WO1997046683A2 - Polynucleotides encoding secreted proteins - Google Patents

Polynucleotides encoding secreted proteins Download PDF

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Publication number
WO1997046683A2
WO1997046683A2 PCT/US1997/009878 US9709878W WO9746683A2 WO 1997046683 A2 WO1997046683 A2 WO 1997046683A2 US 9709878 W US9709878 W US 9709878W WO 9746683 A2 WO9746683 A2 WO 9746683A2
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WIPO (PCT)
Prior art keywords
seq
polynucleotide
protein
amino acid
sequence
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Application number
PCT/US1997/009878
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French (fr)
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WO1997046683A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Cheryl Evans
Michael Bowman
Vikki Spaulding
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Genetics Institute, Inc.
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Publication date
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to JP10500884A priority Critical patent/JP2001501455A/en
Priority to AU33804/97A priority patent/AU3380497A/en
Priority to EP97929838A priority patent/EP0910638A2/en
Priority to CA002256522A priority patent/CA2256522A1/en
Publication of WO1997046683A2 publication Critical patent/WO1997046683A2/en
Publication of WO1997046683A3 publication Critical patent/WO1997046683A3/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
    • 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
    • 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
    • A61P7/04—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
    • 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
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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 ides a composition comp ⁇ sing an isolated polynucleotide selected from the group consisting of
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:2 from nucleotide 351 to nucleotide 506; the nucleotide sequence of the full length protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 5 from nucleotide 23 to nucleotide 517, the nucleotide sequence of the full length protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076
  • the present invention provides a polynucleotide encoding a protein comp ⁇ sing the amino acid sequence of SEQ ID NO 6 from amino acid 35 to amino acid 1 15
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
  • the present invention provides a composition comp ⁇ sing a protein, wherein said protein compnses an amino acid sequence selected from the group consisting of (a) the am o acid sequence of SEQ ID NO 6, 46683 PC ⁇ YUS97/09878
  • the present invention provides a composition comp ⁇ sing an isolated polynucleotide selected from the group consisting of
  • ATCC 98076 (g) a polynucleotide encoding the mature protein encoded by the ⁇ DNA insert of clone AU105_ 14 deposited under accession number ATCC 98076,
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (I) above; and (1) a polynucleotide capable of hyb ⁇ dizing under st ⁇ ngent conditions to any one of the polynucleotides specified in (a)-( ⁇ )
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 8 from nucleotide 288 to nucleotide 629, the nucleotide sequence of SEQ ID NO:8 from nucleotide 441 to nucleotide 629; the nucleotide sequence of the full length protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076
  • the present invention provides a polynucleotide encoding a protein comp ⁇ sing the amino acid sequence of SEQ ID NO'9 from amino acid 25 to amino acid 44
  • the present invention provides a composition comp ⁇ sing a protein, wherein said protein comp ⁇ ses an ammo acid sequence selected from the group consisting of.
  • the present invention provides a composition compnsing an isolated polynucleotide selected from the group consisting of
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 1 1 from nucleotide 164 to nucleotide 298, the nucleotide sequence of the full length protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AS268_1 deposited under accession number A ⁇ TT 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076
  • the present invention provides a composition comp ⁇ sing a protein, wherein said protein comp ⁇ ses an amino acu sequence selected from the group consisting of:
  • the present invention provides a composition comp ⁇ sing an isolated polynucleotide selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15;
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 from nucleotide 254 to nucleotide 681 ; the nucleotide sequence of the full length protein coding sequence of clone D147_17 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone D147_17 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone D147_17 deposited under accession number ATCC 98076.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 73 to amino acid 129.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 16 from amino acid 73 to amino acid 129.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18 from nucleotide 28 to nucleotide 388; the nucleotide sequence of SEQ ID NO: 18 from nucleotide 76 to nucleotide 388; the nucleotide sequence of the full length protein coding sequence of clone 075_9 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone 075_9 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone 075_9 deposited under accession number ATCC 98076.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO: 1
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 19;
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • ⁇ olynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 21 from nucleotide 75 to nucleotide 419, the nucleotide sequence of SEQ ID NO 21 from nucleotide 132 to nucleotide 419, the nucleotide sequence of the full length protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AJ 147_1 deposited under accession number ATCC 98076
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076
  • the present invention provides a composition comprising a protein, wherein said protein comp ⁇ ses 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) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO:24 from nucleotide 69 to nucleotide 377; the nucleotide sequence of SEQ ID NO:24 from nucleotide 120 to nucleotide 377; the nucleotide sequence of the full length protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM262_1 1 deposited under accession number ATCC 98076.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81.
  • 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:25 or the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 26 from nucleotide 1 10 to nucleotide 448, the nucleotide sequence of the full length protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076
  • the present invention provides a polynucleotide encoding a protein comp ⁇ sing the amino acid sequence of SEQ ID NO 27 from ammo acid 15 to ammo acid 78
  • the present invention provides a composition comp ⁇ sing a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of
  • the present invention provides a composition comp ⁇ sing an isolated polynucleotide selected from the group consisting of
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 30 from nucleotide 230 to nucleotide 541 , the nucleotide sequence of the full length protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076 or the nucleotide sequence of the mature protein coding sequence ot clone AS162_1 deposited under accession number ATCC 98076
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO 31 from amino acid 5 to ammo acid 25
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
  • the present invention provides a composition comp ⁇ sing 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 31 , (b) the amino acid sequence of SEQ ID NO:31 from ammo acid 5 to amino acid 25;
  • protein comprises the amino acid sequence of SEQ LD NO:31 or the amino acid sequence of SEQ ID NO:31 from amino acid 5 to amino acid 25.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:34 from nucleotide 202 to nucleotide 467; the nucleotide sequence of SEQ ID NO: 34 from nucleotide 241 to nucleotide 467; the nucleotide sequence of the full length protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 173 to nucleotide 579; the nucleotide sequence of the full length protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comp ⁇ ses the nucleotide sequence of SEQ ID NO 42 from nucleotide 363 to nucleotide 593, the nucleotide sequence of SEQ ID NO 42 from nucleotide 483 to nucleotide 593, the nucleotide sequence of the full length protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076
  • the present invention provides a composition comp ⁇ sing a protein, wherein said protein comp ⁇ ses an amino acid sequence selected from the group consisting of
  • the polynucleotide is operably linked to an expression control sequence
  • the invention also provides a host cell, including bactenal, yeast, insect and mammalian cells, transformed with such polynucleotide compositions Processes are also provided for producing a protein, which comp ⁇ se
  • the protein produced according to such methods is also provided by the present invention.
  • Preferred embodiments include those in which the protein produced by such process is a mature form of the protein Protein compositions of the present invention may further comp ⁇ se a pharmaceutically acceptable earner Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention
  • Methods are also provided for preventing, treating or ameliorating a medical condition which comp ⁇ ses administe ⁇ ng to a mammalian subject a therapeutically effective amount of a composition comp ⁇ sing a protein of the present invention and a pharmaceutically acceptable earner
  • FIGURES Fig 1 is an autoradiograph demonatratmg the expression of D147_17 in COS cells
  • Fig 2 is an autoradiograph deonstrating the expression of AM262_1 1 in COS cells
  • nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids
  • the actual nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods The predicted am o acid sequence (both full length and mature) can then be determined from such nucleotide sequence
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence
  • reported protein sequences include "Xaa” designators These "Xaa” designators indicate either ( 1 ) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined more accurately)
  • 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 m its ammo acid sequence
  • “Secreted” proteins include without limitation proteins secreted wholly (e g , soluble proteins) or partially (e g , receptors) from the cell in which they are expressed "Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplpasmic reticulum
  • a polynucleotide of the present invention has been identified as clone "AZ302_1 ' AZ302_1 was isolated from a human colon (Caco-2 adenocarcinoma) cDNA library using methods which are selective for cDNAs encoding secreted proteins AZ302_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AZ302_1 protein”)
  • nucleotide sequence of the 5' portion of AZ302_1 as presently determined is reported in SEQ ID NO 1
  • SEQ ID NO 2 An additional internal nucleotide sequence from AZ302 . 1 as presently determined is reported in SEQ ID NO 2
  • SEQ ID NO 3 Additional nucleotide sequence from the 3' portion of AZ302_1 , including the polyA tail is reported in SEQ ID NO 4
  • AZ302_1 The nucleotide sequence disclosed herein for AZ302_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AZ302J demonstrated at least some homology with an EST identified as "ye83a03 rl Homo sapiens cDNA clone 124300 5' at accession number R02197 (BlastN) Based upon homology, AZ302_1 proteins and each homologous protein or peptide may share at least some activity
  • AU139_2 A polynucleotide of the present invention has been identified as clone "AU139_2" AU139_2 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins AU139_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AU139_2 protein")
  • the nucleotide sequence of the 5' portion of AU139_2 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 acid sequence of the AU139_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 6
  • Additional nucleotide sequence from the 3' portion of AU139_2, including the polyA tail is reported in SEQ ID NO 7
  • AU139_2 demonstrated at least some homology with three ESTs identified as "EST 16319 Homo sapiens cDNA 5' end" (accession number T30419, BlastN), "EST04080 Homo sapiens cDNA clone HFBDQ07" (accession number T06191 , BlastN), and "FST108441 Rattus sp cDNA 5 ' Based upon homology, AU 139_2 proteins and each homologous protein or peptide may share at least some activity
  • AU105_14 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins
  • AU 10 14 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AU105_14 protein")
  • the nucleotide sequence of the 5' portion of AU 105_14 as presently determined is reported in SEQ ID NO 8 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO 9
  • the predicted acid sequence of the AU105_14 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 9
  • Amino acids 1 to 51 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 52
  • Additional nucleotide sequence from the 3' portion of AU105_14, including the polyA tail is reported in SEQ ID NO 10
  • the EcoRI/NotI rest ⁇ ction fragment obtainable from the deposit containing clone AU105_14 should be approximately 2670 bp
  • the nucleotide sequence disclosed herein for AU105_14 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols No hits were found in the database
  • AS268_1 A polynucleotide of the present invention has been identified as clone "AS268_1 " AS268_1 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins AS268_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS268_1 protein”)
  • the nucleotide sequence of the 5' portion of AS268_1 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 in SEQ ID NO 12
  • the predicted acid sequence of the AS268_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 12
  • Additional nucleotide sequence from the 3' portion of AS268_1 , including the polyA tail, is reported in SEQ ID NO 13
  • the EcoRI/NotI rest ⁇ ction fragment obtainable from the deposit containing clone AS268_1 should be approximately 1800 bp
  • AS268_1 The nucleotide sequence disclosed herein for AS268_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS268J demonstrated at least some homology with the rabbit and mu ⁇ ne ryanodine receptors (BlastN accession number M59743, BlastX accession number X839 3) Ryanodine receptors have recently been shown to be the Ca 2+ releasr channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle Based upon homology. AS268_1 proteins and each homologous protein or peptide may share at least some activity
  • a polynucleotide of the present invention has been identified as clone "D147_I7" DI 47_17 was isolated from a human PBMC cDNA library using methods which are selective for cDNAs encoding secreted proteins D147_17 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as ' D147_17 protein")
  • nucleotide sequence of the 5' portion of D147_17 as presently determined is reported in SEQ ID NO 14
  • An additional internal nucleotide sequence from D147_17 as presently determined is reported in SEQ ID NO 15 What applicants believe is the proper reading frame and the predicted ammo acid sequence encoded by such internal sequence is reported in SEQ ID NO: 16.
  • Additional nucleotide sequence from the 3' portion ot DI 47_17, including the polyA tail, is reported in SEQ ID NO: 17.
  • the nucleotide sequence disclosed herein for D147_17 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
  • a polynucleotide of the present invention has been identified as clone "075_9”.
  • 075_9 was isolated from a human dendritic cells cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • 075_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "075_9 protein").
  • nucleotide sequence of the 5' portion of 075_9 as presently determined is reported in SEQ ID NO: 18 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO: 19.
  • the predicted acid sequence of the 075_9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 19.
  • Amino acids 1 to 16 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 17. Additional nucleotide sequence from the 3' portion of 075_9, including the polyA tail, is reported in SEQ ID NO:20.
  • nucleotide sequence disclosed herein for 075_9 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
  • a polynucleotide of the present invention has been identified as clone "AJ 147_1 ".
  • AJ147_1 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • AJ147_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AJ147_1 protein").
  • the nucleotide sequence of the 5' portion of AJ147_1 as presently determined is reported in SEQ ID NO:21 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ LD NO:22.
  • the predicted acid sequence of the AJ147_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:22.
  • Amino acids 1 to 19 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20.
  • the EcoRI/NotI rest ⁇ ction fragment obtainable from the deposit containing clone AJ147_1 should be approximately 500 bp
  • AJ147_1 The nucleotide sequence disclosed herein for AJ147_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AJ147_1 demonstrated at least some homology with munne calmegin (Meg l )/calnex ⁇ n (BlastN accession number DI 41 17) Calmegin is a Ca 2+ -binding protein that is specifically expressed in spermatogenesis The highly regulated, specific and abundant expression of calmegin suggests that it plays an important role in spermatogenesis Based upon homology, AJ147_1 proteins and each homologous protein or peptide may share at least some activity
  • a polynucleotide of the present invention has been identified as clone "AM262_1 1 "
  • AM262_11 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins
  • AM262_1 1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AM262_1 1 protein”)
  • AM262_1 1 The nucleotide sequence disclosed herein for AM262_1 I was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AM262_1 1 demonstrated at least some identity with the human eotaxm precursor gene and protein (BlastN accession number U34780, this database entry was made subsequent to applicants' isolation of AM262_1 1 ) Based upon homology, AM262_1 1 proteins and each homologous protein or peptide may share at least some activity
  • AR28_1 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins AR28_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AR28_1 protein")
  • nucleotide sequence of the 5' portion of AR28_1 as presently determined is reported in SEQ ID NO 26 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO 27
  • the predicted acid sequence of the AR28_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 27
  • Additional nucleotide sequence from the 3' portion of AR28_1 , including the polyA tail, is reported SEQ ID NO 28 The nucleotide sequence disclosed herein for AR28_1 was searched against the
  • AS162_1 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins
  • AS 1 2_ I is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS162_1 protein")
  • the nucleotide sequence of the 5' portion of AS 162_1 as presently determined is reported in SEQ ID NO 29
  • An additional internal nucleotide sequence from AS162_1 as presently determined is reported in SEQ ID NO 30 What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO 31
  • Additional nucleotide sequence from the 3' portion of AS162_1 including the polyA tail, is reported in SEQ ID NO 32
  • AS162_1 should be approximately 1380 bp
  • AS162_1 The nucleotide sequence disclosed herein for AS162_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS 162J demonstrated at least some identity with an EST identified as ym96e05 si Homo sapiens cDNA clone 166784 3'" (accession number R88809, BlastN) Based upon identity, AS162_1 proteins and each identical protein or peptide may share at least some activity
  • a polynucleotide of the present invention has been identified as clone "AS264_3" AS264_3 w as isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins
  • AS264_ is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS264_3 protein")
  • SEQ ID NO 33 The nucleotide sequence of the 5' portion of AS264_3 as presently determined is reported in SEQ ID NO 33
  • SEQ ID NO 34 An additional internal nucleotide sequence from AS264_3 as presently determined is reported in SEQ ID NO 34 What applicants believe is the proper reading frame and the predicted ammo acid sequence encoded by such internal sequence is reported in SEQ ID NO 35.
  • Amino acids 1 to 13 of SEQ ID NO 35 are a predicted leader/signal sequence, with the predicted mature ammo acid sequence beginning at ammo acid 14
  • the EcoRI/NotI restnction fragment obtainable from the deposit containing clone AS264_3 should be approximately 3300 bp
  • AS264_3 demonstrated at least some weak similanty to collagen Based upon homology, AS264_3 proteins and each homologous protein or peptide may share at least some activity
  • a polynucleotide of the present invention has been identified as clone "AS301_2"
  • AS301_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins
  • AS301_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS301_2 protein")
  • nucleotide sequence of the 5' portion of AS301_2 as presently determined is reported in SEQ ID NO 37
  • SEQ ID NO 38 An additional internal nucleotide sequence from AS301_2 as presently determined is reported in SEQ ID NO 38 What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO.39
  • Additional nucleotide sequence from the 3' portion of AS301_2, including the polyA tail, is reported in SEQ ID NO 40
  • the EcoRI/NotI restnction fragment obtainable from the deposit containing clone AS301_2 should be approximately 2600 bp
  • AS301_2 The nucleotide sequence disclosed herein for AS301_2 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS301_2 demonstrated at least some homology with ESTs identified as "yp82b08 rl Homo sapiens cDNA clone 19391 1 5'" (BlastN accession number R83399), "ye66c02 rl Homo sapiens cDNa clone 122690 5'", and "ym26e09.rl Homo sapiens cDNA clone 49167 5'" (BlastN accession number H 16691 ) Based upon homology, AS301 _2 proteins and each homologous protein or peptide may share at least some activity
  • AS86_1 A polynucleotide of the present invention has been identified as clone "AS86_1 ".
  • AS86_1 was isolated from a human fetal brain cDNA library using methods which are selective forcDNAs encoding secreted proteins.
  • AS86_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS86_1 protein").
  • the nucleotide sequence of the 5' portion of AS86_1 as presently determined is reported in SEQ ID NO:41.
  • An additional internal nucleotide sequence from AS86_1 as presently determined is reported in SEQ LO NO:42.
  • SEQ ID NO:43 Amino acids 1 to 40 of SEQ ID NO:43 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 41. Additional nucleotide sequence from the 3' portion of AS86_1 , including the polyA tail, is reported in SEQ ID NO:44.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone AS86_1 should be approximately 2122 bp.
  • the nucleotide sequence disclosed herein for AS86_1 was searched against the
  • GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
  • Figs. 1 and 2 are autoradiographs evidencing expression of clones of the present invention. All clones were expressed in COS cells.
  • Clones AZ302 , AU139_2, AU105 4, AS268J , D147 7, 075_9, AJ147 , AM262_1 1 , AR28_1, AS162_1, AS264_3, AS301_2 and AS86_1 were deposited on June 6, 1996 with the American Type Culture Collection under accession number ATCC 98076, from which each clone comprising a particular polynucleotide is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit.
  • Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' cite, EcoRI; 3' cite, NotI) to produce the appropriately sized fragment for such clone (approximate clone size fragment are identified below).
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
  • oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
  • the design of the oligonucleotide probe should preferably follow these parameters
  • oligonucleotide should preferably be labeled with g- J2 P ATP (specific activity 6000
  • the bactenal culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a stenle culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well -separated colonies on solid bacte ⁇ ological media containing L-broth containing ampicillin 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 distinct, well-separated colonies can also be employed
  • Standard colony hybndization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88 2 g Na citrate/liter, adjusted to pH 7 0 with NaOH) containing 05% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter)
  • the probe is then added to the hyb ⁇ di/ation 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
  • the positive colonies are picked, grown in culture and plasmid DNA isolated using standard procedures The clones can then be verif ied by restriction analysis, hyb ⁇ dization analysis, or DNA sequencing
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention
  • Fragments of the protein may be in linear form or they may be cyc zed using known methods, for example, as desc ⁇ bed in H U Saragovi, et al , Bio/Technology K), 773-778 ( 1992) and in R S McDowell, et al , J Amer Chem Soc 1 14, 9245-9253 (1992), both of which are inco ⁇ orated herein by reference
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin
  • such a fusion could be to the Fc portion of an IgG molecule
  • Other immunoglobulin isotypes may also be used to generate such fusions
  • the mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell
  • the sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides genes corresponding to the cDNA sequences disclosed herein
  • the corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein Such methods include the preparation of probes or p ⁇ mers from the disclosed sequence information for identification and/or amplification of genes in approp ⁇ ate genomic hbranes or other sources of genomic mate ⁇ als
  • the present invention also provides for soluble forms of such protein In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information
  • Species homologs of the disclosed polynucleotides and proteins are also pro ⁇ ided by the present invention
  • Species homologs may be isolated and identified by making suitable probes or pnmers from the sequences provided herein and screening a suitable nucleic acid source from the desired species
  • the invention also encompasses allelic va ⁇ ants of the disclosed polynucleotides or proteins, that is, natural ly-occur ⁇ ng alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al , Nucleic Acids Res .19, 4485-4490 (1991 ), in order to produce the protein recombinantly
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al , Nucleic Acids Res .19, 4485-4490 (1991 ), 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 En7ymology 185, 537-566 (1990)
  • "operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected)
  • yeast in lower eukaryotes such as yeast or in prokaryotes such as bactena
  • yeast strains include Saccharomyces cerevistae, Schtzosactharomyces pombe Kluvveromyces strains, Candida or any yeast strain capable of expressing heterologous proteins
  • Potentially suitable bacte ⁇ al strains include Eschenchia coli, Bacillus subt ⁇ is.
  • Salmonella typhimurutm or any bacte ⁇ al strain capable of expressing heterologous proteins If the protein is made in yeast or bactena, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the approp ⁇ ate sites, in order to obtain the functional protein Such covalent attachments may be accomplished using known chemical or enzymatic methods
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system
  • suitable control sequences in one or more insect expression vectors
  • 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 descnbed in Summers and Smith, Texas Ag ⁇ cultural Expe ⁇ ment Station Bulletin No 1555 (1987). inco ⁇ orated herein by reference As used her in an insect cell capable of expressing a polynucleotide of the present invention is ' transformed
  • the protein of the invention may be prepared bv culturing transformed host cells under culture conditions suitable to express the recombinant protein
  • the resulting expressed protein may then be punfied from such culture (I e , from culture medium or cell extracts) using known pu ⁇ fication processes, such as gel filtration and ion exchange chromatography
  • the pu ⁇ fication of the protein may also include an affinity column containing agents which will bind to the protein, one or more column steps over such affinity res s as concanavalm A- agarose, hepann-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography
  • the protein of the invention may also be expressed in a form which will facilitate pu ⁇ fication
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin Kits for expression and purification of such fusion proteins
  • the protein can also be tagged with an epitope and subsequently punfied 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
  • the protein of the invention may also be expressed as a product of transgenic animals, e g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are characte ⁇ zed by somatic or germ cells containing a nucleotide sequence encoding the protein
  • the protein may also be produced by known conventional chemical synthesis Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art
  • the synthetically-constructed protein sequences by virtue of sha ⁇ ng pnmary, secondary or tertiary structural and/or contormational characte ⁇ ties with proteins may possess biological properties in common therewith, including protein activity
  • they may be employed as biologically active or immunological substitutes for natural, pu ⁇ fied proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of pu ⁇ fied proteins but into which modification are naturally provided or deliberately engineered
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e g , U S Patent No 4,518,584)
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein
  • Other fragments and de ⁇ vatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled m the art given the disclosures herein
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below Uses or activities desc ⁇ bed for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA)
  • the polynucleotides provided by the present invention can be used by the research community for va ⁇ ous pu ⁇ oses
  • the polynucleotides can be used to express recombinant protein for analysis, characte ⁇ zation or therapeutic use, as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states), as molecular weight markers on Southern gels, as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions, to compare with endogenous DNA sequences in patients to identify potential genetic disorders, as probes to hybridize and thus discover novel, related DNA sequences, as a source of information to de ⁇ ve PCR pnmers for genetic fmge ⁇ ntmg, as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides, for selecting and making oligomers for attachment to a ' gene chip" or other support, including for
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • Many protein factors discovered to date, including all known cytokines have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • a protein of the present invention is evidenced by any one ot a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, TIO, B9, B9/1 1, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI , 123, Tl 165, HT2, CTLL2, TF-1 , Mo7e and CMK
  • the activity of a protein of the invention may, among other means, be measured by the following methods
  • Assays for T-cell or thymocyte proliferation include without limitation those desc ⁇ bed in Current Protocols in 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 Lymphocyte Function 3 1-3 19, Chapter 7, Immunologic studies in Humans), Takai et al , J Immunol 137 3494-3500, 1986, Bertagnolli et al , J Immunol 145 1706-1712, 1990, Bertagnolli et al , Cellular Immunology 133 327-341 , 1991 , Bertagnolli, et al , J Immunol 149 3778-3783, 1992, Bowman et al , J Immunol 152 1756- 1761 , 1994
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in Polyclonal T cell stimulation, Kruisbeek, A M and Shevach, E M In Current Protoc ls 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 Cm tent Protocols in Immunology J E e a Co gan eds Vol 1 pp 6 8 1 -6 8 8, John Wiley and Sons Toronto 1994
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those desc ⁇ bed in Measurement of Human and Mu ⁇ ne Interleukin 2 and Interleukin 4, Bottomly, K , Davis, L S and Lipsky P E In Current Protocols in J E e a Coligan eds Vol
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, he ⁇ esviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asu ma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen- specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft- versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft- versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1 , B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al. Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 59: 1 1 102- 11 105 (1992).
  • murine models of GVHD 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 inapprop ⁇ ate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms
  • blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- characte ⁇ zed animal models of human autoimmune diseases Examples include mu ⁇ ne expenmental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hyb ⁇ d mice, unne autoimmune collagen arthntis, diabetes mel tus in NOD mice and BB rats, and mu ⁇ ne expenmental myasthenia gravts (see Paul ed , Fundamental Immunology, Raven Press, New York, 1989, pp 840-856)
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy
  • Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response
  • enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection
  • systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed
  • APCs either expressing a peptide of the present invention oi together with a stimulatory form of a soluble peptide of the present invention and re troducing the in vitro activated T cells into the patient
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as desc ⁇ bed herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient
  • the infected cells would now be capable of de ve ⁇ ng a costimulatory signal to, and thereby activate, T cells in vivo
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • Tumor cells 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.
  • the tumor cell can be transfected to express a combination of peptides .
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class 1 or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class ⁇ proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class ⁇ proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981 ; Herrmann et al., J. Immunol. 128: 1968- 1974, 1982; Handa et al., J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1 -3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of 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 -
  • lymphocyte survival/apoptosis which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis
  • Assays for lymphocyte survival/apoptosis include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84: 1 1 1-117, 1994; Fine et al., Cellular Immunology 155:1 1 1-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551 , 1991.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81 :2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-591 1, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of bums, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.
  • De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic- plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes.
  • Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, ca ⁇ al tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebro vascular diseases such as stroke.
  • diseases of the peripheral nervous system such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies
  • central nervous system diseases such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
  • Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non- healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular endothelium, and vascular endothelium, and vascular endothelium, and vascular endothelium, and vascular endothelium, and vascular endothelium, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma
  • a protein of the invention may also exhibit angiogenic activity
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in vanous 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 descnbed above from precursor tissues or cells, or for inhibiting the growth of tissues desc ⁇ bed above
  • the activity of a protein of the invention may, among other means, be measured by the following methods
  • Assays for tissue generation activity include, without limitation, those desc ⁇ bed in International Patent Publication No WO95/16035 (bone, cartilage, tendon), International Patent Publication No WO95/05846 (nerve, neuronal), International Patent Publication No WO91/07491 (skin, endothelium )
  • Assays for wound healing activity include, without limitation, those desc ⁇ bed in
  • a protein of the present invention may also exhibit activin- or inhibin-related activities Inhibms are characte ⁇ zed by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are charactered by their ability to stimulate the release of follicle stimulating hormone (FSH)
  • FSH follicle stimulating hormone
  • a protein of the present invention alone or in heterodimers with a member of the inhibm ⁇ family, may be useful as a contraceptive based on the ability of inhibms to decrease fertility in female mammals and d crease spermatogenesis in male mammals Administration of sufficient amounts of other inhibms can induce infertility in these mammals
  • the protein of the invention as a homodimer or as a heterodimer with other protein subunits of the inhibm- ⁇ group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the antenor pitu
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Nature 321 :779-782, 1986; Vale et al., Nature 321 :776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian ceils, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity As a result, such a protein is expected to be useful in treatment of va ⁇ ous coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g , stroke)
  • the activity of a protein of the invention may, among other means, be measured by the following methods
  • Assay for hemostatic and thrombolytic activity include, without limitation, those desc ⁇ bed in Linet et al , J Clin Pharmacol 26 1 1 -140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrey et al , Fib ⁇ nolysis 5 71 -79 (1991 ), Schaub, Prostaglandins 35 467-474, 1988
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integ ⁇ ns and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses)
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction
  • a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions
  • Suitable assays for receptor-ligand activity include without limitation those described in Current Protocols in Immunology, Ed by J.E Coligan, A M Kruisbeek, D.H Margulies, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7 28.1 -7 28 22), Takai et al , Proc Natl Acad Sci USA 84 6864-6868, 1987, Bicrer et al , J Exp Med 168 1145-1 156, 1988, Rosenstem et al., J Exp Med 169:149-160 1989, Stoltenborg et al , J Immunol Methods 175.59-68, 1994, Stitt et al , Cell 80:661 -670, 1995
  • Anti -Inflammatory Activity Proteins of the present invention may also exhibit anti- flammatory activity
  • the anti- inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), lschemia- reperfusion injury, endotoxm lethality, arth ⁇ tis, complement-mediated hyperacute rejection, nephntis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNE or
  • a protein of the invention may exhibit other anti tumor activities
  • a protein may inhibit tumor growth directly or indirectly (such as, tor 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, bactena, viruses, fungi and other parasites, effecting (suppressing or enhancing) bodily characte ⁇ stics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape), effecting biorhythms or ca ⁇ cadic cycles or rhythms, effecting the fertility of male or female subjects, effecting the metabolism, catabolism, anabohsm, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nut ⁇ tional factors or component(s), effecting behavioral charactenstics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders)
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable earner
  • a pharmaceutically acceptable earner Such a composition may also contain (in addition to protein and a earner) diluents, fillers, salts, buffers, stabilizers, solubihzers, and other materials well known in the art
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active mgred ⁇ ent(s) The charactenstics of the earner will depend on the route of administration
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokmes, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, LL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, EL-8, IL-9, IL-10, IL-1 1 , IL-12,
  • a protein of the present invention may be active in multimers (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.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • Altematively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S.
  • Patent No. 4,235,871 U.S. Patent No. 4,501 ,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are inco ⁇ orated herein by reference.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chlo ⁇ de Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chlo ⁇ de Injection, Lactated Ringer's Injection, or other vehicle as known in the art
  • an isotonic vehicle such as Sodium Chlo ⁇ de Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chlo ⁇ de Injection, Lactated Ringer's Injection, or other vehicle as known in the art
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and seventy of the condition being treated, and on the nature of p ⁇ or treatments which the patient has undergone Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient Initially, the attending physician will administer low doses of protein of the present invention and observe the patient s response Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0 01 ⁇ g to about 100 mg (preferably about 0 l ⁇ g 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 seventy of the disease being treated and the condition and potential idiosyncratic response of each individual patient It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration Ultimately the attending physician will decide on the approp ⁇ ate duration of intravenous therapy using the pharmaceutical composition of the present invention
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH) Methods for synthesizing such peptides are known in the art, for example, as in R P Mernfield, J Amer Chem Soc __[,
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein
  • the therapeutic method includes administe ⁇ ng the composition topically, systematically, or locally as an implant or device
  • the therapeutic composition for use this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage
  • Topical administration may be suitable for wound healing and tissue repair
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as desc ⁇ bed above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention
  • the composition would include a matnx capable of de ve ⁇ ng the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body
  • Such matnces may be formed of mate ⁇ als presently in use for other implanted medical applications
  • compositions may be biodegradable and chemically defined calcium sulfate, tncalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhyd ⁇ des
  • Other potential matenals are biodegradable and biologically well-defined, such as bone or dermal collagen
  • Further matnces are compnsed of pure proteins or extracellular matnx components
  • Other potential matnces are nonbiodegradable and chen n ally defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics
  • Matrices may be compnsed of combinations of any of the above mentioned types of mate ⁇ ai, such as polylactic acid and hydroxyapatite or collagen and tncalciumphosphate
  • the bioceramics may be altered in composition, such as in calcium-aluminate
  • a 50 50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and TGF- ⁇ ), and insulin ⁇ like growth factor (IGF).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin ⁇ like growth factor
  • the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • the addition of other known growth factors, such as IGF I (insulin like growth factor I) may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomo ⁇ hometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
  • Patent and literature references cited herein are inco ⁇ orated by reference as if fully set forth.
  • CTTCNTGNNG CAAAAAACAG AAAACTGGGG NTNNGAAACG TGGGCAGTGT GGTCTTNGNG 60
  • NGCACTGTAA ATTTGACTTT GTTTNTNTCA CTGAGCGCAT CAGGNATGTC NATTNGGANG 120
  • GNTGCAGCAA ANCGGGAACC GCGGCCATGG CGACGCGGGA CTCGAGCAGG GCCCGCCTGG 240 CTGTGCGAGG AAAGTAGGCC ATGAAGGCCG 270
  • TAATAGTTAA TAAGGAAGTT CTTCTCTTGC TCAAAGAAGT CATCTACCTC CTTAACTCCA 240
  • GAAGGCAAGC TACAGAAGGC TTTAGAAGAT GCCTTCTTGG CTATTGACGC CAAATTGACC 180
  • GAAGAGGCTA CCATGACTAT TGAAGAGCTG CTGACACGCT ACGGGCAGAA CTGTCACAAG 360
  • Lys Leu Thr Thr Glu Glu Xaa lie Lys Glu Leu Ala Gin He Ala Gly 50 55 60
  • AAGCAACTGC CTTGATAATT CGACAAATTG CTGACCACAG TTTAATGACC TCAAAGAGAG 540
  • MOLECULE TYPE protein
  • CAGAGAGGCT GAGACCAACC CAGAAACCAC CACYTCTCAC GCCAAAGCTC ACACCTTCAG 60
  • AAAAATCCTA GGTCAGACTT ATGCAGTTCC CTATGAAGAC GATCATTATG CAAAAGACCC 360
  • TCCCCATTCA CGGAGTANCA NTCGTANTCN GAGCCTGGGG GCACGGGACA CANTGAGGCC 120
  • GGGCCTGTGT CNACCCTGCT TTTTTCTTTA TTCTTNGTAG TNGTTTAGGA GTGGGGGGCC 240
  • MOLECULE TYPE cDNA
  • GGTANGTCAA CAGTTAGCTG CTTCTCATTG CNGAGTGGCG ATTGGTCCTG TCATGGTTTA 180
  • AAAATCTTAC TCAGAAATCT TCAGAGGTTT GCTAAGGATA CAATTTGATT CTTACACATT 360 TAATGCTCAC CAGCTGCTTA GGCCCACACC ATTTATCCAC CCTGATTTGC TACTGCTCTT 420

Abstract

The invention provides 13 clones 'AZ302-1' isolated from human colon; 'AU139-2', 'AU105-14', and 'AJ147-1' from human adult testes; 'AS268-1', 'AS264-3', 'AS301-2', 'AS162-1' and 'AS86-1' from human fetal brain; 'D147-17' from human PBMC ; '075-9' from human dendritic cells; 'AM262-11' from human fetal kidney and clone 'AR28-1' from human adult retina comprising polynucleotides encoding secreted proteins, using methods selective for cDNAs encoding secreted proteins.

Description

POLY JCLEOTTDE ENCODING SECRETED PROTETNS
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e g , cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly ' in the sense that they rely on information directly related to the discovered protein (l e , partial DNA/amino acid sequence of the protein m the case of hybridization cloning, activity of the protein in the case of expression cloning) More recent "indirect ' cloning techniques such as signal sequence cloning which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stπngency 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 polynucleoi.de encoding them that the present invention is directed
SUMMARY OF THF INVENTION In one embodiment, the present invention
Figure imgf000003_0001
ides a composition compπsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 2,
(b) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 2 from nucleotide 351 to nucleotide 506
(c) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076,
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076, (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:3;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:3 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:2 from nucleotide 351 to nucleotide 506; the nucleotide sequence of the full length protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:2, SEQ ID NO: l or SEQ ID NO:4.
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:3;
(b) fragments of the amino acid sequence of SEQ ID NO:3; and
(c) the amino acid sequence encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:3.
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 23 to nucleotide 517,
(c) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076,
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076,
(e) a polynucleotide compnsing the nucleotide sequence of the mature protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076,
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding a protein compπsing the am o acid sequence of SEQ ID NO 6, (h) a polynucleotide encoding a protein compπsing a fragment of the ammo acid sequence of SEQ ID NO 6 having biological activity,
(l) a polynucleotide which is an allelic vaπant of a polynucleotide of (a)-
(d) above,
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 5 from nucleotide 23 to nucleotide 517, the nucleotide sequence of the full length protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO 6 from amino acid 35 to amino acid 1 15 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO 5 or SEQ ID NO.7
In other embodiments, the present invention provides a composition compπsing a protein, wherein said protein compnses an amino acid sequence selected from the group consisting of (a) the am o acid sequence of SEQ ID NO 6, 46683 PCΪYUS97/09878
(b) the ammo acid sequence of SEQ ID NO:6 from amino acid 35 to amino acid 1 15,
(c) fragments of the ammo acid sequence of SEQ ID NO 6, and
(d) the amino acid sequence encoded by the cDNA insert of clone AU 139_2 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins Preferably such protein compnses the amino acid sequence of SEQ ID NO:6 or the ammo acid sequence of SEQ ID NO.6 from amino acid 35 to amino acid 1 15.
In one embodiment, the present invention provides a composition compπsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO.8;
(b) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 8 from nucleotide 288 to nucleotide 629, (c) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO.8 from nucleotide 441 to nucleotide 629,
(d) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076; (e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076, (f) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AU 105_14 deposited under accession number
ATCC 98076, (g) a polynucleotide encoding the mature protein encoded by the ι DNA insert of clone AU105_ 14 deposited under accession number ATCC 98076,
(h) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO 9,
(i) a polynucleotide encoding a protein compπsing a fragment of the amino acid sequence of SEQ ID N0 9 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 hybπdizing under stπngent conditions to any one of the polynucleotides specified in (a)-(ι)
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 8 from nucleotide 288 to nucleotide 629, the nucleotide sequence of SEQ ID NO:8 from nucleotide 441 to nucleotide 629; the nucleotide sequence of the full length protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO'9 from amino acid 25 to amino acid 44
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO-S or SEQ ID NO lO In other embodiments, the present invention provides a composition compπsing a protein, wherein said protein compπses an ammo acid sequence selected from the group consisting of.
(a) the amino acid sequence of SEQ ID NO 9,
(b) the amino acid sequence of SEQ ID NO 9 from amino acid 25 to amino acid 44,
(c) fragments of the amino acid sequence of SEQ ID N0 9; and
(d) the ammo acid sequence encoded by the cDNA insert of clone AU105_ 14 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins Preferably such protein comprises the amino acid sequence of SEQ ID NO 9 or the ammo acid sequence of SEQ ID
N0 9 from amino acid 25 to amino acid 44
In one embodiment, the present invention provides a composition compnsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO. l l ;
(b) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO. l 1 from nucleotide 164 to nucleotide 298,
(c) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076, (d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076,
(e) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO.12, (h) a polynucleotide encoding a protein compπsing a fragment of the ammo acid sequence of SEQ ID NO 12 having biological activity,
(l) a polynucleotide which is an alle c vaπant of a polynucleotide of (a)-
(d) above,
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 1 1 from nucleotide 164 to nucleotide 298, the nucleotide sequence of the full length protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AS268_1 deposited under accession number A^TT 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 1 1 or SEQ ID NO.13 In other embodiments, the present invention provides a composition compπsing a protein, wherein said protein compπses an amino acu 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
AS268_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins Preferably such protein compπses the amino acid sequence of SEQ ID NO 12
In one embodiment, the present invention provides a composition compπsing an isolated polynucleotide selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 254 to nucleotide 681 ; (c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone D147_17 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone D147_17 deposited under accession number ATCC 98076; (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone D147_17 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone D147_17 deposited under accession number ATCC 98076; (g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)- (d) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 from nucleotide 254 to nucleotide 681 ; the nucleotide sequence of the full length protein coding sequence of clone D147_17 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone D147_17 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone D147_17 deposited under accession number ATCC 98076. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 73 to amino acid 129.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO: 15, SEQ ID NO: 14 or SEQ ID NO: 17. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 16; (b) the amino acid sequence of SEQ ID NO: 16 from amino acid 73 to amino acid 129;
(c) fragments of the amino acid sequence of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone D147_17 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 16 from amino acid 73 to amino acid 129.
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:18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 from nucleotide 28 to nucleotide 388;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 from nucleotide 76 to nucleotide 388;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone 075_9 deposited under accession number ATCC 98076;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone 075_9 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone 075_9 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone 075_9 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 19;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:19 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: 18 from nucleotide 28 to nucleotide 388; the nucleotide sequence of SEQ ID NO: 18 from nucleotide 76 to nucleotide 388; the nucleotide sequence of the full length protein coding sequence of clone 075_9 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone 075_9 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone 075_9 deposited under accession number ATCC 98076. 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 protein, wherein said protein comprises an amino acid sequence selected from the group consisting 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 075_9 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 19.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 ; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:21 from nucleotide 75 to nucleotide 419;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 132 to nucleotide 419; (d) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076,
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076,
(f) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076,
(h) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO 22,
(0 a polynucleotide encoding a protein compπsing a fragment of the amino acid sequence of SEQ ID NO 22 having biological activity, (j) a polynucleotide which is an alleiic vaπant of a polynucleotide of (a)-
(g) above,
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (l) above Preferably, such { olynucleotide compπses the nucleotide sequence of SEQ ID NO 21 from nucleotide 75 to nucleotide 419, the nucleotide sequence of SEQ ID NO 21 from nucleotide 132 to nucleotide 419, the nucleotide sequence of the full length protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AJ 147_1 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 21 or SEQ ID NO 23
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO 22,
(b) fragments of the am o acid sequence of SEQ ID NO 22, and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:22.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:24;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24 from nucleotide 69 to nucleotide 377;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24 from nucleotide 120 to nucleotide 377;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AM262_1 1 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AM262_1 1 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:25;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:25 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. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:24 from nucleotide 69 to nucleotide 377; the nucleotide sequence of SEQ ID NO:24 from nucleotide 120 to nucleotide 377; the nucleotide sequence of the full length protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM262_1 1 deposited under accession number ATCC 98076. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:24.
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:25; (b) the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81 ;
(c) fragments of the amino acid sequence of SEQ ID NO:25; and
(d) the amino acid sequence encoded by the cDNA insert of clone AM262_11 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:25 or the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81.
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:26;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:26 from nucleotide 1 10 to nucleotide 448;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AR28_1 deposited under accession number ATCC
98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AR28_1 deposited under accession number ATCC
98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:27; (h) a polynucleotide encoding a protein compπsing a fragment of the amino acid sequence of SEQ ID NO 27 having biological activity,
(1) a polynucleotide which is an allelic vaπant of a polynucleotide of (a)-
(d) above, (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above, and
(k) a polynucleotide capable of hybπdizing under stπngent conditions to any one of the polynucleotides specified in (a)-(h)
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 26 from nucleotide 1 10 to nucleotide 448, the nucleotide sequence of the full length protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076, or the nucleotide sequence of the mature protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO 27 from ammo acid 15 to ammo acid 78
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 26 or SEQ ID NO 28 In other embodiments, the present invention provides a composition compπsing 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 27,
(b) the amino acid sequence of SFQ ID NO 27 from ammo acid 15 to amino acid 78,
(c) fragments of the amino acid sequence of SEQ ID NO 27, and
(d) the amino acid sequence encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins Preferably such protein compπses the ammo acid sequence of SEQ ID NO 27 or the ammo acid sequence of SEQ ID
NO.27 from amino acid 15 to amino acid 78
In one embodiment, the present invention provides a composition compπsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 30, (b) a polynucleotide comprising the nucleotide sequence ot SEQ ID NO.30 from nucleotide 230 to nucleotide 541 ,
(c) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076,
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076,
(e) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076,
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO 31 , (h) a polynucleotide encoding a protein compπsing a fragment of the amino acid sequence of SEQ ID NO 31 having biological activity,
(0 a polynucleotide which is an allelic vaπant of a polynucleotide of (a)-
(d) above,
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 30 from nucleotide 230 to nucleotide 541 , the nucleotide sequence of the full length protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076 or the nucleotide sequence of the mature protein coding sequence ot clone AS162_1 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO 31 from amino acid 5 to ammo acid 25 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO 30, SEQ ID NO:29 or SEQ ID NO 32
In other embodiments, the present invention provides a composition compπsing 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 31 , (b) the amino acid sequence of SEQ ID NO:31 from ammo acid 5 to amino acid 25;
(c) fragments of the amino acid sequence of SEQ ID NO:31 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone AS 162_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ LD NO:31 or the amino acid sequence of SEQ ID NO:31 from amino acid 5 to amino acid 25.
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:34;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:34 from nucleotide 202 to nucleotide 467; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:34 from nucleotide 241 to nucleotide 467;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076; (e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:35;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:35 having biological activity;
(j) a polynucleotide which is an allelic vaπant of a polynucleotide of (a)-
(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:34 from nucleotide 202 to nucleotide 467; the nucleotide sequence of SEQ ID NO: 34 from nucleotide 241 to nucleotide 467; the nucleotide sequence of the full length protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:34, SEQ ID NO:33 or SEQ ID NO:36.
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:35; (b) fragments of the amino acid sequence of SEQ ID NO:35; and
(c) the amino acid sequence encoded by the cDNA insert of clone
AS264_3 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:35. 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:38;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 173 to nucleotide 579;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS3()1_2 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076; (g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:39;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:39 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(d) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 173 to nucleotide 579; the nucleotide sequence of the full length protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076; or the nucleotide sequence of the mature protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:38. SEQ ID NO:37 or SEQ ID NO:40.
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:39;
(b) fragments of the amino acid sequence of SEQ ID NO:39; and
(c) the amino acid sequence encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:39.
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:42;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:42 from nucleotide 363 to nucleotide 593;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:42 from nucleotide 483 to nucleotide 593; (d) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076,
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076,
(f) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076,
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO 43,
(l) a polynucleotide encoding a protein compπsing a fragment of the amino acid sequence of SEQ ID NO 43 having biological activity, (j) a polynucleotide which is an alle c vaπant 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 hybπd ng under stπngent conditions to any one of the polynucleotides specified in (a)-(ι)
Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 42 from nucleotide 363 to nucleotide 593, the nucleotide sequence of SEQ ID NO 42 from nucleotide 483 to nucleotide 593, the nucleotide sequence of the full length protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076. or the nucleotide sequence of the mature protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO 42, SEQ ID NO 41 or SEQ ID NO 44
In other embodiments, the present invention provides a composition compπsing a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO 43, (b) fragments of the am o acid sequence of SEQ ID NO 43, and (c) the amino acid sequence encoded by the cDNA insert of clone
AS86_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins Preferably such protein compπses the am o acid sequence of SEQ ID NO 43 In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence The invention also provides a host cell, including bactenal, yeast, insect and mammalian cells, transformed with such polynucleotide compositions Processes are also provided for producing a protein, which compπse
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium, and
(b) puπfying the protein from the culture
The protein produced according to such methods is also provided by the present invention
Preferred embodiments include those in which the protein produced by such process is a mature form of the protein Protein compositions of the present invention may further compπse a pharmaceutically acceptable earner Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention
Methods are also provided for preventing, treating or ameliorating a medical condition which compπses administeπng to a mammalian subject a therapeutically effective amount of a composition compπsing a protein of the present invention and a pharmaceutically acceptable earner
BRIEF DESCRIPTION OF FIGURES Fig 1 is an autoradiograph demonatratmg the expression of D147_17 in COS cells Fig 2 is an autoradiograph deonstrating the expression of AM262_1 1 in COS cells
DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids The actual nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods The predicted am o acid sequence (both full length and mature) can then be determined from such nucleotide sequence The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence
For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing Because of the partial ambiguity in reported sequence information, reported protein sequences include "Xaa" designators These "Xaa" designators indicate either ( 1 ) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined more accurately) 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 m its ammo acid sequence "Secreted" proteins include without limitation proteins secreted wholly (e g , soluble proteins) or partially (e g , receptors) from the cell in which they are expressed "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplpasmic reticulum
Clone "AZ302 1 "
A polynucleotide of the present invention has been identified as clone "AZ302_1 ' AZ302_1 was isolated from a human colon (Caco-2 adenocarcinoma) cDNA library using methods which are selective for cDNAs encoding secreted proteins AZ302_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AZ302_1 protein")
The nucleotide sequence of the 5' portion of AZ302_1 as presently determined is reported in SEQ ID NO 1 An additional internal nucleotide sequence from AZ302. 1 as presently determined is reported in SEQ ID NO 2 What applicants believe is the pioper reading frame and the predicted ammo acid sequence encoded by such internal sequence is reported in SEQ ID NO 3 Additional nucleotide sequence from the 3' portion of AZ302_1 , including the polyA tail is reported in SEQ ID NO 4
The nucleotide sequence disclosed herein for AZ302_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AZ302J demonstrated at least some homology with an EST identified as "ye83a03 rl Homo sapiens cDNA clone 124300 5' at accession number R02197 (BlastN) Based upon homology, AZ302_1 proteins and each homologous protein or peptide may share at least some activity
Clone "AU139 2" A polynucleotide of the present invention has been identified as clone "AU139_2" AU139_2 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins AU139_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AU139_2 protein") The nucleotide sequence of the 5' portion of AU139_2 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 acid sequence of the AU139_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 6 Additional nucleotide sequence from the 3' portion of AU139_2, including the polyA tail, is reported in SEQ ID NO 7
The nucleotide sequence disclosed herein for AU 139_2 was searched against the GenBank database using BLASTA/BLASTX and EASTA search protocols AU139_2 demonstrated at least some homology with three ESTs identified as "EST 16319 Homo sapiens cDNA 5' end" (accession number T30419, BlastN), "EST04080 Homo sapiens cDNA clone HFBDQ07" (accession number T06191 , BlastN), and "FST108441 Rattus sp cDNA 5 ' Based upon homology, AU 139_2 proteins and each homologous protein or peptide may share at least some activity
Clone "AU105 14" A polynucleotide of the present invention has been identified as clone "AU105_14"
AU105_14 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins AU 10 14 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AU105_14 protein") The nucleotide sequence of the 5' portion of AU 105_14 as presently determined is reported in SEQ ID NO 8 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO 9 The predicted acid sequence of the AU105_14 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 9 Amino acids 1 to 51 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 52 Additional nucleotide sequence from the 3' portion of AU105_14, including the polyA tail, is reported in SEQ ID NO 10
The EcoRI/NotI restπction fragment obtainable from the deposit containing clone AU105_14 should be approximately 2670 bp The nucleotide sequence disclosed herein for AU105_14 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols No hits were found in the database
Clone "AS268 1 "
A polynucleotide of the present invention has been identified as clone "AS268_1 " AS268_1 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins AS268_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS268_1 protein") The nucleotide sequence of the 5' portion of AS268_1 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 in SEQ ID NO 12 The predicted acid sequence of the AS268_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 12 Additional nucleotide sequence from the 3' portion of AS268_1 , including the polyA tail, is reported in SEQ ID NO 13
The EcoRI/NotI restπction fragment obtainable from the deposit containing clone AS268_1 should be approximately 1800 bp
The nucleotide sequence disclosed herein for AS268_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS268J demonstrated at least some homology with the rabbit and muπne ryanodine receptors (BlastN accession number M59743, BlastX accession number X839 3) Ryanodine receptors have recently been shown to be the Ca2+ releasr channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle Based upon homology. AS268_1 proteins and each homologous protein or peptide may share at least some activity
Clone "D 147 17"
A polynucleotide of the present invention has been identified as clone "D147_I7" DI 47_17 was isolated from a human PBMC cDNA library using methods which are selective for cDNAs encoding secreted proteins D147_17 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as ' D147_17 protein")
The nucleotide sequence of the 5' portion of D147_17 as presently determined is reported in SEQ ID NO 14 An additional internal nucleotide sequence from D147_17 as presently determined is reported in SEQ ID NO 15 What applicants believe is the proper reading frame and the predicted ammo acid sequence encoded by such internal sequence is reported in SEQ ID NO: 16. Additional nucleotide sequence from the 3' portion ot DI 47_17, including the polyA tail, is reported in SEQ ID NO: 17.
The nucleotide sequence disclosed herein for D147_17 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
Clone "075 9"
A polynucleotide of the present invention has been identified as clone "075_9". 075_9 was isolated from a human dendritic cells cDNA library using methods which are selective for cDNAs encoding secreted proteins. 075_9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "075_9 protein").
The nucleotide sequence of the 5' portion of 075_9 as presently determined is reported in SEQ ID NO: 18 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO: 19. The predicted acid sequence of the 075_9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 19. Amino acids 1 to 16 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 17. Additional nucleotide sequence from the 3' portion of 075_9, including the polyA tail, is reported in SEQ ID NO:20.
The nucleotide sequence disclosed herein for 075_9 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
Clone "AJ147 1 "
A polynucleotide of the present invention has been identified as clone "AJ 147_1 ". AJ147_1 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. AJ147_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AJ147_1 protein"). The nucleotide sequence of the 5' portion of AJ147_1 as presently determined is reported in SEQ ID NO:21 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ LD NO:22. The predicted acid sequence of the AJ147_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:22. Amino acids 1 to 19 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20. Additional nucleotide sequence from the 3' portion of AJ147 , including the polyA tail, is reported in SEQ ID NO:23. The EcoRI/NotI restπction fragment obtainable from the deposit containing clone AJ147_1 should be approximately 500 bp
The nucleotide sequence disclosed herein for AJ147_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AJ147_1 demonstrated at least some homology with munne calmegin (Meg l )/calnexιn (BlastN accession number DI 41 17) Calmegin is a Ca2+ -binding protein that is specifically expressed in spermatogenesis The highly regulated, specific and abundant expression of calmegin suggests that it plays an important role in spermatogenesis Based upon homology, AJ147_1 proteins and each homologous protein or peptide may share at least some activity
Clone "AM262 1 1 "
A polynucleotide of the present invention has been identified as clone "AM262_1 1 " AM262_11 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins AM262_1 1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AM262_1 1 protein")
The nucleotide sequence of AM262_1 1 as presently determined is reported in SEQ
ID NO 24 What applicants presently believe to be the proper reading frame and the predicted ammo acid sequence of the AM262_1 1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 25 Amino acids 1 to 17 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18
The nucleotide sequence disclosed herein for AM262_1 I was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AM262_1 1 demonstrated at least some identity with the human eotaxm precursor gene and protein (BlastN accession number U34780, this database entry was made subsequent to applicants' isolation of AM262_1 1 ) Based upon homology, AM262_1 1 proteins and each homologous protein or peptide may share at least some activity
Clone "AR28 1 " A polynucleotide of the present invention has been identified as clone "AR28_1 '
AR28_1 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins AR28_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AR28_1 protein")
The nucleotide sequence of the 5' portion of AR28_1 as presently determined is reported in SEQ ID NO 26 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO 27 The predicted acid sequence of the AR28_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 27 Additional nucleotide sequence from the 3' portion of AR28_1 , including the polyA tail, is reported SEQ ID NO 28 The nucleotide sequence disclosed herein for AR28_1 was searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols No hits were found in the database
Clone "AS 162 1 " A polynucleotide of the present invention has been identified as clone "AS 162_1 "
AS162_1 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins AS 1 2_ I is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS162_1 protein") The nucleotide sequence of the 5' portion of AS 162_1 as presently determined is reported in SEQ ID NO 29 An additional internal nucleotide sequence from AS162_1 as presently determined is reported in SEQ ID NO 30 What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO 31 Additional nucleotide sequence from the 3' portion of AS162_1 , including the polyA tail, is reported in SEQ ID NO 32 The EcoRI/NotI restπction fragment obtainable from the deposit containing clone
AS162_1 should be approximately 1380 bp
The nucleotide sequence disclosed herein for AS162_1 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS 162J demonstrated at least some identity with an EST identified as ym96e05 si Homo sapiens cDNA clone 166784 3'" (accession number R88809, BlastN) Based upon identity, AS162_1 proteins and each identical protein or peptide may share at least some activity
Clone "AS264 3"
A polynucleotide of the present invention has been identified as clone "AS264_3" AS264_3 w as isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins AS264_ is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS264_3 protein")
The nucleotide sequence of the 5' portion of AS264_3 as presently determined is reported in SEQ ID NO 33 An additional internal nucleotide sequence from AS264_3 as presently determined is reported in SEQ ID NO 34 What applicants believe is the proper reading frame and the predicted ammo acid sequence encoded by such internal sequence is reported in SEQ ID NO 35. Amino acids 1 to 13 of SEQ ID NO 35 are a predicted leader/signal sequence, with the predicted mature ammo acid sequence beginning at ammo acid 14 Additional nucleotide sequence from the 3' portion of AS264_3, including the polyA tail, is reported in SEQ ID N0.36.
The EcoRI/NotI restnction fragment obtainable from the deposit containing clone AS264_3 should be approximately 3300 bp
The nucleotide sequence disclosed herein for AS264_3 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS264_3 demonstrated at least some weak similanty to collagen Based upon homology, AS264_3 proteins and each homologous protein or peptide may share at least some activity
Clone "AS301 2"
A polynucleotide of the present invention has been identified as clone "AS301_2" AS301_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins AS301_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS301_2 protein")
The nucleotide sequence of the 5' portion of AS301_2 as presently determined is reported in SEQ ID NO 37 An additional internal nucleotide sequence from AS301_2 as presently determined is reported in SEQ ID NO 38 What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO.39 Additional nucleotide sequence from the 3' portion of AS301_2, including the polyA tail, is reported in SEQ ID NO 40
The EcoRI/NotI restnction fragment obtainable from the deposit containing clone AS301_2 should be approximately 2600 bp
The nucleotide sequence disclosed herein for AS301_2 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols AS301_2 demonstrated at least some homology with ESTs identified as "yp82b08 rl Homo sapiens cDNA clone 19391 1 5'" (BlastN accession number R83399), "ye66c02 rl Homo sapiens cDNa clone 122690 5'", and "ym26e09.rl Homo sapiens cDNA clone 49167 5'" (BlastN accession number H 16691 ) Based upon homology, AS301 _2 proteins and each homologous protein or peptide may share at least some activity
Clone "AS86 1 " A polynucleotide of the present invention has been identified as clone "AS86_1 ". AS86_1 was isolated from a human fetal brain cDNA library using methods which are selective forcDNAs encoding secreted proteins. AS86_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "AS86_1 protein"). The nucleotide sequence of the 5' portion of AS86_1 as presently determined is reported in SEQ ID NO:41. An additional internal nucleotide sequence from AS86_1 as presently determined is reported in SEQ LO NO:42. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such intemal sequence is reported in SEQ ID NO:43. Amino acids 1 to 40 of SEQ ID NO:43 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 41. Additional nucleotide sequence from the 3' portion of AS86_1 , including the polyA tail, is reported in SEQ ID NO:44.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone AS86_1 should be approximately 2122 bp. The nucleotide sequence disclosed herein for AS86_1 was searched against the
GenBank database using BLASTA/BLASTX and FASTA search protocols. No hits were found in the database.
Figs. 1 and 2 are autoradiographs evidencing expression of clones of the present invention. All clones were expressed in COS cells.
Deposit of Clones
Clones AZ302 , AU139_2, AU105 4, AS268J , D147 7, 075_9, AJ147 , AM262_1 1 , AR28_1, AS162_1, AS264_3, AS301_2 and AS86_1 were deposited on June 6, 1996 with the American Type Culture Collection under accession number ATCC 98076, from which each clone comprising a particular polynucleotide is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' cite, EcoRI; 3' cite, NotI) to produce the appropriately sized fragment for such clone (approximate clone size fragment are identified below). Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence AZ302_1 SEQ ID NO:45
AU139_2 SEQ ID NO:46
AU105_14 SEQ ID NO:47
AS268 SEQ ID NO:48
D147_17 SEQ ID NO:49 075_9 SEQ ID NO:50
AJ147_1 SEQ ID NO:51
AM262_1 1 SEQ ID NO:52
AR28_I SEQ ID NO:53
AS162J SEQ ID NO:54 AS264_3 SEQ ID NO:55
AS301_2 SEQ ID NO:56
AS86_1 SEQ ID N0.57
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-J2P ATP (specific activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for 1 A ^mg oligonucleotides Other labeling techniques can also be used Unincorporated label should preferably be removed by gel filtration chromatography or other established methods The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bactenal culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a stenle culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well -separated colonies on solid bacteπological media containing L-broth containing ampicillin 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 distinct, well-separated colonies can also be employed
Standard colony hybndization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88 2 g Na citrate/liter, adjusted to pH 7 0 with NaOH) containing 05% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter) Preferably, the probe is then added to the hybπdi/ation 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 IX SSC/0 5% SDS at 65°C for 30 minutes to 1 hour is optional The filter is then preferably dπed and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film Other known hybπdization 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 verif ied by restriction analysis, hybπdization analysis, or DNA sequencing
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention Fragments of the protein may be in linear form or they may be cyc zed using known methods, for example, as descπbed in H U Saragovi, et al , Bio/Technology K), 773-778 ( 1992) and in R S McDowell, et al , J Amer Chem Soc 1 14, 9245-9253 (1992), both of which are incoφorated herein by reference Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule Other immunoglobulin isotypes may also be used to generate such fusions For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention The present invention also provides both full-length and mature forms of the disclosed proteins The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the cDNA sequences disclosed herein The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein Such methods include the preparation of probes or pπmers from the disclosed sequence information for identification and/or amplification of genes in appropπate genomic hbranes or other sources of genomic mateπals
Where the protein of the present invention is membrane-bound (e g , is a receptor), the present invention also provides for soluble forms of such protein In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information
Species homologs of the disclosed polynucleotides and proteins are also pro\ ided by the present invention Species homologs may be isolated and identified by making suitable probes or pnmers from the sequences provided herein and screening a suitable nucleic acid source from the desired species
The invention also encompasses allelic vaπants of the disclosed polynucleotides or proteins, that is, natural ly-occurπng alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al , Nucleic Acids Res .19, 4485-4490 (1991 ), in order to produce the protein recombinantly Many suitable expression control sequences are known in the art General methods of expressing recombinant proteins are also known and are exemplified in R Kaufman, Methods in En7ymology 185, 537-566 (1990) As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence A number of types of cells may act as suitable host cells for expression of the protein Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed pπmate cell lines, normal diploid cells, cell strains deπved from in vitro culture of pnmary tissue, pnmary 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 bactena Potentially suitable yeast strains include Saccharomyces cerevistae, Schtzosactharomyces pombe Kluvveromyces strains, Candida or any yeast strain capable of expressing heterologous proteins Potentially suitable bacteπal strains include Eschenchia coli, Bacillus subtύis. Salmonella typhimurutm, or any bacteπal strain capable of expressing heterologous proteins If the protein is made in yeast or bactena, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropπate sites, in order to obtain the functional protein Such covalent attachments may be accomplished using known chemical or enzymatic methods
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e g , Invitrogen, San Diego, California, U S A (the MaxBac® kit), and such methods are well known in the art, as descnbed in Summers and Smith, Texas Agπcultural Expeπment Station Bulletin No 1555 (1987). incoφorated herein by reference As used her in an insect cell capable of expressing a polynucleotide of the present invention is ' transformed
The protein of the invention may be prepared bv culturing transformed host cells under culture conditions suitable to express the recombinant protein The resulting expressed protein may then be punfied from such culture (I e , from culture medium or cell extracts) using known puπfication processes, such as gel filtration and ion exchange chromatography The puπfication of the protein may also include an affinity column containing agents which will bind to the protein, one or more column steps over such affinity res s as concanavalm A- agarose, hepann-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography
Alternatively, the protein of the invention may also be expressed in a form which will facilitate puπfication For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively The protein can also be tagged with an epitope and subsequently punfied by using a specific antibody directed to such epitope One such epitope ("Flag") is commercially available from Kodak (New Haven, CT)
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e g , silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein Some or all of the foregoing puπfication steps, in vaπous combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein The protein thus puπfied is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein "
The protein of the invention may also be expressed as a product of transgenic animals, e g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are characteπzed by somatic or germ cells containing a nucleotide sequence encoding the protein
The protein may also be produced by known conventional chemical synthesis Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art The synthetically-constructed protein sequences, by virtue of shaπng pnmary, secondary or tertiary structural and/or contormational characteπ ties with proteins may possess biological properties in common therewith, including protein activity Thus, they may be employed as biologically active or immunological substitutes for natural, puπfied proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of puπfied proteins but into which modification are naturally provided or deliberately engineered For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e g , U S Patent No 4,518,584) Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein Other fragments and deπvatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled m the art given the disclosures herein Such modifications are believed to be encompassed by the present invention
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below Uses or activities descπbed for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA)
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research community for vaπous puφoses The polynucleotides can be used to express recombinant protein for analysis, characteπzation or therapeutic use, as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states), as molecular weight markers on Southern gels, as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions, to compare with endogenous DNA sequences in patients to identify potential genetic disorders, as probes to hybridize and thus discover novel, related DNA sequences, as a source of information to deπve PCR pnmers for genetic fmgeφπntmg, as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides, for selecting and making oligomers for attachment to a ' gene chip" or other support, including for examination of expression patterns, to raise anti-protein antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit another immune response Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that descnbed in Gyuns et al , Cell 75 791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press. Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one ot a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, TIO, B9, B9/1 1, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI , 123, Tl 165, HT2, CTLL2, TF-1 , Mo7e and CMK
The activity of a protein of the invention may, among other means, be measured by the following methods
Assays for T-cell or thymocyte proliferation include without limitation those descπbed in Current Protocols in 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 Lymphocyte Function 3 1-3 19, Chapter 7, Immunologic studies in Humans), Takai et al , J Immunol 137 3494-3500, 1986, Bertagnolli et al , J Immunol 145 1706-1712, 1990, Bertagnolli et al , Cellular Immunology 133 327-341 , 1991 , Bertagnolli, et al , J Immunol 149 3778-3783, 1992, Bowman et al , J Immunol 152 1756- 1761 , 1994
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in Polyclonal T cell stimulation, Kruisbeek, A M and Shevach, E M In Current Protoc ls 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 Cm tent Protocols in Immunology J E e a Co gan eds Vol 1 pp 6 8 1 -6 8 8, John Wiley and Sons Toronto 1994 Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those descπbed in Measurement of Human and Muπne Interleukin 2 and Interleukin 4, Bottomly, K , Davis, L S and Lipsky P E In Current Protocols in J E e a Coligan eds Vol 1 pp 6 1 -6 3 12, John Wiley and Sons, Toronto 1991 , deVπes et al , J Exp Med 173 1205-121 1 , 1991 Moreau et al , Nature 336 690-692, 1988, Greenberger et al , Proc Natl Acad Sci U S A 80 2931 2938, 1983, Measurement of mouse and human interleukin 6 - Nordan, R In Current Protocols in Immunology J E e a Coligan eds Vol 1 pp 6 6 1-6 6 5, John Wiley and Sons, Toronto 1991 , Smith et al , Proc Natl Acad Sci U S A 83 1857-1861 , 1986, Measurement of human Interleukin 1 1 - Bennett, F , Giannotti, J , Clark, S C and Turner, K J In Current Protocols in Immunology J E e a Coligan eds Vol 1 pp 6 15 1 John Wiley and Sons, Toronto 1991 , Measurement of mouse and human Interleukin 9 - Ciarletta, A , Giannotti, J , Clark, S C and Turner, K J In Current Protocols in Immunology J E e a Coligan eds Vol 1 pp 6 13 1 , John Wiley and Sons, Toronto 1991
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuπng proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 1 1 :405-41 1 , 1981 ; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, heφesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asu ma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen- specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft- versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1 , B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al. Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 59: 1 1 102- 11 105 (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 inappropπate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms Administration of reagents which block costimulation of T cells by disrupting receptor. ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-denved cytokines which may be involved in the disease process Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- characteπzed animal models of human autoimmune diseases Examples include muπne expenmental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybπd mice, unne autoimmune collagen arthntis, diabetes mel tus in NOD mice and BB rats, and muπne expenmental myasthenia gravts (see Paul ed , Fundamental Immunology, Raven Press, New York, 1989, pp 840-856)
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed
APCs either expressing a peptide of the present invention oi together with a stimulatory form of a soluble peptide of the present invention and re troducing the in vitro activated T cells into the patient Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as descπbed herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient The infected cells would now be capable of de veπng a costimulatory signal to, and thereby activate, T cells in vivo
In another application, up regulation or enhancement of antigen function (preferably
B lymphocyte antigen function) may be useful in the induction of tumor immunity Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides . For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class 1 or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class π proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1 , B7-2, B7- 3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981 ; Herrmann et al., J. Immunol. 128: 1968- 1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981 ; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61 : 1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341 , 1991 ; Brown et al., J. Immunol. 153:3079-3092, 1994. Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1 -3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991 ; Macatonia et al., Journal of Immunology 154:5071 -5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961 -965, 1994; Macatonia et al., Journal of Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631 -
640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer
Research 53: 1945-1951 , 1993; Itoh et al., Cell 66:233-243, 1991 ; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891 -897, 1993; Gorczyca et al., International Journal of Oncology 1 :639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84: 1 1 1-117, 1994; Fine et al., Cellular Immunology 155:1 1 1-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551 , 1991.
Hematopoiesis Regulating Activity A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81 :2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-591 1, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of bums, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic- plastic surgery. A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, caφal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebro vascular diseases such as stroke.
Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non- healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate A protein of the invention may also exhibit angiogenic activity
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in vanous 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 descnbed above from precursor tissues or cells, or for inhibiting the growth of tissues descπbed above
The activity of a protein of the invention may, among other means, be measured by the following methods
Assays for tissue generation activity include, without limitation, those descπbed in International Patent Publication No WO95/16035 (bone, cartilage, tendon), International Patent Publication No WO95/05846 (nerve, neuronal), International Patent Publication No WO91/07491 (skin, endothelium ) Assays for wound healing activity include, without limitation, those descπbed in
Winter, Epidermal Wound Healing, pps 71 -1 12 (Maibach, HI and Rovee, DT, eds ), Year Book Medical Publishers, Inc , Chicago, as modified by Eaglstem 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 Inhibms are characteπzed by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are charactered by their ability to stimulate the release of follicle stimulating hormone (FSH) Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibm α family, may be useful as a contraceptive based on the ability of inhibms to decrease fertility in female mammals and d crease spermatogenesis in male mammals Administration of sufficient amounts of other inhibms can induce infertility in these mammals Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibm-β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the antenor pituitary See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Nature 321 :779-782, 1986; Vale et al., Nature 321 :776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian ceils, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1 -6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994. Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity As a result, such a protein is expected to be useful in treatment of vaπous coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g , stroke)
The activity of a protein of the invention may, among other means, be measured by the following methods
Assay for hemostatic and thrombolytic activity include, without limitation, those descπbed in Linet et al , J Clin Pharmacol 26 1 1 -140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrey et al , Fibπnolysis 5 71 -79 (1991 ), Schaub, Prostaglandins 35 467-474, 1988
Receptor Lieand Activity
A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integπns and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses) Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions
The activity of a protein of the invention may, among other means, be measured by the following methods Suitable assays for receptor-ligand activity include without limitation those described in Current Protocols in Immunology, Ed by J.E Coligan, A M Kruisbeek, D.H Margulies, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7 28.1 -7 28 22), Takai et al , Proc Natl Acad Sci USA 84 6864-6868, 1987, Bicrer et al , J Exp Med 168 1145-1 156, 1988, Rosenstem et al., J Exp Med 169:149-160 1989, Stoltenborg et al , J Immunol Methods 175.59-68, 1994, Stitt et al , Cell 80:661 -670, 1995
Anti -Inflammatory Activity Proteins of the present invention may also exhibit anti- flammatory activity The anti- inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), lschemia- reperfusion injury, endotoxm lethality, arthπtis, complement-mediated hyperacute rejection, nephntis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNE or IL-1 Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or mateπai
Tumor Inhibition Activity
In addition to the activities descπbed 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, tor 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, bactena, viruses, fungi and other parasites, effecting (suppressing or enhancing) bodily characteπstics, 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 caπcadic cycles or rhythms, effecting the fertility of male or female subjects, effecting the metabolism, catabolism, anabohsm, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutπtional factors or component(s), effecting behavioral charactenstics, 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 endocnne activity, in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases, treatment of hypeφro ferative disorders (such as, for example, psoriasis), lmmunoglobuhn-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 mateπai or entity which is cross-reactive with such protein
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source deπved, including without limitation from recombinant and non -recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable earner Such a composition may also contain (in addition to protein and a earner) diluents, fillers, salts, buffers, stabilizers, solubihzers, and other materials well known in the art The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active mgredιent(s) The charactenstics of the earner will depend on the route of administration The pharmaceutical composition of the invention may also contain cytokines, lymphokmes, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, LL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, EL-8, IL-9, IL-10, IL-1 1 , 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 ag 'it which either enhance the activity of the protein or compliment its activity or use in treatment Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Altematively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S.
Patent No. 4,235,871 ; U.S. Patent No. 4,501 ,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incoφorated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co- administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloπde Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloπde Injection, Lactated Ringer's Injection, or other vehicle as known in the art The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art
The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and seventy of the condition being treated, and on the nature of pπor treatments which the patient has undergone Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient Initially, the attending physician will administer low doses of protein of the present invention and observe the patient s response Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0 01 μg to about 100 mg (preferably about 0 lμg 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 seventy of the disease being treated and the condition and potential idiosyncratic response of each individual patient It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration Ultimately the attending physician will decide on the appropπate duration of intravenous therapy using the pharmaceutical composition of the present invention Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH) Methods for synthesizing such peptides are known in the art, for example, as in R P Mernfield, J Amer Chem Soc __[,
2149-2154 (1963), J L Krstenansky, et al , FEBS Lett 2_±, 10 (1987) Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administeπng the composition topically, systematically, or locally as an implant or device When administered, the therapeutic composition for use this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage Topical administration may be suitable for wound healing and tissue repair Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as descπbed above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention Preferably for bone and/or cartilage formation, the composition would include a matnx capable of de veπng the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body Such matnces may be formed of mateπals 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 matnces for the compositions may be biodegradable and chemically defined calcium sulfate, tncalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydπdes Other potential matenals are biodegradable and biologically well-defined, such as bone or dermal collagen Further matnces are compnsed of pure proteins or extracellular matnx components Other potential matnces are nonbiodegradable and chen n ally defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics Matrices may be compnsed of combinations of any of the above mentioned types of mateπai, such as polylactic acid and hydroxyapatite or collagen and tncalciumphosphate The bioceramics may be altered in composition, such as in calcium-aluminate-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 glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin¬ like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic puφoses.
Patent and literature references cited herein are incoφorated by reference as if fully set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John LaVallie, Edward Racie, Lisa Merberg, David Treacy, Maurice Evans, Cheryl Bowman, Michael Spaulding, Vikki
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(iii) NUMBER OF SEQUENCES: 57
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Brown, Scott A.
(B) REGISTRATION NUMBER: 32,724
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8224
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
CTTCNTGNNG CAAAAAACAG AAAACTGGGG NTNNGAAACG TGGGCAGTGT GGTCTTNGNG 60
NGCACTGTAA ATTTGACTTT GTTTNTNTCA CTGAGCGCAT CAGGNATGTC NATTNGGANG 120
GGGGATCNAC ATTCNGGTCC ACAGATACNG ATCTCGGCTT GGGGCGGTCC TCCTCCTGCT 180
GNTGCAGCAA ANCGGGAACC GCGGCCATGG CGACGCGGGA CTCGAGCAGG GCCCGCCTGG 240 CTGTGCGAGG AAAGTAGGCC ATGAAGGCCG 270
(2) INFORMATION FOR SEQ ID NO:2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 506 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :
TANACTTTCC TTAGTTTTTC AAATAGCTCA NCAACCTTCA ATANNTACTT TTTGATGACT 60
GTGGNNTCTT NTAAANCCAG GNTATGTAAN CAGGCTNCGG TGTGNATATA TTCATCGNCA 120
ACATTTNTAT AAAATCTGKT CATTTTKTCA GCTTTMACAC AAGAATCTTT GATCCTATTG 180
TAATAGTTAA TAAGGAAGTT CTTCTCTTGC TCAAAGAAGT CATCTACCTC CTTAACTCCA 240
GTAAAAAGGA CTTCATCAGC ACTTTTCACC ACACTTTTGA AGAAGCCACC AAACATTTYT 300
TTAGTATTTT TCCGCCTAAC ACTTAGATCC TGATCATATT CCAGGAAAAC ATGAAAGTTG 360
CGATCTTTAC TGAGAACAGG GTGAGAAGAA AGCCGCTGAA GAAAGACTTT CATGGGAGGA 420
CACAGTTYTT CTTAAACACA GCGAGATACT CAGCTTCCAG TTCTTGTTTC ATCTGGGCTT 480
ATTATTCCAC CTTCTCCCAG TTTCTG 506 (2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
( C ) STRANDEDNESS :
( D) TOPOLOGY : l inear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :
Met Lys Val Ala lie Phe Thr Glu Asn Arg Val Arg Arg Lys Pro Leu 1 5 10 15
Lys Lys Asp Phe His Gly Arg Thr Gin Phe Phe Leu Asn Thr Ala Arg 20 25 30
Tyr Ser Ala Ser Ser Ser Cys Phe lie Trp Ala Tyr Tyr Ser Thr Phe 35 40 45
Ser Gin Phe 50
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : TTTTTAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAAAAAAA AAAAA 85
(2) INFORMATION FOR SEQ ID NO: 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 517 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CAATTGCAGA NTTNGAATTC GGCTTTCATG GCATACGGCY TTCATGGCCT AGGGGAGGAA 60 GTTGCCTTGT ACTGTGCCAA ATATCTTCCT GATATCATCA AAGATCAGAA GGCCTACAAG 120
GAAGGCAAGC TACAGAAGGC TTTAGAAGAT GCCTTCTTGG CTATTGACGC CAAATTGACC 180
ACTGAAGAAN TCATTAAAGA GCTGGCACAG ATTGCAGGGC GACCCACTGA GGATGAAGAT 240
GAAAAAGAAA AAGTAGCTGA TGAAGATGAT GTGGACAATG AGGAGGCTGC ACTGCTGCAT 300
GAAGAGGCTA CCATGACTAT TGAAGAGCTG CTGACACGCT ACGGGCAGAA CTGTCACAAG 360
GGCCCTCCCC ACAGCAAATN TGGAGGTGGG ACAGGCGAGG AACCAGGGTC CCAGGGCCTC 420
AATGGGGAGG CAGGACCTGA GGACTCAACT AGGGAAACTC CTTCACAAGA AAATGGCCCC 480
ACAGCCAAGG CCTACACAGG CTTTTCCTCC AACTCGG 517 (2) INFORMATION FOR SEQ ID NO: 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 115 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 :
Met Ala Tyr Gly Xaa His Gly Leu Gly Glu Glu Val Ala Leu Tyr Cys 1 5 10 15
Ala Lys Tyr Leu Pro Asp lie lie Lys Asp Gin Lys Ala Tyr Lys Glu 20 25 30
Gly Lys Leu Gin Lys Ala Leu Glu Asp Ala Phe Leu Ala lie Asp Ala 35 40 45
Lys Leu Thr Thr Glu Glu Xaa lie Lys Glu Leu Ala Gin He Ala Gly 50 55 60
Arg Pro Thr Glu Asp Glu Asp Glu Lys Glu Lys Val Ala Asp Glu Asp 65 70 75 80
Asp Val Asp Asn Glu Glu Ala Ala Leu Leu His Glu Glu Ala Thr Met 85 90 95
Thr He Glu Glu Leu Leu Thr Arg Tyr Gly Gin Asn Cys His Lys Gly 100 105 110
Pro Pro His 115 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TTAGATTGTT TTTNGGCCTT CNGGACCTGA GATTGAGTTT TNTTTTTTTC CTTTAGCNTT 60
AGCAGTGGGN ATGAGGTGNG CAGGGGGAGN TGGGTGGTTN AATCCGCCCA TTCCAAAGAG 120
GGTTNTCCTT CNANANTGCA GCNGGGAGCT TTTGANGTCN TTCCCAGCCG CTTTTGTTCN 180
TNGGGTTNAT NACCGGTTNT GNGCCTGTGT TNTGTTGTGT TGGAGGGAAG GACTGGCGGT 240
TCTGGTTTTT ACTCTGTGAA CTTTATTTAA GGACATTTTT TTTTATTGGG GGGTCCATGG 300
CCCTCGGCCG CTKGCACCCG YTTTTTGTTG WACACTTTCA ATCAACACTT TTTCAGANTA 360
AAGGCCAAAA CCTAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA 406 (2) INFORMATION FOR SEQ ID NO: 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 629 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :
CAGTTTTAGA AAGAAGCACC TTGCTGGATA GATTTGGAGG TTTTCTTTTG GAAATTCANA 60
TTCCATATGT GTTTTTTGCA TCTGAAGGAC TTCTTAATAC TCCAGACATA CTTCAGCTGC 120
TAGAATCCAA CTATAACATC TCACTAGTAG AGAGAGGCTG CAGTGAGTCA TTGAAACTCT 180
TTGGAAGTTC AGAGTGTTAT GTAGTGGTGA CAATTGATGA ACACACTGCC ATAATTTTGC 240
AGGATCTARA AGAATTGAAT TGTGAGAAGG CATCAGACAA TATCATTATG AGGCTGATGG 300
CATTATCATT ACAGTACAGA TATTGTTGGA TAATTTTATA TACCAAAGAA ACATTAAATT 360 CAGAGTATCC GCTTACAGAA AAGACACTTC ATCACCTAGC ACTGATTTAT GCAGCTTTGG 420
TTTCATTTGG GCTAAACTCT GAAGAACTGG ATGTAAAGCT TATAATTGCC CCAGGAGTAG 480
AAGCAACTGC CTTGATAATT CGACAAATTG CTGACCACAG TTTAATGACC TCAAAGAGAG 540
ATCCTCATGA ATGGTTGGAT AAATCCTGGC TTAAAGTTTC ACCATCTGAG GAAGAAATGT 600
ACTTACTTGA TTTTNCCATG TATTAACCC 629 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 9 :
Met Arg Leu Met Ala Leu Ser Leu Gin Tyr Arg Tyr Cys Trp He He
1 5 10 15
Leu Tyr Thr Lys Glu Thr Leu Asn Ser Glu Tyr Pro Leu Thr Glu Lys 20 25 30
Thr Leu His His Leu Ala Leu He Tyr Ala Ala Leu Val Ser Phe Gly 35 40 45
Leu Asn Ser Glu Glu Leu Asp Val Lys Leu He He Ala Pro Gly Val 50 55 60
Glu Ala Thr Ala Leu He He Arg Gin He Ala Asp His Ser Leu Met 65 70 75 80
Thr Ser Lys Arg Asp Pro His Glu Trp Leu Asp Lys Se trp Leu Lys 85 90 95
Val Ser Pro Ser Glu Glu Glu Met Tyr Leu Leu Asp Phe 100 105
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 280 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TCAAGAAAGT TAAAACTTAG GACAAAATNG AAGTTNGAAA ATTTCCAACT TAAAGTATCA 60
TTTTCTGTAA ACACAATTTA AGAACAAATT ANTAAGAGGA AATATTTGCA ACCCAGATAA 120
TAGGAAAAAA AGTTNACATT TNTCATATAT AAAGAATTCC TACAAATTGA TAGAAAGAAG 180
ACAACNTGAT AGAAGAACGG GCAAAATATA TGAACAGATA TTTCCTCAGA AAAAAACAAA 240
AATTGTCAAT AAACATTTGA AACACAAAAA AAAAAAAAAA 280 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GGAAWAAGAA GAGAAAGCCA AGGAAGACAA GGGCAAACAA AAGTTGAGGC AGCTTCACAC 60
ACACAGATAC GGAGAACCAG AAGTGCCAGA GTCAGCATTC TGGAAGAAAA TCATAGCATA 120
TCAACAGAAA CTTCTAAACT ATTTTGCTCG CAACTTTTAC AACATGAGAA TGTTAGCCTT 180
ATTTGTCGCA TTTGCTATCA ATTTCATCTT GCTCTTTTAT AAGGTCTCCA CTTCTTCTGT 240
GGTTGAAGGA AAGGAGCTCC CCACGAGAAG TTCAAGTGAA AATGCCAAAG TGACAAGC 298 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 Met Arg Met Leu Ala Leu Phe Val Ala Phe Ala He Asn Phe He Leu 1 5 10 15
Leu Phe Tyr Lys Val Ser Thr Ser Ser Val Val Glu Gly Lys Glu Leu 20 25 30
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 165 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GGAAACATTT TGCTGATTTT GNGAATTGCC AGCGTTGTGT GTTTTCTGGG AGCATNGAAG 60 CTCTGTTTCG GAAGAGCTGT TTCCTCCCCC CACCTTTTGT ATTTACTTTG AGACTAAAGA 120 CNGAAGAATA ATCTAAATTC ATACTCAGAC AAAAAAAAAA AAAAA 165
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCTGGGCTCG GGCTCANCTC GACTGGGCTC GGCGGGCGGC GGCGGCGGCG CCCGCGGCTG 60
GCGGAAGAAG GAGGGCGAGG GCGGGCGCGG GCCGGCGGGC GGGCGGAAAA AGGAGGAAAG 120
GCGCGGGGAG CCAGGCCTCG GGGCCTCGGA NCAACCACCC GAGCAGACGG AGTACACGGA 180
GCAGCGGCCC CGGCCCCGCC AACGCTGCCG CCGGGATGCT CCAA 224
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 595 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTGGCCGCCA GGTAGAGCGT TGGTTCCGTC GCCGCCGCAA CCAGGACCGG CCCAGTNTCN 60
TCAAGAAGTT CCGAGAAGCC AGCTGGAGAT TCACATTTTA CNTGATTGCN TTCATTGCCG 120
GCATGGCCGT CATTGTGGAT AAACCCTGGT TCTATGACAT GAAGAAAGTT TGGGAGGGAT 180
ATCCCATACA GAGCACTATC CNTTCCCAGT ATTGGTACNA CATGATTGAA CTTTCCTTNT 240
ACTGGTGCSC TGCTCTTCAG CATTGCCTCT GATGTCAAGC GAAAGGATTT CAAGGAACAG 300
ATCATCCACC ATGTGKCCAC CATCATTCTC ATCAGCTTTT CCTGGGTTTG CCAATTACAT 360
CCGAGCTGGG ACTCTAATCA TGGCTCTGCA TGACTCTTCC GATTACCTGC TGGAGTCAGC 420
CAAGATGTTT AACTACGCGG GATGGAAGAA CACCTGCAAC AACATCTTCA TCGTCTTCGC 480
CATTGTTTTT ATCATCACCC GACTGGTCNT CCTGCCCTTC TGGNTCCTGC ATTGCACCCT 540
GGTGTNCCCN CTGGAGCTCT ATCCTGCCTT CTTTGGCTNT TACTTCTTCN ATTCC 595 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ser Ser Ala Leu Pro Leu Met Ser Ser Glu Arg He Ser Arg Asn Arg 1 5 10 15
Ser Ser Thr Met Xaa Pro Pro Ser Phe Ser Ser Ala Phe Pro Gly Phe 20 25 30
Ala Asn Tyr He Arg Ala Gly Thr Leu He Met Ala Leu His Asp Ser 35 40 45 Ser Asp Tyr Leu Leu Glu Ser Ala Lys Met Phe Asn Tyr Ala Gly Trp 50 55 60
Lys Asn Thr Cys Asn Asn He Phe He Val Phe Ala He Val Phe He 65 70 75 80
He Thr Arg Leu Val He Leu Pro Phe Trp He Leu His Cys Thr Leu 85 90 95
Val Tyr Pro Leu Glu Leu Tyr Pro Ala Phe Phe Gly Tyr Tyr Phe Phe 100 105 110
Asn Ser Met Met Gly Val Leu Gin Leu Leu His He Phe Trp Ala Tyr 115 120 125
Leu
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GGTTTTAAAG CCAGAATTAC GGNTAGCACC TAGCATTTCA GCAGAGGGAC CATTTTAGAC 60
CAAAATGTAC TGTTAANGGG TTTTTTTTTA AAATTAAAAG ATTAAATAAA AAATATTAAA 120
TAAAACANGA AAAAAAAAAA AAAAA 145 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 398 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAATTCGGCC AAANANGCCT AGGCAGAATG GGACTCCAAG CCTGCCTCCT AGGGCTCTTT 60 GCCCTCATCC TCTCTGGCAA ATGCAGTTAC AGCCCGGAGC CCGACCAGCG GAGGACGCTG 120
CCCCCAGGCT GGGTGTCCCT GGGCCGTGCG GACCCTGAGG AAGAGCTGAG TCTCACCTTT 180
GCCCTGAGAC AGCAGAATGT GGAAAGACTC TCGGAGCTGG TGCANGCTGT GTCGGATCCC 240
AGCTCTCCTC AATACGGAAA ATACCTGACC CTAGAAAAAT GTGGCTGATC TGGTGAGGCC 300
ATCCCCACTG ACCCTCCACA CGGTGCAAAA ATGGCTCTTG GCAGCCCGGA NCCCAAAAAT 360
TGCCATTCTG TGATCACACA GGAACTTTCT GACTTGCT 398 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Gly Leu Gin Ala Cys Leu Leu Gly Leu Phe Ala Leu He Leu Ser 1 5 10 15
Gly Lys Cys Ser Tyr Ser Pro Glu Pro Asp Gin Arg Arg Thr Leu Pro 20 25 30
Pro Gly Trp Val Ser Leu Gly Arg Ala Asp Pro Glu Glu Glu Leu Ser 35 40 45
Leu Thr Phe Ala Leu Arg Gin Gin Asn Val 50 55
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 437 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TGCTTCCTTG GGGGTGAGTT TTCATTCACT ATGTGGGGAG GGACCTTACG GGAAAACCCA 60 TGTTGTAAGG TTCCCCCAAA TTCCTACCAG CTTTCCACAG GCCTTGGCCC CCCATGTGGA 120
CTTTGTGGGG GGACTGCACC GTTTTTCCCC CAACATCATC CCTGAGGCAA CGTCCTGAGC 180
CGCAGGTGAC AGGGACTGTA GGCCTGCATC TGGGGGTAAC CCCCTCTGTG ATCCGTAAGC 240
GATACAACTT GACCTCACAA GACGTGGGCT CTGGCACCAG CAATAACAGC CAAGCCTGTG 300
CCCAGTTCCT GGAGCAGTAT TTCCATGACT CAGACCTGGC TCAGTTCATG CGCCTCTTCG 360
GTGGCAACTT TGCACATCAG GCATCAGTAG CCCGTGTGGT TGGACAACAG GGCCGGGGCC 420
GGCCGGCGCA TCTCGAG 437 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ I NO:21:
CGGGGACCWA GTGGCAACGA CTTGGACATC TGAGCTGTCA CTGCCGAAAA CAGGCCGCAA 60
GAGAGATAAT CAATATGCAT TTCCAAGCCT TTTGGCTATG TTTGGGTCTT CTGTTCATCT 120
CAATTAATGC AGAATTTATG GATGATGATG TTGAGACGGA AGACTTTGAA GAAAATTCAG 180
AAGAAATTGA TGTTAATGAA AGTGAACTTT CCTCAGAGAT TAAATATAAG ACACCTCAAC 240
CTATAGGAGA AGTATATTTT GCAGAAACTT TTGATAGTGG AAGGTTGGCT GGATGGGTCT 300
TATCMAAARC AAAGAAAGAT GACATGGATG AGGAAATTTC AATATWCGAT GGAAGATGGG 360
AAATTGAAGA GTTGAMAGAA AACCAGGTAC CTGGTGACAG AGGACTGGTA TTAAAATCT 419 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met His Phe Gin Ala Phe Trp Leu Cys Leu Gly Leu Leu Phe He Ser 1 5 10 15
He Asn Ala Glu Phe Met Asp Asp Asp Val Glu Thr Glu Asp Phe Glu 20 25 30
Glu Asn Ser Glu Glu He Asp Val Asn Glu Ser Glu Leu Ser Ser Glu 35 40 45
He Lys Tyr Lys Thr Pro Gin Pro He 50 55
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60
AAAAAAAAAA AAAAAAAAAA AAAAAAGCGG CCGC 94 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CAGAGAGGCT GAGACCAACC CAGAAACCAC CACYTCTCAC GCCAAAGCTC ACACCTTCAG 60
CCTCCAACAT GAAGGTCTCC GCAGCACTTC TGTGGCTGCT GCTCATAGCA GYTGCCTTCA 120
GCCCCCAGGG GCTCGCTGGG CCAGCTTCTG TCCCAACCAC CTGCTGCTTT AACCTGGCCA 180
ATAGGAAGAT ACCCCTTCAG CGACTAGAGA GCTACAGGAG AATCACCAGT GGCAAATGTC 240 CCCAGAAAGC TGTGATCTTC AAGACCAAAC TGGCCAAGGA TATHTGTGCC GACCCCAAGA 300
AGAAGTGGGT GCAGGATTCC CATGAAGTAT CTGGACCAAA AATCTCCAAC TCCAAAGCCA 360 TAAATAATCA CCATTTTNGA AACCAAAAAA AAAAAAAAA 399
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Met Lys Val Ser Ala Ala Leu Leu Trp Leu Leu Leu He Ala Xaa Ala
1 5 10 15
Phe Ser Pro Gin Gly Leu Ala Gly Pro Ala Ser Val Pro Thr Thr Cys 20 25 30
Cys Phe Asn Leu Ala Asn Arg Lys He Pro Leu Gin Arg Leu Glu Ser 35 40 45
Tyr Arg Arg He Thr Ser Gly Lys Cys Pro Gin Lys Ala Val He Phe 50 55 60
Lys Thr Lys Leu Ala Lys Asp He Cys Ala Asp Pro LyF Lys Lys Trp 65 70 75 80
Val Gin
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 448 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GATTTCGAAT TCGGCCAAAG AGGCCTACAA GGGACTAGCA GGCCTAGGGA TACCCTTCCT 60 CTATGGCTCC AGTGTCCCAG CTGCCCCCGC TGCCTACCAT GGCAGGAGCA TGCTCCCTGC 120
CGGTGACCTG CATTTTCACA GAAGCACCCK CAGAAACCTT CAGGGAAACC CCATGCTAGC 180
GGCAACTGCA CCACACTTTG AGGAGAGCTG GGGGCAGAGA TGTNGTCGAC TCAGGAAAAA 240
TACAGGGAAT CAAAAAGCTC TAGACAGTGA TGCTGAGAGT TCCAAAAGTC AAGCAGAAGA 300
AAAAATCCTA GGTCAGACTT ATGCAGTTCC CTATGAAGAC GATCATTATG CAAAAGACCC 360
AGACATTGAA GCACCCAGCA ACCAGAAGTC AAGTGAAACG AATGAAAAGC CAACGACAGC 420
TCTTGCCAAC ACCTGTGGAG AGCTCGAG 448 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Met Leu Pro Ala Gly Asp Leu His Phe His Arg Ser Thr Xaa Arg Asn 1 5 10 15
Leu Gin Gly Asn Pro Met Leu Ala Ala Thr Ala Pro His Phe Glu Glu 20 25 30
Ser Trp Gly Gin Arg Cys Xaa Arg Leu Arg Lys Asn Thr Gly Asn Gin 35 40 45
Lys Ala Leu Asp Ser Asp Ala Glu Ser Ser Lys Ser Gin Ala Glu Glu 50 55 60
Lys He Leu Gly Gin Thr Tyr Ala Val Pro Tyr Glu Asp Asp His Tyr 65 70 75 80
Ala Lys Asp Pro Asp He Glu Ala Pro Ser As Gin Lys Ser Ser Glu 85 90 95
Thr Asn Glu Lys Pro Thr Thr Ala Leu Ala Asn Thr Cys Gly Glu Leu 100 105 110
Glu
(2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 287 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.28
GGCTCACGGC TGTAATCCCA ACACTTTGGG AGTCTGAGGC GGGNGAATCA TGAGGTCAGG 60
AGATTAAGAC CAGCTTGGCC AACATGGTGA AACCCCGTNT NTACTAAAAA TACAAAAAAA 120
TTAGCTAGGC CTGGTGGTGC GCGAATGTAG TCCCAGCTAC TCGGGAGGCT GAAGCAGGAG 180
AATTGCTTGA ACCTGGGAAG CGGAGGCTAC AGTGAGCTGA GATCGTGCCA CTGCACTCCA 240 GCCTGGGTGA CAGAGCAGGA CTCTGTNTCA AAAAAAAAAA AAAAAAA 287
(2) INFORMATION FOR SEQ ID NO:29
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 228 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY linear
(ii) MOLECULE TYPE cDNA
(xi) SEQUENCE DESCRIPTION. SEQ ID NO:29.
GGANAGCTGT TCAGCTATGG GTGCTGTCAC ANATTANCTG TATCTTTGNC AGTTTAGCTG 60
GATGCTCATT CAGTCTGTGA ATTTCTGGTA NGTGCTGGTG ATGAATGATG AGCACACAGA 120
GAGGGGATAT CTGCTGTTTT TCCTTCTGAG TTGGGGANTA CCAGCTTTTG TGGTGATTCT 180
CCTCATAGTT ATTTTGAAAG GAATCTATCA TCAGAGCATG TCANAGAT 228 (2) INFORMATION FOR SEQ ID NO: 30
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GACCTGTNTT TTATTCCAAA CGTCTATNCT GCTTTNTTCA CTGCAGCTNC TTGTTCCTTT 60
GACNTNCCTC NTGNTGGTNT TCNTGGTNTT CATCCATGCC TACCAGGTGA ANCCACANTG 120
GAAAGCATAT GATGATNTNT TCAGAGGAAG GACAAATGCT GCAGAAATTC CACTGATTTT 180
ATATCTCTTT GCTCTGATTT CCNTGACATG GCTTTGGGGA GGACTACACA TGGCCTACAG 240
GCACTTCTGG ATGTTGGTTC TCTTTGTCAT TTTCAACAGT CTGCAGGGAC TTTATGTTTT 300
CATGGTTTAT TTCATTTTWC ACAACCAAAT GTGTTGCCCT ATGAAGGCCA GTTACACTGT 360
GGAAATGAAT GGGCATCCTG GACCCAGCAC AGCCTTTTTC ACGCCCGGGA GTGGAATGCC 420
TCCTGCTGGA GGGGAAATCA GCAAGTCCAC CCAGAATCTC ATCGGTGCTA TGGAGGAGGT 480
GCCACCTGAC TGGGAGAGAG CATCCTTCCA ACAGGGCAGT CAGGCCAGCC CTGATTTAAA 540
G 541 (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 104 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met Ala Tyr Arg His Phe Trp Met Leu Val Leu Phe Val He Phe Asn 1 5 10 15
Ser Leu Gin Gly Leu Tyr Val Phe Met Val Tyr Phe He Xaa His Asn 20 25 30
Gin Met Cys Cys Pro Met Lys Ala Ser Tyr Thr Val Glu Met Asn Gly 35 40 45
His Pro Gly Pro Ser Thr Ala Phe Phe Thr Pro Gly Ser Gly Met Pro 50 55 60
Pro Ala Gly Gly Glu He Ser Lys Ser Thr Gin Asn Leu He Gly Ala 65 70 75 80 Met Glu Glu Val Pro Pro Asp Trp Glu Arg Ala Ser Phe Gin Gin Gly 85 90 95
Ser Gin Ala Ser Pro Asp Leu Lys 100
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CCTGTGAATT NTACTGGATG ATTAATACAA ACGTGATTGT TGTATTTGGA GTATAAATTA 60
CTGATTGTAT GTNACCTGAA AATTCACTGC TATAAGAAAG GTGGANTCAG TTTGTATCAN 120
TTAATAGGAT TTTCATATTC CAAGGATATT AGTTGTTTTT TTAATCATCC TATATGGCTA 180
ACATTGTTTA ATGAAAGTAA TAATCAATAA AGCAATAGAA TCTAAAAAAA AAAAAAAA 238 (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
{xi ) SEQUENCE DESCRIPTION: SEQ ID NO:33:
TTGCTGTTGC GCGGGATGGT GGATGACTTG TCAAACTCGG GCGGGCCCTC GCTGTCCGCA 60
TCCCCATTCA CGGAGTANCA NTCGTANTCN GAGCCTGGGG GCACGGGACA CANTGAGGCC 120
CANGGCCCAN GTGGCCCCTT GCCCCCAGCC CACCAGGGTG AGCACAGAGG GGGAAGGACG 180
GGGCCCTCCT GGATGGCTAA NTCCCANCTG TCCCTGGTCC CACCCCANCC CCGCGGGCCT 240
GCCTTGGGAA GGGATGGTGT CCTCA 265 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 467 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION- SEQ ID NO 34
GCAGGAAATC TGGGGATCTA AATCCAGAAT GATTCAGAGA GCTGGAGGAA GGGGGCTGCC 60
CTGGCTTAAC TTGGTTCATT CCCAGGCCAA CACCTCACCG TGATCCACGT CCCCACTGCT 120
GTGCTGAAGC TGGNGTNTGC CCCAGGGAAC CCTGCCGGTC ACACATGYTC AGGATTTCAT 180
GGGCCTGTGT CNACCCTGCT TTTTTCTTTA TTCTTNGTAG TNGTTTAGGA GTGGGGGGCC 240
TCGCAGAACA CNTAGTCCAG CCCACTGCCC AGAGCAGGTG TGTCCCTTTC ATAMTTCAGT 300
CCACTTTAAA ACAGCCTTCC CCCACCCCCT TYTATGGTAG CAGTTYTCCT CGGGGTYTCC 360
ATGGACACCC TGTGCCCCAA GCCGATGGCC CCACCCAGCA GCATCAGCAC AGCTGCCCCC 420
CTTYTCCGCA GAGCAGGCTY TCCTTTACGG GAMTYTCCTY TTCCCTC 467 (2) INFORMATION FOR SEQ ID NO:35
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH. 28 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE protein
(xi) SEQUENCE DESCRIPTION. SEQ ID NO.35
Phe Phe Phe He Leu Xaa Ser Xaa Leu Gly Va l Gly Gly Leu Ala Glu 1 5 10 15
His Xaa Val Gin Pro Thr Ala Gin Ser Arg Cys Val 20 25
(2) INFORMATION FOR SEQ ID NO:36 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GGGAGGCTGA NGCATAAGGA TCACTTGAGC CCAGGAGTTC ANGACCAGCC TAACATAGTG 60
AGACCCCTGA NTTTACAAAA AATTAAAGTT AGCCAGGTGT GGTGGNACAN GCCTGTGGTC 120
CCAGCTACAC AGGAAGCTGA GGCAGGAGGA TATNTTGAGC CTAGGAATTC AAGGCTGCAG 180
NGAGCTGTGA TNACACCACT GAACTCCTGA ACTCCAGTGT GCGTGACAGA GCAAGACTCT 240
GTNTCNAAAN AAAAANAAAA AAAAAAAAAA GCGGCCGCT 279 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 233 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
AAGCCCCCCA CGCCCCGAGC CCACCCTGCT CACCGGCCTC TGCCCGAGTT CCCCGCATAG 60
TGTGGGAGTG TGGANCATCC TANCTTTTCC CCAGCGCCCA GTTCTTTCAC TCTCACTGGA 120
GTCCTGCAGG GACAGCTCGG GCACCATGTA NGCCCGGGTG GGCGTGGGGG CTCACCTANC lp''
TCGGTGGTGA ACAGCTGGCA CGTCTCTGGG TTGCGGACGG TAAAGGCCAC GTA 33 (2) INFORMATION FOR SEQ ID NO:38 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 579 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ACGNGNGNTC CATCGCCAGA ATTCCNGTNG TNTCAGGAAT GTTCAATAGA TGGAATCCTG 60
TGTGGCCTGA GANGAGTGTT TTTCATGCCG NGTGACACCC TNGAGGCCCG NGCAACTGTT 120
GGTANGTCAA CAGTTAGCTG CTTCTCATTG CNGAGTGGCG ATTGGTCCTG TCATGGTTTA 180
TTCAGCCATG NGGNGGATGG CTACTTGTTT TNTAAGCCAC TTGCCTTCTG ATCGCTGGAC 240
NGACTCTYTC GCCYTYTCTT GGTGCAGTCC TCAGGAGGCT CGGTCACAYT CTCCAAGAGC 300
ACAGCCATCA TCTCCCACGG TACCACAGGC CTGGTCACAT GGGATGCCGC CCTYTACCTT 360
GCAGAATGGG CCATCGAGAA CCCGGCAGCC TTCATTAACA GGTGACCTCG GGGCACAGGG 420
CAGGGCACCG AGGCAGGCTT ACCCTGGTGC AGTCGAAAAC ACGGTCCCCT TTCCTCCCGC 480
CAGGACTGTC CTAGAGCTTG GCAGTGGTGC CGGCCTCACA GGCCTTGCCA TYTGCAAGAN 540 GTGCCGCCCC CGGGCATACA TYTTCAGCGA CCCTCACAG 579
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Met Val Tyr Ser Ala Met Xaa Xaa Met Ala Thr Cys Phe Xaa Ser His 1 5 10 15
Leu Pro Ser Asp Arg Trp Thr Asp 20
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0: AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAAAAAAG CGGCCGC 87
(2) INFORMATION FOR SEQ ID NO:41:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GCTTTTTTTT TTTTTTTTGT ATCTTTCTAG TTCATTCTGT CCATTGNTAN TTTTTTATAA 60
ANAAAATTTC ACTACCATAT ACTTTCTGTT CCANANACAG GAAACTGTTT GCAGGTCCCT 120
GAACCTACCT TCATTTTCTA GTGCTGTGCA TTTCCTCATT TCTTTCATTT GGAAANTGGT 180
GAAAANGTCC TCTAACTTGC TTCTTGCCCT CATTTCTCTA AGCA 224 (2) INFORMATION FOR SEQ ID NO: 42:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 593 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
TGTTCTTCNG GCACTTCCCA TCACCGNGAT AGCATATTTC ACTGGGGTGG TTTCCATTCC 60
CATTTCCTCT ACTATAGTGA GCCACTGGAG GCGAGAACCC TNGTATTTCC AACATTGGTG 120
TACTCAGCAT CTTGCGTCGA GGACTCAGTA AGTATTATAT TTGAATTCCC ACTGCACCGC 180
TCTAATTAGA ATTTTAAAAA TCACTTTCTA TGTGGATTGT NACACACTTT TTTTCCCCTT 240
AATTCATTTT TCTCCANGNA CTACCCATAT GCATCCTATA TAAATTTACC AGCACTCATA 300
AAAATCTTAC TCAGAAATCT TCAGAGGTTT GCTAAGGATA CAATTTGATT CTTACACATT 360 TAATGCTCAC CAGCTGCTTA GGCCCACACC ATTTATCCAC CCTGATTTGC TACTGCTCTT 420
TGAAATACAA CCAGTGTTTC AGCCAGACTG TTTTCCTGCT TCTGCTCCCC TTCTCCTCCT 480
CCCAGCACAT CTGTGAATTC TTTGACTGGT TTACCACTCC CAMACTCCTC CCCAGCAATG 540
CAGATCTTCT ACACCCTTTA GGATCTAAGC TAAGTCTGCT TCCCAGATAT CCT 593 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Met Leu Thr Ser Cys Leu Gly Pro His His Leu Ser Thr Leu He Cys 1 5 10 15
Tyr Cys Ser Leu Lys Tyr Asn Gin Cys Phe Ser Gin Thr Val Phe Leu 20 25 30
Leu Leu Leu Pro Phe Ser Ser Ser Gin His He Cys Glu Phe Phe Asp 35 40 45
Trp Phe Thr Thr Pro Xaa Leu Leu Pro Ser Asn Ala Asp Leu Leu His 50 55 60
Pro Leu Gly Ser Lys Leu Ser Leu Leu Pro Arg Tyr Pro 65 70 75
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: TCNTGNATTT AATTATTTAA GCTATATTAA AAAAATTGAA ACCCTTAGAT GTTAATTAAT 60 TTTAAAAACT ANTGATNGAT GCAGNTAAGC TAGAATGATT GGATCAAATC TCACACACAA 120
ATGAGTTTAT TCTTTAAAAA AAAATTTTTT TTTTAGAGAC GGNTTCTTGC TATGTTCCCC 180
AGGATGTTCT TGAACTCATG ACCTCAAGCA ATCCTCCTCC CTCACCCTAC CTGAATTAAA 240
AAAAAAAAAA AAAAAA 256 (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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:45: AATGATCAGG ATCTAAGTGT TAGGCGGA 28
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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:46: GGTTTCCCTA GTTGAGTCCT CAGGTCCT 28
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 47 : TATCAAGGCA GTTGCTTCTA CTCCTGGG 28
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 48: ACAAGATGAA ATTGATAGCA AATGCGAC 28
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 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:49: ACCTGCTGGA GTCAGCCAAG ATGTTTA 27
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 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: 50: GAATGGGACT CCAAGCCTGC CTCCTA 26
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 51: TGAGATGAAC AGAAGACCCA AACATAGC 28
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 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:52: TCTCACGCCA AAGCTCACAC CTTCAGC 27
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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:53 TTGGGTCTTT TGCATAATGA TCGTCTTC 28
(2) INFORMATION FOR SEQ ID NO:54:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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:54. GAACTGGCCT TCATAGGGCA ACACATTT 28
(2) INFORMATION FOR SEQ ID NO: 55:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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:55 AACCCCGAGG AGAACTGCTA CCATAGAA (2) INFORMATION FOR SEQ ID NO: 56:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 56- TCTCGATGGC CCATTCTGCA AGGTAGAG 28 (2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 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: 57: GTTGTATTTC AAAGAGCAGT AGCAAATC

Claims

What is claimed is:
1. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 from nucleotide 351 to nucleotide 506;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AZ302_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:3;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:3 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
2. A composition of claim 1 wherein said polynucleotide is operably linked to an expression control sequence.
3. A host cell transformed with a composition of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein, which comprises: (a) growing a culture of the host cell of claim 3 in a suitable culture medium, and
(b) puπfying the protein from the culture
6 A protein produced according to the process of claim 5
7 The protein of claim 6 compnsing a mature protein
8 A composition compnsing a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO 3,
(b) fragments of the amino acid sequence of SEQ ID NO 3, and
(c) the ammo acid sequence encoded by the cDNA insert of clone AZ302_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins
9 The composition of claim 8, wherein said protein compπses the amino acid sequence of SEQ ID NO 3
10 The composition of claim 8, wherein said protein compπses the amino acid sequence of SEQ ID NO 3 from ammo acid to ammo acid
1 1 The composition of claim 8, further comprising a pharmaceutically acceptable earner
12 A method for preventing, treating or ameliorating a medical condition which compnses administeπng to a mammalian subject a therapeutically effective amount of a composition of claim 1 1
13 The gene corresponding to the cDNA sequence of SEQ ID NO 2, SEQ ID NO 1 or SEQ ID NO 4
14 A composition compπsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 5, (b) a polynucleotide compnsing the nucleotide sequence of SEQ ID NO'5 from nucleotide 23 to nucleotide 517,
(c) a polynucleotide compπsing the nucleotide sequence of the full length protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076,
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076,
(e) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AU139_2 deposited under accession number ATCC 98076,
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AU139_2 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding a protein comprising the ammo acid sequence of SEQ ID NO-6,
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO.6 having biological activity,
(l) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above,
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above , and
(k) a polynucleotide capable of hybridizing under stπngent conditions to any one of the polynucleotides specified in (a)-(h)
15 A composition compπsing a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO 6,
(b) the amino acid sequence of SEQ ID NO 6 from amino acid 35 to amino acid 1 15,
(c) fragments of the amino acid sequence of SEQ ID NO-6, and
(d) the amino acid sequence encoded by the cDN A insert of clone AU 139_2 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins
16 The gene corresponding to the cDNA sequence of SEQ ID NO.5 or SEQ ID NO:7.
17. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 288 to nucleotide 629;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 441 to nucleotide 629;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AU105_14 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:9;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:9 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) .
18. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:9;
(b) the amino acid sequence of SEQ ID NO:9 from amino acid 25 to amino acid 44;
(c) fragments of the amino acid sequence of SEQ ID NO:9; and
(d) the amino acid sequence encoded by the cDNA insert of clone AU105_14 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
19. The gene corresponding to the cDNA sequence of SEQ ID NO:8 or SEQ ID NO:10 .
20. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 1 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 1 from nucleotide 164 to nucleotide 298;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS268_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 12;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
21. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 12;
(b) fragments of the amino acid sequence of SEQ ID NO: 12; and (c) the amino acid sequence encoded by the cDNA insert of clone AS268_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
22. The gene corresponding to the cDNA sequence of SEQ ID NO: 1 1 or SEQ ID NO:13 .
23. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 75 to nucleotide 419;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21 from nucleotide 132 to nucleotide 419;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ 147_1 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ147_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:22;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:22 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).
24 A composition compπsing a protein, wherein said protein compnses an amino acid sequence selected from the group consisting of
(a) the amino acid sequence of SEQ ID NO.22,
(b) fragments of the amino acid sequence of SEQ ID NO 22; and
(c) the ammo acid sequence encoded by the cDNA insert of clone AJ147_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
25 The gene corresponding to the cDNA sequence of SEQ ID NO 21 or SEQ ID NO:23
26 A composition compnsing an isolated polynucleotide selected from the group consisting of
(a) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 24,
(b) a polynucleotide compπsing the nucleotide sequence of SEQ ID NO 24 from nucleotide 69 to nucleotide 377,
(c) a polynucleotide compnsing the nucleotide sequence of SEQ ID NO 24 from nucleotide 120 to nucleotide 377,
(d) a polynucleotide compnsing the nucleotide sequence of the full length protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076,
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AM262_11 deposited under accession number ATCC 98076,
(f) a polynucleotide compnsing the nucleotide sequence of the mature protein coding sequence of clone AM262_1 1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AM262_11 deposited under accession number ATCC 98076,
(h) a polynucleotide encoding a protein compnsing the ammo acid sequence of SEQ ID NO 25,
(i) a polynucleotide encoding a protein compnsing a fragment of the am o acid sequence of SEQ ID NO.25 having biological activity,
(j) a polynucleotide which is an allelic vaπant 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).
27. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:25;
(b) the amino acid sequence of SEQ ID NO:25 from amino acid 14 to amino acid 81;
(c) fragments of the amino acid sequence of SEQ ID NO:25; and
(d) the amino acid sequence encoded by the cDNA insert of clone AM262_11 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
28. The gene corresponding to the cDNA sequence of SEQ ID NO:24.
29. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide cot ■ ;sing the nucleotide sequence of SEQ ID NO:26;
(b) a polynucleotide compnsing the nucleotide sequence of SEQ ID NO:26 from nucleotide 1 10 to nucleotide 448;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AR28_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:27;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:27 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
30. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 27;
(b) the amino acid sequence of SEQ ID NO: 27 from amino acid 15 to amino acid 78;
(c) fragments of the amino acid sequence of SEQ ID NO:27; and
(d) the amino acid sequence encoded by the cDNA insert of clone AR28_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
31. The gene corresponding to the cDNA sequence of SEQ ID NO:26 or SEQ ID NO:28 .
32. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:30;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:30 from nucleotide 230 to nucleotide 541 ;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS162_1 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS162_1 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:31 ; (h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:31 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above .
33. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:31 ;
(b) the amino acid sequence of SEQ ID NO: 31 from amino acid 5 to amino acid 25;
(c) fragments of the amino acid sequence of SEQ ID NO:31 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone AS 162_1 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
34. The gene corresponding to the cDNA sequence of SEQ ID NO:30, SEQ ID NO:29 or SEQ ID NO: 32 .
35. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:34;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:34 from nucleotide 202 to nucleotide 467;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:34 from nucleotide 241 to nucleotide 467;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS264_3 deposited under accession number ATCC 98076; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:35;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:35 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
36. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:35;
(b) fragments of the amino acid sequence of SEQ ID NO:35; and
(c) the amino acid sequence encoded by the cDNA insert of clone AS264_3 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
37. The gene corresponding to the cDNA sequence of SEQ ID NO:34, SEQ ID NO:33 or SEQ ID NO:36.
38. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 173 to nucleotide 579;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076; (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AS301_2 deposited under accession number ATCC 98076;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:39;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:39 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
39. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:39;
(b) fragments of the amino acid sequence of SEQ ID NO: 39; and
(c) the amino acid sequence encoded by the cDNA insert of clone AS301_2 deposited under accession number ATCC 98076; the protein being substantially free from other mammalian proteins.
40. The gene corresponding to the cDNA sequence of SEQ ID NO:38, SEQ ID NO:37 or SEQ ID NO:40 .
41. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:42;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:42 from nucleotide 363 to nucleotide 593;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:42 from nucleotide 483 to nucleotide 593;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076; (e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076,
(f) a polynucleotide compπsing the nucleotide sequence of the mature protein coding sequence of clone AS86_1 deposited under accession number ATCC 98076,
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076,
(h) a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO:43,
(I) a polynucleotide encoding a protein compπsing a fragment of the ammo acid sequence of SEQ ID NO 43 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
(I) a polynucleotide capable of hybπdizmg under stπngent conditions to any one of the polynucleotides specified in (a)-(ι)
42 A composition compnsing a protein, wherein said protein compnses an amino acid sequence selected from the group consisting of
(a) the am o acid sequence of SEQ ID NO 43,
(b) fragments of the am o acid sequence of SEQ ID NO 43, and
(c) the amino acid sequence encoded by the cDNA insert of clone AS86_1 deposited under accession number ATCC 98076, the protein being substantially free from other mammalian proteins
43 The gene corresponding to the cDNA sequence of SEQ ID NO 42, SEQ ID NO 41 or SEQ ID NO 44
PCT/US1997/009878 1996-06-07 1997-06-06 Polynucleotides encoding secreted proteins WO1997046683A2 (en)

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EP97929838A EP0910638A2 (en) 1996-06-07 1997-06-06 Polynucleotide encoding secreted proteins
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072771A3 (en) * 2000-03-29 2003-12-04 Dgi Biotechnologies L L C Insulin and igf-1 receptor agonists and antagonists
US6875741B2 (en) 1998-09-02 2005-04-05 Renuka Pillutla Insulin and IGF-1 receptor agonists and antagonists
US7005509B1 (en) 1995-02-17 2006-02-28 Incyte Corporation Chemokine PANEC-1 polynucleotides and compositions and methods related thereto
US7173005B2 (en) 1998-09-02 2007-02-06 Antyra Inc. Insulin and IGF-1 receptor agonists and antagonists
US7265201B1 (en) 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002500035A (en) * 1998-01-07 2002-01-08 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド 36 human secreted proteins
CN111334469A (en) * 2020-03-27 2020-06-26 郑州鲲鹏健康科技有限公司 PBMC (peripheral blood mononuclear cell) in-vitro 3D (three-dimensional) methylcellulose agarose hydrogel culture medium and preparation method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536637A (en) * 1993-04-07 1996-07-16 Genetics Institute, Inc. Method of screening for cDNA encoding novel secreted mammalian proteins in yeast

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536637A (en) * 1993-04-07 1996-07-16 Genetics Institute, Inc. Method of screening for cDNA encoding novel secreted mammalian proteins in yeast

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CALLARD R E ET AL: "AIMS OF THE BOOK" CYTOKINE FACTSBOOK, 1994, CALLARD R E;GEARING A J H, pages 2/3, 31-38, 64/65, 75/76, 97/98, 148 - 151, 252/253, XP002039160 *
ENG MONG LIM ET AL: "IDENTIFICATION OF MYCOBACTERIUM TUBERCULOSIS DNA SEQUENCES ENCODING EXPORTED PROTEINS BY USING PHOA GENE FUSIONS" JOURNAL OF BACTERIOLOGY, vol. 177, no. 1, 1 January 1995, pages 59-65, XP000560419 *
J. NATHANS: "69C1 human retina cDNA Tsp509I-cleaved sublibrary Homo sapiens cDNA not directional" EMBL DATABASE ENTRY HSW22546, ACCESSION NUMBER W22546, 9 May 1996, XP002046017 *
JACOBS K ET AL: "A NOVEL METHOD FOR ISOLATING EUKARYOTIC CDNA CLONES ENCODING SECRETED PROTEINS" JOURNAL OF CELLULAR BIOCHEMISTRY - SUPPLEMENT, vol. 21A, 10 March 1995, page 19 XP002027246 *
K. A. JACOBS ET AL: "A genetic selection for isolating cDNAs encoding secreted proteins" GENE., vol. 198, 1 October 1997, AMSTERDAM NL, pages 289-296, XP002045919 *
L. HILLIER ET AL: "The WashU-Merck EST project. zo29g02.r1 stratagene colon(#937204) Homo sapiens cDNA clone 588338 5'" EMBL DATABASE ENTRY HSAA51630 , ACCESSION NUMBER AA151630, 15 December 1996, XP002046018 *
M. YOKOYAMA-KOBAYASHI ET AL: "A signal sequence detection system using secreted protease activity as an indicator" GENE., vol. 163, 1995, AMSTERDAM NL, pages 193-196, XP002046435 *
TASHIRO K ET AL: "SIGNAL SEQUENCE TRAP: A CLONING STRATEGY FOR SECRETED PROTEINS AND TYPE I MEMBRANE PROTEINS" SCIENCE, vol. 261, 30 July 1993, pages 600-603, XP000673204 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7005509B1 (en) 1995-02-17 2006-02-28 Incyte Corporation Chemokine PANEC-1 polynucleotides and compositions and methods related thereto
US7265201B1 (en) 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
US7285620B2 (en) 1995-06-23 2007-10-23 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
US7423129B2 (en) 1995-06-23 2008-09-09 Millennium Pharmaceuticals, Inc. Antibodies to human eotaxin
US7858326B2 (en) 1995-06-23 2010-12-28 Millennium Pharmaceuticals, Inc. Method of diagnosis of inflammatory disease using eotaxin antibodies
US6875741B2 (en) 1998-09-02 2005-04-05 Renuka Pillutla Insulin and IGF-1 receptor agonists and antagonists
US7173005B2 (en) 1998-09-02 2007-02-06 Antyra Inc. Insulin and IGF-1 receptor agonists and antagonists
WO2001072771A3 (en) * 2000-03-29 2003-12-04 Dgi Biotechnologies L L C Insulin and igf-1 receptor agonists and antagonists

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WO1997046683A3 (en) 1998-05-07
CA2256522A1 (en) 1997-12-11
AU3380497A (en) 1998-01-05
AU3568697A (en) 1998-01-05
WO1997046682A3 (en) 1998-03-12
EP0910638A2 (en) 1999-04-28
JP2000515726A (en) 2000-11-28
JP2001501455A (en) 2001-02-06

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