAGGTAAT TGGTATGTGA AAAAGTGTCT AAACGACGTT GGAATTTGCA AGAAGAAGTG 180
CAAACCTGAA GAGATGCATG TAAAGAATGG TTGGGCAATG TGCGGCAAAC AAAGGGACTG 240
CTGTGTTCCA GCTGACAGAC GTGCTAATTA TCCTGTTTTC TGTGTCCAGA CAAAGACTAC 300
AAGAATTTCA ACAGTAACAG CAACAACAGC AACAACAACT TTGATGATGA CTACTGCTTC 360
GATGTCTTCG ATGGCTCCTA CCCGTTTCTC CCACTGGTTG AACATTCCAG CCTCTGTCTC 420
CTGCTCTAGG 430 (2) INFORMATION FOR SEQ ID NO : 2 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: prote
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Lys Ser Leu Leu Phe Thr Leu Ala Val Phe Met Leu Leu Ala Gin 1 5 10 15
Leu Val Ser Gly Asn Trp Tyr Val Lys Lys Cys Leu Asn Asp Val Gly 20 25 30 lie Cys Lys Lys Lys Cys Lys Pro Glu Glu Met His Val Lys Asn Gly 35 40 45
Trp Ala Met Cys Gly Lys Gin Arg Asp Cys Cys Val Pro Ala Asp Arg
50 55 60
Arg Ala Asn Tyr Pro Val Phe Cys Val Gin Thr Lys Thr Thr Arg lie 65 70 75 80
Ser Thr Val Thr Ala Thr Thr Ala Thr Thr Thr Leu Met Met Thr Thr 85 90 95 Ala Ser Met Ser Ser Met Ala Pro Thr Arg Phe Ser His Trp Leu Asn 100 105 110 lie Pro Ala Ser Val Ser Cys Ser Arg 115 120
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : TTTCCTGNTT TNGGATCCCC GATTCATTAA AGCAANGGGG NTTNAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 112
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
NCTACCCCAA CCTGTGTGGC TGGGCCGCGG TCTCCCCTCA AGGGCCTGGG GCCGTGCCTC 60
GGGTGTACNC GTANGGGTCT GTGTGCTGGG GGTGGCTCAC CGGGCAGCGT GGGTGAGCGG 120
CGCANCGGCG GCAGCGGAGA ACGAGAGAGG GGAGCAGANA CAGAATCGCC TAAGCTGAAG 180
TGTATTGGCG CCATCATGGC TCACTGCGGC CTCCGGCTCC TTGGCTCGGG TGATTCTCCT 240
GCCTGAGCCT CCCTAGTAGC TAGGACTACA GTGCTGTAGA AGAAAATCAC ATGATTGGTG 300
CCCTCAAAAA ATTGGTGCCA CTTG 324 (2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 794 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
CATTATTTCA TCACCAGAGA ATACACATGC AGCAAATAGC ATTGTGAGTC AAACTATTCC 60
NAAAGCACAG ATTCAGCAAT CNACACACAC TCATCTGGAT ATCTCACNNN "NNNNNNNNN 120
TTAACTGATG AAAAAAGTAA TGGAACAATT GCCCTTGTGG ATGATTCTGA GGATCCTGGA 180
GCCNATGTAT CTAACATACA GCTTCAGCAA AAAATTTCAA GTCTGGAGAT TAAACTCAAA 240
GTATCTGAAG AAGAAAAACA GAGAATTAAA CAGGATGTGG AAKCATTGAT GGAAAAGCAT 300
AATGTCTTAG AAAAAGGCTT TCTAAAAGAA AAAGAGCAAG AGGCCATTTC TTTTCAAGAT 360
AGATACAAAG AACTTCAGGA AAAACATAAA CAAGAATTGG AAGACATGAG GAAAGCTGGT 420
CACGAAGCCC CAGCATTAT TGTGGATGAA TATAAGGCAC TACTGCAGTC TCAGTTAAG 480
CAACAAGTAG AAGCTATTGA AAAACAGTAC ATTTCTGCAA TTGAGAAACA GGCACACAAG 540
TGTGAGGAGT TGCTAAATGC TCAGCATCAG AGGCTCCTTG AAGTGCTAGA TACAGAGAAG 600
GAACTGTTAA AAGAAAAAAT AAAGGAAGCT TTGATTCAGC AATCTCAAGA ACAGAAGGAA 660
ATATTGGAAA AGTGTTTGGA GGAAGAAAGG CAAAGAAATA AAGAGGCATT AGTATCCGCT 720
GCAAAGCTTG AAAAAGAACC AGTGAAGGAT GCANTTTTAA AATTCGTAGA AGAAGAAAGA 780
AAAAAAAAAA AAAA 794
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids (3) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : Met Glu Lys His Asn Val Leu Glu Lys Gly Phe Leu Lys Glu Lys Glu 1 5 10 15
Gin Glu Ala lie Ser Phe Gin Asp Arg Tyr Lys Glu Leu Gin Glu Lys 20 25 30
His Lys Gin Glu Leu Glu Asp Met Arg Lys Ala Gly His Glu Ala Leu 35 40 45
Ser lie lie Val Asp Glu Tyr Lys Ala Leu Leu Gin Ser Ser Val Lys 50 55 60
Gin Gin Val Glu Ala lie Glu Lys Gin Tyr lie Ser Ala lie Glu Lys 65 70 75 80
Gin Ala His Lys Cys Glu Glu Leu Leu Asn Ala Gin His Gin Arg Leu 85 90 95
Leu Glu Val Leu Asp Thr Glu Lys Glu Leu Leu Lys Glu Lys lie Lys 100 105 110
Glu Ala Leu lie Gin Gin Ser Gin Glu Gin Lys Glu lie Leu Glu Lys 115 120 125
Cys Leu Glu Glu Glu Arg Gin Arg Asn Lys Glu Ala Leu Val Ser Ala 130 135 140
Ala Lys Leu Glu Lys Glu Pro Val Lys Asp Ala Xaa Leu Lys Phe Val 145 150 155 160
Glu Glu Glu Arg
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1494 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
CGTTTGTCGA CGCCGCTGCC ACCGCCTGCC TGAGAGAAGT CGTCGCGGCC GACCCCGTCG 60
CCTCCGCCGG CTACCATGTC CGCCCAGGCG CAGATGCGGG CCCTGCTGGA CCAGCTCATG 120
GGCACGGCTC GGGACGGAGA CGAAACCAGA CAGAGGGTCA AGTTTACAGA TGACCGTGTC 180
TGCAAGAGTC ACCTTCTGGA CTGCTGCCCC CATGACATCC TGGCTGGGAC GCGCATGGAT 240 TTAGGAGAAT GTACCAAAAT CCACGACTTG GCCCTCCGAG CAGATTATGA GATTGCAAGT 300
AAAGAAAGAG ACCTGTTTTT TGAATTAGAT GCAATGGATC ACTTGGAGTC CTTTATTGCT 360
GAATGTGATC GGAGAACTGA GCTCGCCAAG AAGCGGCTGG CAGAAACACA GGAGGAAATC 420
AGTGCGGAAG TTTCTGCAAA GGCAGGAAAA GTACATGAGT TAAATGAAGA AATAGGAAAA 480
CTCCTTGCTA AAGCCGAACA GCTAGGGGCT GAAGGTAATG TGGATGAATC CCAGAAGATT 540
CTTATGGAAG TGGAAAAAGT TCGTGCGAAG AAAAAAGAAG CTGAGGAAGA ATACAGAAAT 600
TCCATGCCTG CATCCAGTTT TCAGCAGCAA AAGCTGCGTG TCTGCGAGGT CTGTTCAGCC 660
TACCTTGGTC TCCATGACAA TGACCGTCGC CTGGCAGACC ACTTCGGTGG CAAGTTACAC 720
TTGGGGTTCA TTCAGATCCG AGAGAAGCTT GATCAGTTGA GGAAAACTGT CGCTGAAAAG 780
CAGGAGAAGA GAAATCAGGA TCGCTTGAGG AGGAGAGAGG AGAGGGAACG GGAGGAGCGT 840
CTGAGCAGGA GGTCGGGATC AAGAACCAGA GATCGCAGGA GGTCACGCTC CCGGGATCGG 900
CGTCGGAGGC GGTCAAGATC TACCTCCCGA GAGCGACGGA AATTGTCCCG GTCCCGGTCC 960
CGAGATAGAC ATCGGCGCCA CCGCAGCCGT TCCCGGAGCC ACAGCCGGGG ACATCGTCGG 1020
GCTTCCCGGG ACCGAAGTGC GAAATACAAG TAACTACTCT GACTCCTTCG GTAGCTGCAA 1080
CCAGGAGTTC TCCAGAGAGC GGGCATCCAG AGAGGAGTCC TGGGAGAGCG GGCGGAGCGA 1140
GCGAGGGCCC CCGGACTGGA GGCTTGAGAG CTCCAACGGG AAGATGGCTT CACGGAGGTC 1200
AGAAGAGAAG GAGGCCGGCG AGATCTGAAC CCGTCTCCCG GGTGCTGTAA ATAGTCTGAT 1260
AAACGTTCAC ACAGTCTAAA ATTACCCTTT ATATTTGCTG AATACAACTC ATCTTTTGTA 1320
GTTTAAAATT TCTATTGTTT TGGAGCTAGC TGTGAGTTTC TAGAAGTGTA CAGAGTTGCT 1380
CCTGTGTTCC CGGGTCATGT TGAGTAGGAA TAAATAAATC TGATGCTGCC TCCTGGAAAA 1440
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 1494 (2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 325 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Ser Ala Gin Ala Gin Met Arg Ala Leu Leu Asp Gin Leu Met Gly 1 5 10 15
Thr Ala Arg Asp Gly Asp Glu Thr Arg Gin Arg Val Lys Phe Thr Asp 20 25 30
Asp Arg Val Cys Lys Ser His Leu Leu Asp Cys Cys Pro His Asp lie 35 40 45
Leu Ala Gly Thr Arg Met Asp Leu Gly Glu Cys Thr Lys lie His Asp 50 55 60
Leu Ala Leu Arg Ala Asp Tyr Glu lie Ala Ser Lys Glu Arg Asp Leu 65 70 75 80
Phe Phe Glu Leu Asp Ala Met Asp His Leu Glu Ser Phe lie Ala Glu 85 90 95
Cys Asp Arg Arg Thr Glu Leu Ala Lys Lys Arg Leu Ala Glu Thr Gin 100 105 110
Glu Glu lie Ser Ala Glu Val Ser Ala Lys Ala Gly Lys Val His Glu 115 120 125
Leu Asn Glu Glu lie Gly Lys Leu Leu Ala Lys Ala Glu Gin Leu Gly 130 135 140
Ala Glu Gly Asn Val Asp Glu Ser Gin Lys lie Leu Met Glu Val Glu 145 150 155 160
Lys Val Arg Ala Lys Lys Lys Glu Ala Glu Glu Glu Tyr Arg Asn Ser 165 170 175
Met Pro Ala Ser Ser Phe Gin Gin Gin Lys Leu Arg Val Cys Glu Val 180 185 190
Cys Ser Ala Tyr Leu Gly Leu His Asp Asn Asp Arg Arg Leu Ala Asp 195 200 205
His Phe Gly Gly Lys Leu His Leu Gly Phe lie Gin lie Arg Glu Lys 210 215 220
Leu Asp Gin Leu Arg Lys Thr Val Ala Glu Lys Gin Glu Lys Arg Asn 225 230 235 240
Gin Asp Arg Leu Arg Arg Arg Glu Glu Arg Glu Arg Glu Glu Arg Leu 245 250 255
Ser Arg Arg Ser Gly Ser Arg Thr Arg Asp Arg Arg Arg Ser Arg Ser 260 265 270
Arg Asp Arg Arg Arg Arg Arg Ser Arg Ser Thr Ser Arg Glu Arg Arg 275 280 285 Lys Leu Ser Arg Ser Arg Ser Arg Asp Arg His Arg Arg His Arg Ser 290 295 300
Arg Ser Arg Ser His Ser Arg Gly His Arg Arg Ala Ser Arg Asp Arg 305 310 315 320
Ser Ala Lys Tyr Lys 325
(2) INFORMATION FOR SEQ ID NO : 9 :
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 1761 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : CAAGGGAAGT TCTGAGGGCT GAGAGGTTGC TCATTCGTCA GAGCGTGCTG CCCACCCTCC 60
ACCCCTGCAT GGCAGAAACT GTGCAGGGGA CGAGGCCAAG GAATCAGGAG ACCCAGAGGC 120
AGGGGTGGCC CGGAGACGGT GAAGAAACCA AGACGCAGAG AGGCCAAGCC CCTTGCCTTG 180
GGTCACACAG CCAAAGGAGG CAGAGCCAGA ACTCACAACC AGATCCAGAG GCAACAGGGA 240
CATGGCCACC TGGGACGAAA AGGCAGTCAC CCGCAGGGCC AAGGTGGCTC CCGCTGAGAG 300
GATGAGCAAG TTCTTAAGGC ACTTCACGGT CGTGGGAGAC GACTACCATG CCTGGAACAT 360
CAACTACAAG AAATGGGAGA ATGAAGAGGA GGAGGAGGAG GAGGAGCAGC CACCACCCAC 420
ACCAGTCTCA GGCGAGGAAG GCAGAGCTGC AGCCCCTGAC GTTGCCCCTG CCCCTGGCCC 480
CGCACCCAGG GCCCCCCTTG ACTTCAGGGG CATGTTGAGG AAACTGTTCA GCTCCCACAG 540
GTTTCAGGTC ATCATCATCT GCTTGGTGGT TCTGGATGCC CTCCTGGTGC TTGCTGAGCT 600
CATCCTGGAC CTGAAGATCA TCCAGCCCGA CAAGAATAAC TATGCTGCCA TGGTATTCCA 660
CTACATGAGC ATCACCATCT TGGTCTTTTT TATGATGGAG ATCATCTTTA AATTATTTGT 720
CTTCCGCCTG GAGTTCTTTC ACCACAAGTT TGAGATCCTG GATGCCGTCG TGGTGGTGGT 780
CTCATTCATC CTCGACATTG TCCTCCTGTT CCAGGAGCAC CAGTTTGAGG CTCTGGGCCT 840
GCTGATTCTG CTCCGGCTGT GGCGGGTGGC CCGGATCATC AATGGGATTA TCATCTCAGT 900
TAAGACACGT TCAGAACGGC AACTCTTAAG GTTAAAACAG ATGAATGTAC AATTGGCCGC 960 CAAGATTCAA CACCTTGAGT TCAGCTGCTC TGAGAAGGAA CAAGAAATTG AAAGACTTAA 1020
CAAACTATTG CGACAGCATG GACTTCTTGG TGAAGTGAAC TAGACCCGGA CCAGCTCCCC 1080
TCAAAAAGAA GACACTGTCT CATGGGCCTG TGCTGTCACG AGAGGAACAG CTGCCCCTCC 1140
TGGGCCGCTT GGTGAGAGGT TTGGTTTGAT ACCTCTGCCT CCCTCCTGCC AGCATGGATT 1200
CTGGGTGGAC ACAGCCTTGT GGAAGGTCCA GTACCACCAA GAGCTGCCCA TCCACTCCCA 1260
CCCCACACTG TATCAAATGT ATCACATTTT CTCATGTTGA ACACTTTAGC CTTAATTGAA 1320
AATGAGCAAC AAAGCTGGAC AATTGCTAGT TGTATATAAA ATTTAATCTC ACCGAATGTA 1380
CAGTTTTCAA ATTTCACGTG TATATTAAGG AACTGATGCA TCTGAGCATT CTGAAAGAAA 1440
GAAAAAGAAG CTACTTTAGC TGCCACCCCA TTCTAGAAAA GTCTCTTATT TTCAAGCTGT 1500
TCTAAATAGC TTCGTCTCAG TTTCCCCAAA AGGGGTACCC AGGCCCCTCC TCTGTGTGCC 1560
CCAGCTGCAT CAGCCAGCTT CTAGGTGGCT CCATTGTTTT CTGCCACCTG ACAACATTTT 1620
TCCTCAATTA CTGTACAACT ACTGTATAAA ATAAAACAAC TACTGTATAA AATAAACTCT 1680
CTCTTTTCCC TGGAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1740
AAAAAAAAAA AAAAAAAAAA A 1761 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 ammo acids
(B) TYPE: am o acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ala Thr Trp Asp Glu Lys Ala Val Thr Arg Arg Ala Lys Val Ala
1 5 10 15
Pro Ala Glu Arg Met Ser Lys Phe Leu Arg His Phe Thr Val Val Gly
20 25 30
Asp Asp Tyr His Ala Trp Asn lie Asn Tyr Lys Lys Trp Glu Asn Glu
35 40 45
Glu Glu Glu Glu Glu Glu Glu Gin Pro Pro Pro Thr Pro Val Ser Gly
50 55 60 Glu Glu Gly Arg Ala Ala Ala Pro Asp Val Ala Pro Ala Pro Gly Pro 65 70 75 80
Ala Pro Arg Ala Pro Leu Asp Phe Arg Gly Met Leu Arg Lys Leu Phe 85 90 95
Ser Ser His Arg Phe Gin Val lie lie lie Cys Leu Val Val Leu Asp 100 105 110
Ala Leu Leu Val Leu Ala Glu Leu lie Leu Asp Leu Lys lie lie Gin 115 120 125
Pro Asp Lys Asn Asn Tyr Ala Ala Met Val Phe His Tyr Met Ser lie 130 135 140
Thr lie Leu Val Phe Phe Met Met Glu lie lie Phe Lys Leu Phe Val 145 150 155 160
Phe Arg Leu Glu Phe Phe His His Lys Phe Glu lie Leu Asp Ala Val 165 170 175
Val Val Val Val Ser Phe lie Leu Asp lie Val Leu Leu Phe Gin Glu 180 185 190
His Gin Phe Glu Ala Leu Gly Leu Leu lie Leu Leu Arg Leu Trp Arg 195 200 205
Val Ala Arg lie lie Asn Gly lie lie lie Ser Val Lys Thr Arg Ser 210 215 220
Glu Arg Gin Leu Leu Arg Leu Lys Gin Met Asn Val Gin Leu Ala Ala 225 230 235 240
Lys lie Gin His Leu Glu Phe Ser Cys Ser Glu Lys Glu Gin Glu lie 245 250 255
Glu Arg Leu Asn Lys Leu Leu Arg Gin His Gly Leu Leu Gly Glu Val 260 265 270
Asn
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 928 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AATCGGGAGT CCTGGAAAGT TAATTCCAAT GTCATGACGT CAAACTTAAA ATGGTCGTCT 60
GCCACCATGA AGATTGATAA CCCTCACATA ACATATACTG CCAGGGTCCC AGTCGATGTC 120
CATGAATACA ACCTAACGCA TCTGCAGCCT TCCACAGATT ATGAAGTGTG TCTCACAGTG 180
TCCAATATTC ATCAGCAGAC TCAAAAGTCA TGCGTAAATG TCACAACCAA AAATGCCGCC 240
TTCGCAGTGG ACATCTCTGA TCAAGAAACC AGTACAGCCC TTGCTGCAGT AATGGGGTMT 300
ATGTTTGCCG TCATTAGCCT TGCGTCCATT GCTGTGGTAC TTTGCCAAAA GATTTAAGAG 360
AAAAAANTAC CACCACTCAT TAAAAAAGTA TATGCAAAAA ACCTCTTCAA TCCCACTAAA 420
TGAGCTGTAC CCACCACTCA TTAACCTNTG GGAAGGTGAC AGCGAGNAAG ACAAAGATGG 480
TTTTGCAGAC ACCAAGCCAA CCCAGGTNGA CACATCCAGA AGGTATTACA TGTGGTAANT 540
CAGAGGATAT TTTGCTTCTG GTAGTAAGGA GCACAAAGAC GTTTTTGCTT TATTCTGCAA 600
AAGTGAACAA GTTGAAGACT TTTGTATTTT TGACTTTGCT AGTTTGTGGC AGAGTGGAGA 660
GGACGGGTGG ATATTTCAAA TTTTTTTAGT ATAGCGTATC GCAAGGGTTT GACACGGCTG 720
CCAGCGACTC TAGGCTTCCA GTCTGTGTTT GGTTTTTATT CTTATCATTA TTATGATTGT 780
TATTATATTA TTATTTTATT TTAGTTGTTG TGCTAAACTC AATAATGCTG TTCTAACTAC 840
AGTGCTCAAT AAAATGATTA ATGACAGGAT GGGGTTCCCC TGTGCTTTTA CCAGTAGCAT 900
GACCCTTCCT GAAGCCATCC GTAGAAAG 928 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met lie Val lie lie Leu Leu Phe Tyr Phe Ser Cys Cys Ala Lys Leu 1 5 10 15
Asn Asn Ala Val Leu Thr Thr Val Leu Asn Lys Met lie Asn Asp Arg 20 25 30 Met Gly Phe Pro Cys Ala Phe Thr Ser Ser Met Thr Leu Pro Glu Ala 35 40 45 lie Arg Arg Lys 50
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AAATTAAANA AAAAAAAAAA AAAAAAAAAA AAATAAAGAA AAAAAAAAA 49
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 597 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
ATTCTACAAG ATAACTTCCC AGTACTTTAA AAAAGTCTCA AAGTCATAAA CAAGAAAGAA 60
CTGAGGGACT ATTGCATATT GGAGCGATCT AAAGAAGTAT TACAATTTGT GGAATTCTTG 120
ATTAAATCCT GGACCAGCAA AAGGACATTA GTGGGAAAAT TGATGAAATT CAAATGAGAT 180
CTTATATTGA AGTTAATTGT GTCAGTGTAC ATTTCCTGGT TTTCATAATT GCAAGTGATT 240
ATGTAAGGTT TGTTAATATT AGGAGCAGCT GGGTAAAGGT TATACAAAAA CTCTATACTA 300
TTTTTGCATT TTTTTCTGTA AGTTTAAAAC ATTTTCCAAC TAAAAAGTTG AAAACACATG 360
TATTAGAGAC ACATGCGTAT GTGTCTCTAA TAATCTTAAA TATATTTAAG ATGATAGAAG 420
GAATTCTTGA GATAGTAAAA TGAAGTCACC AAAAAACAAA CAAAGAAACA AAACGAAATC 480
ACCAAAATCT ATCAATAAAT TTCAGGTAAT ACTTTTGGCA GATTCATTCC TTTGAGATGG 540 AGTCTCACTC CCAGTCTGGG CAACGAGCGA AACTCCGTCT AAAAAAAAAA AAAAAAA 597 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Arg Ser Tyr lie Glu Val Asn Cys Val Ser Val His Phe Leu Val 1 5 10 15
Phe lie lie Ala Ser Asp Tyr Val Arg Phe Val Asn lie Arg Ser Ser 20 25 30
Trp Val Lys Val lie Gin Lys Leu Tyr Thr lie Phe Ala Phe Phe Ser 35 40 45
Val Ser Leu Lys His Phe Pro Thr Lys Lys Leu Lys Thr His Val Leu 50 55 60
Glu Thr His Ala Tyr Val Ser Leu lie lie Leu Asn lie Phe Lys Met 65 70 75 80
He Glu Gly He Leu Glu He Val Lys 85
[ 2 ) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGAGTTCATT AGGCCAGTGG CACTGATTTT TCCTTCCCAT CATAGCTATT TATTCTAGTA 60
AGAGGATAAT AAAAGAAATT TCCTATACAA GAACTGAAAT TTTCCATTTG TATGGATCTA 120
GTCACTTTCA GATTTCAATT TGAGGTTAAG TATATAAAGC ACATCCCAAT TTTATATGCT 180 GCCTTGAGAA AATTACAGGA TGCACGGCAA TTTGTAGGAA TTTCAAATGG GATCATTTAA 240
ACATTTGAAA AATTATTTTA AAAACCATCT AGTTTGCTTT TGGATTTTAG ACATTAAAGC 300
CTATGTTGTC TTGTTAACAG GGGTGGAATG TATAACCATC AGATTCAGCA TGTGATTTCA 360
CCTTTGAATC TGAGTATTTC TTCCCTATCT TCTTTGAGTC ATTTTTGGAG CAGACTGTCA 420
CCAGTATTGA TAACTAAGCA TTAAAGGGAA AAGTTGCATT GCAACTATGC ATTGGTTTCC 480
TGGAAGAACT TTTCTTTTGT TTTAGTGAAT GAAGAGGCTT GATGGGATCA CTTACTGTAA 540
CTCCTTCTAC ATAAGGACCC CTTCTGCAAG CAGAACACAA AAGAACATGC TCAAGGAGTA 600
TCCCATTTTC TGGATAAATT GAAGAAGTTT GCTAGTAATG TCTTTATACT AGCGTCTTCC 660
TTGTATCCCT TTGCTGGCAA GGGAATACAA GGCGTCAAGA CCACAGATCA AAACACCCCA 720
CATTTGAGTG GAGTCTTATT TTTACTCCAA GAGCAGTTAT TCCCTTCTAG TCTAAAATTG 780
GCAGTTTTTT CTTTTTTTTA ATAAAATTTT TAAAATATTC CCAAACCAGT GGAACACAGA 840
CACTGGCTGC ACTTAGTACT GCCAAAAGCC AAGGTCATTT GCACATATTC CATCAACCTG 900
TCGAGAATTA GGCCTCACTT TATAACCCAA GGCATGGAAG TGCATGCATT CTCTTAGCTG 960
GGCAAACAAT TATACTGTAG TTGTGATACA ACACATGTGG CTTTTATTTG TACTGCACAT 1020
ATCCACTGTA CAGCCACTTG GGAGTATCGT GGTTAGCTTG CAGCAACTGC TGTCTGCATT 1080
TATACTGTTT ATTGCATATT CTTTTCCCTG GAAGTGAAAG AGAAATGTTT TTCTTGTTGC 1140
ATTGATTACA TTTTATAAAT TTGCTTAGCT GGAAAGTTTG GGAAAAGAGG CCTGTTTGTC 1200
AATTGTACAA CCGATTGTGA AGCTCTAGTG TGAATATTTT TACGTCTGTA TTAGACATTT 1260
TCTTTGCAAA TCTATTGTTC GATTGAAATG TAAATGAAAT TAAAGATGGT GTACACCCAT 1320
CATGTAAAAA GCAGGCACCA TCTCTAAGAT GGATTTAATG CTCATTTTTA AGGCATATAC 1380
TCAGCTTCTA TTTAAAACTA TAATTTAAAA TAATTCTGTA CAATGAAATG GGGAATATAT 1440
ATGGGAATAA ATTCTATTCC ATTTATTTCA ATTTGAATTT CCAAATTGTA ATGTTTCCCT 1500
TTGTGCTATA GGAATAGGAT TAAATGGGGG AAGACTAGGA TTTATAAGGC CTGTATATGG 1560
GGGGAGGGCA GAGATGGAAC AATGAGGGTT GTGATGATAG TGAATAGCAA AGAGTGAATT 1620
CTGTGTGTTT TTGCTGTAGC ACTGAAGTGA AGAGATATTA GCTTTGGCTG TTCACAAAAT 1680
AGAGCATCAT GATTTTCAGT GTTTGAGAGA AAATTGATGG AAAAAGTTTG CAGTACTTGA 1740
CATGTATTTG CATGCACAAA ATAAAATTAT TTGTCCACCT TAAAAAAAAA AAANAAAAAA 1800
AAAA 1804 ( 2 ) INFORMATION FOR SEQ ID NO : 17 :
( i ) SEQUENCE CHARACTERISTICS :
( A ) LENGTH : 37 amino acids
( B ) TYPE : amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Lys Arg Leu Asp Gly He Thr Tyr Cys Asn Ser Phe Tyr He Arg 1 5 10 15
Thr Pro Ser Ala Ser Arg Thr Gin Lys Asn Met Leu Lys Glu Tyr Pro 20 25 30
He Phe Trp He Asn 35
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 360 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AGAATACCAA GACTGTGTGT ACACGCAGAT GTCAGTGGCA GAGAATGAAG ATCAGCTTCG 60
TGCAAAGGGT TATGACAAAA CACCAGACTT CATTTTACAA GTACCAGTTG CTGTAGAAGG 120
GCACATAATT CACTGGATTG AAAGCAAAGC CTCATTTGGT GATGAATGTA GCCACCACGC 180
CTACCTGCAT GACCAGTTCT GGAGCTACTG GAATAGGGTC CCAATATAAC AGACAAATGG 240
TGAAACAGAG GGATACTCAC TAGGAAACAG ATTTGGGCCA GGCTTAGTCA TCTATTGGTA 300
TGGATTTATC CAGGAGCTGG ACTGCAACCG GGAAAGGGGC ATCCTGCTCA AAGCCTGTTT 360
(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
In) MOLECULE TYPE: prote
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Asp Leu Ser Arg Ser Trp Thr Ala Thr Gly Lys Gly Ala Ser Cys 1 5 10 15
Ser Lys Pro Val 20
(2) INFORMATION FOR SEQ ID NO: 20.
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 202 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 120
AAAAAAAAAA AAAAANTNAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 180
AAAAAAAAAA AAAAAAAAAA AA 202 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 1189 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGCCTTCATG GCCTAATCAG ATTTTAGTGG CAAGACAGGT GATGATAGGG AGAAAGAAAG 60
AGTCCTGCCT GTCAAAAGTG GCTGTGCTAT ATARATGAAC CCTCACAGGT AGCACCTCTC 120
AGAGAGAATA RATGGTAAAT GTTTCTTTTC AGGTCTTTAA AAGTGTCAGG CTATCAGTTA 180
ACCTCTCCTA GATCTCAGAA ATGCCTAGAA AGAGAAGTCC TGGCTACATC AATGGAAATT 240
CTCCACAGAT GCAAATTTTC TCTACAAAAG ATGGCTTTGC AGAGCCACCT CAGTCTGTTG 300
TCCCTGTAGC AGCCATTTCA AATTATGTCA AAGAGATATA TTTTGGGGTA AAATATTTTG 360
ATTATCTTCA TTAGTATATC TCAATTTTGT CAATACAAAC CTGAGAGTTA TAGTCAGAGG 420
TTGAATTTTC ATTTCAAAAT GTTTTCCTAG TTTTTTTTCT CTTTTTTGTT TTATTGTAAG 480
TTGACAATTT ATAATTGTAT AAAAGTATGA GGTACAAAGT GATGTTATAG CTTAAGAATA 540
CAGTATGGTA TGATTAAATC AAGTTATTAA CCTATCCTTC ACGTTAAATG CTTAAATTTT 600
TTGATGAGAA CATTTGAAAT TTACTCTTGG AAGGTAAAAA AAAATCTCAG GACCCCCCAA 660
ATTAAAGCCA TGAAGCTGAA TTGTGCAACA TCCTCTTCCA AATGGAAGCT TGTCTTCCAG 720
GTACAGAACA AAAACAAGAC TCATTTCTTC ACCTGCCTAA AGATGTGCAC ATAATTGGCT 780
CCTCCTTTAC TCCCTTTTTC TCTTCTAACA TTCATTATAT CTTGTGTAAA ATGTAGATTT 840
ACTGGACACT AACTAAAATT TCACAGGGTT GTACCCATTT GCCTTACTGC CTACCTACCT 900
GTYTTCCTAC GTACCTTYTC CCCACTTTAA GGAATGCATA CATATTAAAC CTCCCAAAAA 960
CYTYTTTAGA AAAATAGCCA CAGGTTTATC TGTGGCTGGT GTGTTTCCCT AGATGCACTY 1020
TAAAGCTGGC TTAATAAACC TCAGTGATTG AAACTTATGC CTCAATCACT CATTTTGGTT 1080
GTCACTGTCT TACCAATTTG AAATGTAAAA TATTYTACTA CTAATTATAT TTACCACATT 1140
GTGCAACAGA ACTCNAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA 1189 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2. Met Arg Thr Phe Glu He Tyr Ser Trp Lys Val Lys Lys Asn Leu Arg 1 5 10 15
Thr Pro Gin He Lys Ala Met Lys Leu Asn Cys Ala Thr Ser Ser Ser 20 25 30
Lys Trp Lys Leu Val Phe Gin Val Gin Asn Lys Asn Lys Thr His Phe 35 40 45
Phe Tnr Cys Leu Lys Met Cys Thr 50 55
(2) INFORMATION FOR SEQ ID NO: 23:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CATAACAGCG TCAGAGAGAA AGAACTGACT GAAACGTTTG AGATGAAGAA AGTTCTCCTC 60
CTGATCACAG CCATCTTGGC AGTGGCTGTT GGTTTCCCAG TCTCTCAAGA CCAGGAACGA 120
GAAAAAAGAA GTATCAGTGA CAGCGATGAA TTAGCTTCAG GGTTTTTTGT GTTCCCTTAC 180
CCATATCCAT TTCGCCCACT TCCACCAATT CCATTTCCAA GATTTCCATG GTTTAGACGT 240
AATTTTCCTA TTCCAATACC TGAATCTGCC CCTACAACTC CCCTTCCTAG CGAAAAGTAA 300
ACAAGAAGGA AAAGTCACGA TAAACCTGGT CACCTGAAAT TGAAATTGAG CCACTTCCTT 360
GAAGAATCAA AATTCCTGTT AATAAAAGAA AAACAAATGT AATTGAAATA GCACACAGCA 420
TTCTCTAGTC AATATCTTTA GTGATYTTYT TTAATAAACA TGRAAGCAAA GRAAAAAAAA 480
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 525 (2) INFORMATION FOR SEQ ID NO: 24:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protem (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Met Lys Lys Val Leu Leu Leu He Thr Ala He Leu Ala Val Ala Val 1 5 10 15
Gly Phe Pro Val Ser Gin Asp Gin Glu Arg Glu Lys Arg Ser He Ser 20 25 30
Asp Ser Asp Glu Leu Ala Ser Gly Phe Phe Val Phe Pro Tyr Pro Tyr 35 40 45
Pro Phe Arg Pro Leu Pro Pro He Pro Phe Pro Arg Pne Pro Trp Phe 50 55 60
Arg Arg Asn Phe Pro He Pro He Pro Glu Ser Ala Pro Thr Thr Pro 65 70 75 80
Leu Pro Ser Glu Lys 85
(2) INFORMATION FOR SEQ ID NO : 25 :
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 base pairs
(B) TYPE, nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION, /desc = "oligonucleotide1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25 ANCAGGAGGCA GTGGGTTCTA TAGAGTCC 29
(2) INFORMATION FOR SEQ ID NO: 26-
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CNCTTGTGTGC CTGTTTCTCA ATTGCAGA 29
(2) INFORMATION FOR SEQ ID NO: 27:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 27- CNCTAGCTGTT CGGCTTTACC AAGGAGTT 29
(2) INFORMATION FOR SEQ ID NO : 28 :
(l) 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 ANCACGCTCTG ACGAATGAGC AACCTCTC 29
(2) INFORMATION FOR SEQ ID NO: 29:
(l) 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: CNAACACAGAC TGGAAGCCTA GAGTCGCT 29 (2) INFORMATION FOR SEQ ID NO : 30 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 30 :
GNAAACCAGGA AATGTACACT GACACAAT 29
(2) INFORMATION FOR SEQ ID NO : 31 :
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (3) TYPE: nucleic aciα
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 31 : TNATCAATACT GGTGACAGTC TGCTCCAA 29
(2) INFORMATION FOR SEQ ID NO : 32 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GNCATAACCCT TTGCACGAAG CTGATCTT 29
(2) INFORMATION FOR SEQ ID NO: 33: (ι) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = " oligonucleotide ,:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: ANATTTAAGCA TTTAACGTGA AGGATAGG 29
(2) INFORMATION FOR SEQ ID NO: 34:
(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:34: TNAGAGACTGG GAAACCAACA GCCACTGC 29

Claims

What is claimed is.
1. A composition comprising an isolated polynucleohde selected from the group consisting of.
(a) a polynucleohde compπsing the nucleohde sequence of SEQ ID NO:l,
(b) a polynucleohde comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 68 to nucleohde 430;
(c) a polynucleohde comprising the nucleohde sequence of SEQ ID NO:l from nucleohde 128 to nucleohde 430;
(d) a polynucleohde comprising the nucleotide sequence of the full- length protem coding sequence of clone AJ20_2 deposited under accession number ATCC 98261;
(e) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261;
(f) a polynucleohde comprising the nucleohde sequence of the mature protem coding sequence of clone AJ20_2 deposited under accession number ATCC 98261,
(g) a polynucleohde encoding the mature protem encoded by the cDNA insert of clone AT20_2 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding a protein comprising the ammo acid sequence of SEQ ID NO.2,
(l) a polynucleohde encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO.2 having biological activity,
(j) a polynucleohde which is an allelic variant ot a polynucleo de of
(a)-(g) above;
(k) a polynucleohde which encodes a species homologue of the protein of (h) or (l) above ; and
(1) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleohdes specified in (a)-(i).
2. A composition of claim 1 wherein said polynucleohde is operably linked to at least one expression control sequence.
3. A host cell transformed with a composition of claim 2.
4 The host cell of claim 3, wherem said cell is a mammalian cell
5 A process for producing a protem encoded by a composition 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 protem from the culture
6 A protein produced according to the process of claim 5
7 The protein of claim 6 comprising a mature protein
8 A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of
(a) the amino acid sequence ot SEQ ID NO 2,
(b) fragments of the ammo acid sequence of SEQ ID NO 2, and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ20_2 deposited under accession number ATCC 98261, the protem being substantially free from other mammalian proteins
9 The composition of claim 8 wherem said protem comprises the amino acid sequence of SEQ ID NO 2
10 The composition of claim 8, further comprising a pharmaceutically acceptable carrier
11 A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effechve amount of a composition of claim 10
12 An isolated gene corresponding to the cDNA sequence of SEQ ID NO 1 or SEQ ID NO 3
13. A composition comprising an isolated polynucleotide selected from the group consisting of
(a) a polynucleohde comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleohde comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 289 to nucleohde 780;
(c) a polynucleohde comprising the nucleotide sequence of the full- length prote coding sequence of clone AR440_1 deposited under accession number ATCC 98261,
(d) a polynucleohde encoding the full-length protem encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261,
(e) a polynucleohde comprising the nucleotide sequence of the mature protem coding sequence of clone AR440_1 deposited under accession number ATCC 98261,
(f) a polynucleohde encodmg the mature protem encoded by the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261,
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO 6,
(h) a polynucleohde encoding a prote comprising a fragment of the ammo acid sequence of SEQ ID NO 6 having biological activity;
(1) a polvnucleohde which is an allelic variant of a polynucleohde of
(a)-(f) above,
(j) a polynucleohde which encodes a species homologue of the protem of (g) or (h) above , and
(k) a polynucleo de capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(h)
14. A composition comprising a protem, wherem said protem comprises an ammo acid sequence selected from the group consishng of
(a) the ammo acid sequence of SEQ ID NO:6,
(b) the ammo acid sequence of SEQ ID NO 6 from amino acid 1 to amino acid 160,
(c) fragments of the ammo acid sequence of SEQ ID NO:6; and (d) the amino acid sequence encoded bv the cDNA insert of clone AR440_1 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins
15 An isolated gene corresponding to the cDNA sequence of SEQ ID NO 5 or SEQ ID NO 4
16 A composition comprising an isolated polynucleohde selected from the group consisting of
(a) a polynucleotide comprising the nucleohde sequence of SEQ ID NO 7,
(b) a polynucleotide comprising the nucleohde sequence of SEQ ID NO 7 from nucleohde 76 to nucleohde 1050,
(c) a polynucleohde comprising the nucleo de sequence of SEQ ID NO 7 from nucleotide 331 to nucleo de 567,
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone AS164_1 deposited under accession number ATCC 98261,
(e) a polynucleohde encodmg the full-length protein encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261,
(f) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone AS164_1 deposited under accession number ATCC 98261,
(g) a polynucleotide encoding the mature protem encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO 8,
(l) a polynucleotide encoding a protem comprising a fragment of the amino acid sequence of SEQ ID NO 8 having biological activity,
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above,
(k) a polynucleotide which encodes a species homologue of the protem of (h) or (l) above , and (1) a polynucleotide capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(i).
17. 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 87 to amino acid 164;
(c) fragments of the amino acid sequence of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone AS164_1 deposited under accession number ATCC 98261; the protein being substantially free from other mammalian proteins.
18. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
19. 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 242 to nucleotide 1060;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 596 to nucleotide 1060;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 10 to nucleotide 373;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261;
(g) a polynucleotide comprising the nucleohde sequence of the mature protein coding sequence of clone AX8_1 deposited under accession number ATCC 98261; (h) a polynucleotide encoding the mature protem encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261,
(1) a polynucleotide encoding a protein compπsing the ammo acid sequence of SEQ ID NO:10,
(j) a polynucleotide encodmg a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protem of (l) or (j) above ; and
(m) a polynucleohde capable ot hybridizing under stringent condihons to any one of the polynucleohdes specified m (a)-(j).
20. A composition comprising a protem, wherem said protem comprises an ammo acid sequence selected from the group consisting of-
(a) the amino acid sequence of SEQ ID NO.10,
(b) the amino acid sequence of SEQ ID NO.10 from ammo acid 1 to amino acid 44;
(c) fragments of the ammo acid sequence of SEQ ID NO 10; and
(d) the amino acid sequence encoded by the cDNA insert of clone AX8_1 deposited under accession number ATCC 98261, the protem being substantially free from other mammalian protems
21. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
22. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleohde sequence of SEQ ID NO:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 773 to nucleotide 928;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 815 to nucleohde 928; (d) a polynucleotide comprising the nucleohde sequence of the full- length protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261;
(e) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261;
(f) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone BD176_3 deposited under accession number ATCC 98261;
(g) a polynucleohde encoding the mature protein encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261;
(h) a polynucleohde encoding a protein compπsing the ammo acid sequence of SEQ ID NO: 12;
(1) a polynucleohde encoding a protem comprising a fragment of the ammo acid sequence of SEQ ID NO:12 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (l) above , and
(1) a polynucleohde capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(ι)
23. A composition comprising a prote , wherem said prote comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 12,
(b) fragments of the ammo acid sequence of SEQ ID NO.12; and
(c) the amino acid sequence encoded by the cDNA insert of clone BD176_3 deposited under accession number ATCC 98261; the protem being substantially free from other mammalian protems
24. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:ll or SEQ ID NO:13.
25. A composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:14,
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO-14 from nucleotide 174 to nucleotide 440,
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.14 from nucleotide 1 to nucleotide 313;
(d) a polynucleohde compπsing the nucleotide sequence of the full- length protem coding sequence of clone BD339_1 deposited under accession number ATCC 98261,
(e) a polynucleohde encoding the full-length protein encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261;
(f) a polynucleohde comprising the nucleohde sequence of the mature prote coding sequence of clone BD339_1 deposited under accession number ATCC 98261,
(g) a polynucleohde encoding the mature protein encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO.15,
(l) a polynucleohde encoding a protein comprising a fragment of the ammo acid sequence of SEQ ID NO.15 having biological activity,
(j) a polynucleotide which is an allelic variant of a polynucleo de of
(a)-(g) above;
(k) a polvnucleohde which encodes a species homologue of the protem of (h) or (l) above , and
(1) a polynucleohde capable of hybridizing under strmgent condihons to any one of the polynucleohdes specified m (a)-(ι).
26 A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consishng of-
(a) the amino acid sequence of SEQ ID NO:15,
(b) the amino acid sequence of SEQ ID NO 15 from ammo acid 1 to
Figure imgf000091_0001
(c) fragments of the amino acid sequence of SEQ ID NO:15; and (d) the amino acid sequence encoded by the cDNA insert of clone BD339_1 deposited under accession number ATCC 98261; the protein being substantially free from other mammalian proteins.
27. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:14.
28. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16 from nucleotide 509 to nucleohde 619;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16 from nucleotide 1 to nucleotide 580;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BD427_1 deposited under accession number ATCC 98261;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BD427_1 deposited under accession number ATCC 98261;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 17;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:17 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleohde which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleohde capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
29. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:17;
(b) the amino acid sequence of SEQ ID NO:17 from amino acid 1 to amino acid 24;
(c) fragments of the amino acid sequence of SEQ ID NO: 17; and
(d) the amino acid sequence encoded by the cDNA insert of clone BD427_1 deposited under accession number ATCC 98261; the protein being substantially free from other mammalian proteins.
30. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:16.
31. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 from nucleotide 300 to nucleotide 360;
(c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BL229_22 deposited under accession number ATCC 98261;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261;
(e) a polynucleohde comprising the nucleohde sequence of the mature protein coding sequence of clone BL229_22 deposited under accession number ATCC 98261;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 19;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:19 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above; (j) a polynucleohde which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleohde capable of hybridizing under stringent condihons to any one of the polynucleotides specified in (a)-(h).
32 A composition comprising a protein, wherem said protem comprises an amino acid sequence selected from the group consishng of:
(a) the amino acid sequence of SEQ ID NO:19;
(b) fragments of the amino acid sequence of SEQ ID NO 19; and
(c) the amino acid sequence encoded by the cDNA insert of clone BL229_22 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins
33 An isolated gene corresponding to the cDNA sequence of SEQ ID NO:18 or SEQ ID NO.20.
34 A composition comprising an isolated polynucleotide selected from the group consisting of.
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO:21,
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.21 from nucleotide 604 to nucleotide 771,
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.21 from nucleotide 1 to nucleotide 684;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protem coding sequence of clone BV123_16 deposited under accession number ATCC 98261,
(e) a polynucleotide encoding the full-length protem encoded by the cDNA msert of clone BV123_16 deposited under accession number ATCC 98261;
(f) a polynucleohde compπsmg the nucleohde sequence of the mature protein coding sequence of clone BV123_16 deposited under accession number ATCC 98261,
(g) a polynucleotide encoding the mature protein encoded by the cDNA msert of clone BV123_16 deposited under accession number ATCC 98261; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22,
(1) a polynucleotide encoding a protein comprising a fragment of the ammo acid sequence of SEQ ID NO.22 having biological activity,
(j) a polynucleotide which is an allelic variant of a polynucleohde of
(a)-(g) above;
(k) a polynucleohde which encodes a species homologue of the protem of (h) or (1) above ; and
(1) a polynucleohde capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(ι)
35 A composition compπsing a protein, wherein said protem comprises an amino acid sequence selected from the group consisting of
(a) the amino acid sequence ot SEQ ID NO.22,
(b) the amino acid sequence of SEQ ID NO.22 from ammo acid 1 to ammo acid 27,
(c) fragments of the ammo acid sequence of SEQ ID NO.22, and
(d) the amino acid sequence encoded by the cDNA msert of clone BV123_16 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins
36. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:21
37 A composition comprising an isolated polynucleotide selected from the group consisting of-
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.23,
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO.23 from nucleohde 43 to nucleohde 297;
(c) a polynucleotide comprising the nucleohde sequence of SEQ ID NO.23 from nucleohde 94 to nucleotide 297;
(d) a polynucleotide comprising the nucleohde sequence of SEQ ID NO:23 from nucleohde 1 to nucleotide 379; (e) a polynucleohde compπsing the nucleohde sequence of the full- length protein coding sequence of clone CH377_1 deposited under accession number ATCC 98261,
(f) a polynucleohde encoding the full-length protem encoded by the cDNA msert of clone CH377_1 deposited under accession number ATCC 98261,
(g) a polynucleohde comprising the nucleohde sequence of the mature protem coding sequence of clone CH377_1 deposited under accession number ATCC 98261,
(h) a polynucleotide encoding the mature protem encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261;
(l) a polynucleotide encoding a protem comprising the amino acid sequence of SEQ ID NO.24;
(j) a polynucleohde encodmg a prote comprising a fragment of the amino acid sequence of SEQ ID NO 24 having biological activity;
(k) a polynucleohde which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleohde which encodes a species homologue of the protein of (l) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent condihons to any one of the polynucleohdes specified in (a)-(j)
38 A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of
(a) the ammo acid sequence of SEQ ID NO:24;
(b) fragments of the amino acid sequence of SEQ ID NO.24; and
(c) the amino acid sequence encoded by the cDNA insert of clone CH377_1 deposited under accession number ATCC 98261, the protein being substantially free from other mammalian proteins.
39. An isolated gene correspondmg to the cDNA sequence of SEQ ID NO:23.
PCT/US1997/020740 1996-11-15 1997-11-14 Secreted proteins and polynucleotides encoding them WO1998021332A2 (en)

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