WO1998041539A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998041539A2
WO1998041539A2 PCT/US1998/005474 US9805474W WO9841539A2 WO 1998041539 A2 WO1998041539 A2 WO 1998041539A2 US 9805474 W US9805474 W US 9805474W WO 9841539 A2 WO9841539 A2 WO 9841539A2
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WO
WIPO (PCT)
Prior art keywords
amino acid
polynucleotide
seq
protein
sequence
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Application number
PCT/US1998/005474
Other languages
French (fr)
Other versions
WO1998041539A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
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Genetics Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to CA002284109A priority Critical patent/CA2284109A1/en
Priority to EP98912986A priority patent/EP0971954A2/en
Priority to JP54082198A priority patent/JP2001517941A/en
Priority to AU67650/98A priority patent/AU6765098A/en
Publication of WO1998041539A2 publication Critical patent/WO1998041539A2/en
Publication of WO1998041539A3 publication Critical patent/WO1998041539A3/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
    • 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
    • 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 provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 463 to nucleotide 606; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 501; the nucleotide sequence of the full-length protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364.
  • 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: (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
  • a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364;
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bk95_3 deposited under accession number ATCC 98364;
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bk95_3 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 2 to amino acid 102.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:6 from amino acid 2 to amino acid 102.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:6;
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:8 from nucleotide 156 to nucleotide 902; the nucleotide sequence of SEQ ID NO:8 from nucleotide 225 to nucleotide 902; the nucleotide sequence of SEQ ID NO: 8 from nucleotide 237 to nucleotide 654; the nucleotide sequence of the full-length protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:9 from amino acid 28 to amino acid 166.
  • 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:9 or the amino acid sequence of SEQ ID NO:9 from amino acid 28 to amino acid 166.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364.
  • 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: (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
  • a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13 (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13 having biological activity, the fragment comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:13; (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:12 from nucleotide 506 to nucleotide 1096; the nucleotide sequence of SEQ ID NO:12 from nucleotide 656 to nucleotide 1096; the nucleotide sequence of SEQ ID NO: 12 from nucleotide 2 to nucleotide 1078; the nucleotide sequence of the full-length protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13 from amino acid 1 to amino acid 191.
  • 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:
  • a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:15 having biological activity, the fragment comprising the amino acid sequence from amino acid 15 to amino acid 24 of SEQ ID NO:15;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:14 from nucleotide 1563 to nucleotide 1685; the nucleotide sequence of SEQ ID NO:14 from nucleotide 1100 to nucleotide 1646; the nucleotide sequence of the full-length protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 15 from amino acid 1 to amino acid 28.
  • 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:15 or the amino acid sequence of SEQ ID NO:15 from amino acid 1 to amino acid 28.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364.
  • 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: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 19 from amino acid 61 to amino acid 175.
  • 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: 19 or the amino acid sequence of SEQ ID NO:19 from amino acid 61 to amino acid 175.
  • 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:20;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:20 from nucleotide 3746 to nucleotide 4027; the nucleotide sequence of SEQ ID NO:20 from nucleotide 3815 to nucleotide 4027; the nucleotide sequence of SEQ ID NO:20 from nucleotide 3640 to nucleotide 3940; the nucleotide sequence of the full-length protein coding sequence of clone fe366_l deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone fe366_l deposited under accession number ATCC 98364.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:21 from amino acid 1 to amino acid 65.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • polynucleotide is operably linked to an expression control sequence.
  • the invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
  • Processes are also provided for producing a protein, which comprise:
  • the protein produced according to such methods is also provided by the present invention.
  • Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
  • Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier.
  • Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
  • Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
  • nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application.
  • the nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence.
  • "Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum. Clone "bd!64 7"
  • a polynucleotide of the present invention has been identified as clone "bdl64_7".
  • bdl64_7 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bdl64_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bdl64_7 protein").
  • nucleotide sequence of bdl64_7 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bdl64_7 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Another potential bdl64_7 reading frame and predicted amino acid sequence is encoded by basepairs 610 to 762 of SEQ ID NO:l and is reported in SEQ ID NO:32.
  • the EcoRI /Notl restriction fragment obtainable from the deposit containing clone bdl64_7 should be approximately 1950 bp.
  • bdl64_7 demonstrated at least some similarity with sequences identified as AF001540 (Human clone alphal mRNA, partial sequence), C05823 (similar to none), G22994 (human STS WI-30658), H03651 (yj37el2.sl Homo sapiens cDNA clone 150958 3'), H26492 (EST51a22 Homo sapiens cDNA clone 51a22), H90721 (yv96f02.rl Homo sapiens cDNA clone 250587 5'), N58545 (yv73d07.sl Homo sapiens cDNA clone 2483653'), R10191 (yf35d07.rl Homo sapiens cDNA clone 1288455'),
  • bil29_2 A polynucleotide of the present invention has been identified as clone "bil29_2".
  • bil29_2 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bil29_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bil29_2 protein").
  • nucleotide sequence of bil29_2 as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bil29_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Amino acids 91 to 103 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 104, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone bil29_2 should be approximately 1100 bp.
  • bil29_2 demonstrated at least some similarity with sequences identified as H88684 (yw23b01.rl Homo sapiens cDNA), R59623 (yh02g07.sl Homo sapiens cDNA clone 42126 3'), T17199 (NIB515 Homo sapiens cDNA 3'end), T24786 (Human gene signature HUMGS06869), T65550 (yc76bl2.sl Homo sapiens cDNA clone 21611 3'), and T65617 (yc76bl2.rl Homo sapiens cDNA clone 21611 5').
  • the predicted amino acid sequence disclosed herein for bil29_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bil29_2 protein demonstrated at least some similarity to sequences identified as AF016712 (testicular condensing enzyme [Mus musculus]) and U43375 (Similar to sugar transporter (Caenorhabditis elegans cosmid K09C4)). Based upon sequence similarity, bil29_2 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts six potential transmembrane domains within the bil29_2 protein sequence, centered around amino acids 11, 36, 69, 100, 131, and
  • a polynucleotide of the present invention has been identified as clone "bk95_3".
  • bk95_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bk95_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bk95_3 protein").
  • nucleotide sequence of the 5' portion of bk95_3 as presently determined is reported in SEQ ID NO:5. What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO:6.
  • the predicted amino acid sequence of the bk95_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • Amino acids 87 to 99 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 100, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of bk95_3/ including the polyA tail, is reported in SEQ ID NO:7.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone bk95_3 should be approximately 2400 bp.
  • bk95_3 demonstrated at least some similarity with sequences identified as AA521036 (aa71b06.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:826355 3' similar to SW:SYB2_XENLA P47193 SYNAPTOBREVIN 2), N29686 (yw78a05.sl Homo sapiens cDNA clone 258320 3' similar to SP:SW:SYB2_XENLA P47193 SYNAPTOBREVIN 2), T33715 (Cellubrevin-2 coding sequence), U14567 (***ALU WARNING Human Alu-J subfamily consensus sequence), and U60150 (Mus musculus vesicle-associated membrane protein VAMP-2 mRNA
  • the predicted amino acid sequence disclosed herein for bk95_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bk95_3 protein demonstrated at least some similarity to sequences identified as L14270 (synaptobrevin [Drosophila melanogaster]), M36205 (synaptobrevin 2 (SYB2) [Homo sapiens]), U60961 (cellubrevin [Mus musculus]), U64520 (synaptobrevin-3 [Homo sapiens]), W04181 (Cellubrevin-2), and X76199 (synaptobrevin [Bos taurus]). Based upon sequence similarity, bk95_3 proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of bk95_3 indicates that it may contain an Alu repetitive element.
  • cgl60_6 A polynucleotide of the present invention has been identified as clone "cgl60_6".
  • cgl60_6 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cgl60_6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cgl60_6 protein").
  • nucleotide sequence of cgl60_6 as presently determined is reported in SEQ ID NO:8. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cgl60_6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:9. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone cgl60_6 should be approximately 1400 bp.
  • cgl60_6 The nucleotide sequence disclosed herein for cgl60_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cgl60_6 demonstrated at least some similarity with sequences identified as AA405957 (zu66c07.rl Soares testis NHT Homo sapiens cDNA clone 742956 5') and T19219 (f02011t Testis 1 Homo sapiens cDNA clone f02011 5' end). Based upon sequence similarity, cgl60_6 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts three additional potential transmembrane domains within the cgl60_6 protein sequence, centerd around amino acids 148, 195, and 236 of SEQ ID NO:9, respectively.
  • cw775 1 A polynucleotide of the present invention has been identified as clone "cw775_l".
  • cw775_l was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cw775_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
  • nucleotide sequence of cw775_l as presently determined is reported in SEQ ID NO: 10. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cw775_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:ll.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone cw775_l should be approximately 4200 bp.
  • the nucleotide sequence disclosed herein for cw775_l was searched against the
  • cw775_l demonstrated at least some similarity with sequences identified as AA104324 (mo50d06.rl Life Tech mouse embryo 10 5dpc 10665016 Mus musculus cDNA clone 557003 5'), AA373350 (EST85423 HSC172 cells I Homo sapiens cDNA 5' end), H30439 (ym58fl0.rl Homo sapiens cDNA clone 52688 5'), N28734 (yx67cl0.rl Homo sapiens cDNA clone 2668025'), and N57005 (yy56h03.sl Homo sapiens cDNA clone 277589 3'). Based upon sequence similarity, cw775_l proteins and each similar protein or peptide may share at least some activity.
  • dn740_3 A polynucleotide of the present invention has been identified as clone "dn740_3".
  • dn740_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • dn740_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dn740_3 protein").
  • nucleotide sequence of dn740_3 as presently determined is reported in SEQ ID NO:12. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dn740_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:13.
  • Amino acids 38 to 50 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 51, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone dn740_3 should be approximately 1650 bp.
  • dn740_3 demonstrated at least some similarity with sequences identified as AA053844 (zf53h07.rl Soares retina N2b4HR Homo sapiens cDNA clone 380701 5'), AA056525 (zl65g08.rl Stratagene colon (#937204) Homo sapiens cDNA clone 5095345'), H70470 (yr91c07.sl Homo sapiens cDNA clone 2126523'), N53038 (yv53d09.sl Homo sapiens cDNA clone 246449 3'), R56318 (yg90e03.rl Homo sapiens cDNA clone 40653 5'), and W73718 (zd50f06.sl Soares fetal
  • the predicted amino acid sequence disclosed herein for dn740_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted dn740_3 protein demonstrated at least some similarity to sequences identified as M34651 (ORF-3 protein [Suid herpesvirus 1]), U15306 (NFXl [Homo sapiens]), and Z81103 (M04G12.1 [Caenorhabditis elegans]). Based upon sequence similarity, dn740_3 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts two potential transmembrane domains within the dn740_3 protein sequence, centerd around amino acids 110 and 180 of SEQ ID NO:13, respectively.
  • the nucleotide sequence of dn740_3 indicates that it may contain a simple AT repeat sequence.
  • dn904_2 A polynucleotide of the present invention has been identified as clone "dn904_2".
  • dn904_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • dn904_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dn904_2 protein").
  • nucleotide sequence of dn904_2 as presently determined is reported in SEQ ID NO:14. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dn904_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:15.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone dn904_2 should be approximately 2700 bp.
  • the nucleotide sequence disclosed herein for dn904_2 was searched against the
  • dn904_2 demonstrated at least some similarity with sequences identified as N66026 (za28g05.sl Homo sapiens cDNA clone 2939123' similar to contains Alu repetitive element;contains element MER6 repetitive element) and U67221 (Human clone HS4.14 Alu-Ya5 sequence).
  • the predicted amino acid sequence disclosed herein for dn904_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted dn904_2 protein demonstrated at least some similarity to sequences identified as U79260 (unknown [Homo sapiens]).
  • dn904_2 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a potential transmembrane domain within the dn904_2 protein sequence centered around amino acid 15 of SEQ ID NO:15.
  • the nucleotide sequence of dn904_2 indicates that it may contain an Alu repetitive element.
  • a polynucleotide of the present invention has been identified as clone "do568_ll".
  • do568_ll was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • do568_ll is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "do568_ll protein").
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone do568_ll should be approximately 2300 bp.
  • the nucleotide sequence disclosed herein for do568_ll was searched against the
  • do568_ll demonstrated at least some similarity with sequences identified as AA399248 (zt57d07.sl Soares testis NHT Homo sapiens cDNA clone 726445 3'), AA552222 (nk06a07.sl NCI_CGAP_Co2 Homo sapiens cDNA clone AGE:1012692), H41337 (yn91d06.rl Homo sapiens cDNA clone), H56978 (yr07a01.rl Homo sapiens cDNA clone 204552 5'), J05096 (Human Na,K-ATPase subunit alpha 2 (ATP1A2) gene, complete cds), N95160 (zb52c09.sl Soares fetal lung NbHL19W Homo sapiens cDNA clone 307216 3'similar to contains
  • do568_ll proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts two potential transmembrane domains within the do568_ll protein sequence, one at the amino terminus and another centered around amino acid 230 of SEQ ID NO:17.
  • ek626_3 A polynucleotide of the present invention has been identified as clone "ek626_3".
  • ek626_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • ek626_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ek626_3 protein").
  • the nucleotide sequence of ek626_3 as presently determined is reported in SEQ ID NO: A polynucleotide sequence of ek626_3 as presently determined is reported in SEQ ID NO: ek626_3 protein
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone ek626_3 should be approximately 1900 bp.
  • ek626_3 The nucleotide sequence disclosed herein for ek626_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ek626_3 demonstrated at least some similarity with sequences identified as AAl 12543 (zm28al2.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526942 5'), AAl 60534 (zo73f06.sl Stratagene pancreas (#937208) Homo sapiens cDNA clone 592547 3'), AA160629 (zo73f06.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 5925475'), AA168779 (ms37g07.rl Stratagene mouse heart (#937316) Mus musculus cDNA clone 613788 5'), AA211632
  • the predicted amino acid sequence disclosed herein for dn904_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted dn904_2 protein demonstrated at least some similarity to sequences identified as R99052 (Spider dragline variant, DP-1A.9 monomer) and Z97342 (nuclear antigen homolog [Arabidopsis thaliana]). Based upon sequence similarity, ek626_3 proteins and each similar protein or peptide may share at least some activity.
  • fe366_l A polynucleotide of the present invention has been identified as clone "fe366_l”.
  • fe366_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • fe366_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fe366_l protein").
  • nucleotide sequence of fe366_l as presently determined is reported in SEQ ID NO:20. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the fe366_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:21. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone fe366_l should be approximately 3100 bp.
  • nucleotide sequence disclosed herein for fe366_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
  • fe366_l demonstrated at least some similarity with sequences identified as AA139623 (mq40b07.rl Barstead MPLRB1 Mus musculus cDNA clone 581173 5' similar to WP:F43E2.7 CE07243), AA306766 (EST177699 Jurkat T-cells VI Homo sapiens cDNA 5' end), AA663899 (ae74d05.sl Stratagene schizo brain Sll Homo sapiens cDNA clone 969897 3'), H29956 (yp44b03.rl Homo sapiens cDNA clone 190253 5'), H93431
  • fe366_l proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of fe366_l indicates that it may contain one or more of the following: CAA repeat, Alu repetitive element.
  • Clones bdl64_7, bil29_2, bk95_3, cgl60_6, cw775_l, dn740_3, dn904_2, do568_l 1, ek626_3, and fe366_l were deposited on March 19, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98364, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. ⁇ 1.808(b).
  • Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/Notl digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1.
  • the pED6dpc2 vector (“pED6" was derived from pED ⁇ dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et ah, 1991, Nucleic Acids Res.
  • the pNOTs vector was derived from pMT2 (Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site.
  • the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate.
  • the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, Notl will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector.
  • the cDNA may also be expressed from the vectors in which they were deposited.
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
  • oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
  • the design of the oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
  • the oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • 6X SSC 20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio /Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • a bivalent form of the protein such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a protein - IgM fusion would generate a decavalent form of the protein of the invention.
  • the present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone.
  • the mature form(s) of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • the sequence(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
  • Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
  • the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997,
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein).
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
  • Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue” is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide.
  • polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustek ⁇ ison, Canis familiar is, Oryctolagus cuniculus, Bos taunts, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species
  • allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
  • allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybrid length is that anticipated for the hybridized reg ⁇ on(s) of the hybridizing polynucleotides When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleoUdes and idenhfying the region or regions of optimal sequence complementarity
  • SSPE (lxSSPE is 0 ⁇ .5M NaCl, lOmM NaH 2 P0 4 , and 1 25mM EDTA, pH 74) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
  • T m melting temperature
  • each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide /expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
  • Ovary (CHO) cells human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA im
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cytokine
  • cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al, J. Immunol.
  • Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SOD severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
  • tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of 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.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
  • blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lprfipr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
  • an antigen function preferably a B lymphocyte antigen function
  • Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.
  • enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection.
  • systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells ⁇ e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject.
  • the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation /chemotherapy to stimulate the production of erythroid precursors and /or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
  • a protein of the invention may also exhibit angiogenic activity.
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
  • a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • FSH follicle stimulating hormone
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al, Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
  • the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule.
  • the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes.
  • Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by retiirning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth.
  • reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
  • Fragments of proteins of the present invention with cadherin activity can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No.4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.
  • the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question.
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • the addition of other known growth factors, such as IGF I (insulin like growth factor I) may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • GGGAGAACAT CCGTTACCGG ATCTGCGTGG GGCTGGTGGT GGTTGGTGTC CTGCTCATCA 300
  • Leu Leu lie Leu lie Val Leu Leu Val Val Phe Leu Pro Gin Ser 85 90 95
  • GCCCACATCA GCTGCTCACT CAACCTCGAA AGACTGAAAA TGGGGCCACC TGTCTTCCCA 840
  • CTCTATGAAC CAAAGAAGGC TGGGCCCCAG AGAGGTGGGG GGCCAGGGAG CAGGCAACAC 780
  • ATTTACTGGA TATAATATGA TTATCTCTGT AGGGAGTTGA TTTCCATCTC CTCAATTACT 600
  • AATAAAGTTA AAAATCTTTT CATATGTTTTTT ATTGCCATTT TTATTTCTTC TGTAAAGTAC 660
  • CTACTCATGG CTTTTTCTCA TTTTTTGTTT GTCATCATTG AATTATAGGA GTTTTGAGAG 720
  • AAATGTTCTT CAAAAGAGTT CCTGCAAACG TTTTTGTTTT TATTTCCTAC TGTTCCCTTC 1620
  • ATATTGTTCA TCTTTGGTAG CATTAAACAA TGAATACAGT GTTTTTTACT TAATAGATAT 1860
  • CAGTTTCACA TATGTTTTTG CATCACTGTC TCTTTTTTTC TTGAGCTTAT TCCAGAGTGT 2040
  • ACCCAAGCAC AATATACTGA TTTGCACCTC TGCCTTTGTT CATGCCCCTT GTTCAGGAGA 2280 ACTGCTTTCA TGTGCTACTG TCCATAGATC TTCTCTATCC TTACAGATTA ATTTCTTCCT 2340
  • ACCTGTTCCC CAGGACTCAC CCCAGCCCCT GCCTGCCCCT GAGGAAGAAG AGGCACTCAC 1200
  • TAGTCTGTGT AACCTTCACT GCATCCTTGC CCCATTCAGC CCGGCCTTTC ATGATGCAGG 2040
  • MOLECULE TYPE protein
  • ACTTTGTGAT TCCATAATAA CCATCTATCG GGAAGAGGGC ATTCTAGGAT TTTTCGCGGG 540 TCTTGTTCCT CGCCTTCTAG GTGACATCCT TTCTTTGTGG CTGTGTAACT CACTGGCCTA 600
  • CAAAGTAATA AAATGTCACC TACAGGGAGC CTCTGAGCCT ACTCTAGTTC AAGAGGCTAC 1560

Abstract

Polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,XXX
(converted to a provisional application from non-provisional application 08/820,493), filed March 19, 1997, which is incorporated by reference herein.
FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE P VENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed. SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 463 to nucleotide 606;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 501; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364; (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 19 to amino acid 28 of SEQ ID NO:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 463 to nucleotide 606; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 501; the nucleotide sequence of the full-length protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:l.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 19 to amino acid 28 of SEQ ID NO:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 202 to nucleotide 849;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 511 to nucleotide 849;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364; (g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 103 to amino acid 112 of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:3 from nucleotide 202 to nucleotide 849; the nucleotide sequence of SEQ ID NO:3 from nucleotide 511 to nucleotide 849; the nucleotide sequence of the full-length protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209; (c) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 103 to amino acid 112 of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 51 to nucleotide 356; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 348 to nucleotide 356;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bk95_3 deposited under accession number ATCC 98364; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bk95_3 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bk95_3 deposited under accession number ATCC 98364; (g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone bk95_3 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 46 to amino acid 55 of SEQ ID NO:6;
(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:5 from nucleotide 51 to nucleotide 356; the nucleotide sequence of SEQ ID NO:5 from nucleotide 348 to nucleotide 356; the nucleotide sequence of the full-length protein coding sequence of clone bk95_3 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone bk95_3 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone bk95_3 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 2 to amino acid 102. 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 comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 2 to amino acid 102;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising the amino acid sequence from amino acid 46 to amino acid 55 of SEQ ID NO:6; and (d) the amino acid sequence encoded by the cDNA insert of clone bk95_3 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 2 to amino acid 102. 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 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 156 to nucleotide 902;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 225 to nucleotide 902; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:8 from nucleotide 237 to nucleotide 654;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364; (g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364; (h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:9;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:9 having biological activity, the fragment comprising the amino acid sequence from amino acid 119 to amino acid 128 of SEQ ID NO:9;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:8 from nucleotide 156 to nucleotide 902; the nucleotide sequence of SEQ ID NO:8 from nucleotide 225 to nucleotide 902; the nucleotide sequence of SEQ ID NO: 8 from nucleotide 237 to nucleotide 654; the nucleotide sequence of the full-length protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:9 from amino acid 28 to amino acid 166.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:8.
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:9;
(b) the amino acid sequence of SEQ ID NO:9 from amino acid 28 to amino acid 166; (c) fragments of the amino acid sequence of SEQ ID NO:9 comprising the amino acid sequence from amino acid 119 to amino acid 128 of SEQ ID NO:9; and
(d) the amino acid sequence encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:9 or the amino acid sequence of SEQ ID NO:9 from amino acid 28 to amino acid 166.
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:10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 10 from nucleotide 400 to nucleotide 2454;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:10 from nucleotide 1454 to nucleotide 1787;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cw775_l deposited under accession number ATCC 98364; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cw775_l deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:ll; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:ll having biological activity, the fragment comprising the amino acid sequence from amino acid 337 to amino acid 346 of SEQ ID NO:ll;
(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:10 from nucleotide 400 to nucleotide 2454; the nucleotide sequence of SEQ ID NO:10 from nucleotide 1454 to nucleotide 1787; the nucleotide sequence of the full-length protein coding sequence of clone cw775_l deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone cw775_l deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:10. 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:ll; (b) fragments of the amino acid sequence of SEQ ID NO: 11 comprising the amino acid sequence from amino acid 337 to amino acid 346 of SEQ ID NO:ll; and
(c) the amino acid sequence encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:ll.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 506 to nucleotide 1096;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 656 to nucleotide 1096;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 2 to nucleotide 1078;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13 having biological activity, the fragment comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:13; (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:12 from nucleotide 506 to nucleotide 1096; the nucleotide sequence of SEQ ID NO:12 from nucleotide 656 to nucleotide 1096; the nucleotide sequence of SEQ ID NO: 12 from nucleotide 2 to nucleotide 1078; the nucleotide sequence of the full-length protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13 from amino acid 1 to amino acid 191.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:12.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 13; (b) the amino acid sequence of SEQ ID NO:13 from amino acid 1 to amino acid 191;
(c) fragments of the amino acid sequence of SEQ ID NO:13 comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:13; and (d) the amino acid sequence encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:13 or the amino acid sequence of SEQ ID NO:13 from amino acid 1 to amino acid 191. 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:14; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:14 from nucleotide 1563 to nucleotide 1685;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:14 from nucleotide 1100 to nucleotide 1646;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dn904_2 deposited under accession number
ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:15;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:15 having biological activity, the fragment comprising the amino acid sequence from amino acid 15 to amino acid 24 of SEQ ID NO:15; (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:14 from nucleotide 1563 to nucleotide 1685; the nucleotide sequence of SEQ ID NO:14 from nucleotide 1100 to nucleotide 1646; the nucleotide sequence of the full-length protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 15 from amino acid 1 to amino acid 28.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:14. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:15;
(b) the amino acid sequence of SEQ ID NO:15 from amino acid 1 to amino acid 28;
(c) fragments of the amino acid sequence of SEQ ID NO:15 comprising the amino acid sequence from amino acid 15 to amino acid 24 of SEQ ID NO: 15; and
(d) the amino acid sequence encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:15 or the amino acid sequence of SEQ ID NO:15 from amino acid 1 to amino acid 28.
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:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16 from nucleotide 359 to nucleotide 1369; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:16 from nucleotide 1547 to nucleotide 1868;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone do568_ll deposited under accession number ATCC 98364; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone do568_ll deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:17; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 17 having biological activity, the fragment comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:17;
(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:16 from nucleotide 359 to nucleotide 1369; the nucleotide sequence of SEQ ID NO:16 from nucleotide 1547 to nucleotide 1868; the nucleotide sequence of the full-length protein coding sequence of clone do568_ll deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone do568_ll deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:16. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 17; (b) fragments of the amino acid sequence of SEQ ID NO:17 comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:17; and
(c) the amino acid sequence encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:17.
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 85 to nucleotide 1263;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:18 from nucleotide 265 to nucleotide 608;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364;
(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, the fragment comprising the amino acid sequence from amino acid 191 to amino acid 200 of
SEQ ID NO:19;
(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 85 to nucleotide 1263; the nucleotide sequence of SEQ ID NO:18 from nucleotide 265 to nucleotide 608; the nucleotide sequence of the full-length protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 19 from amino acid 61 to amino acid 175.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:18.
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) the amino acid sequence of SEQ ID NO:19 from amino acid 61 to amino acid 175;
(c) fragments of the amino acid sequence of SEQ ID NO:19 comprising the amino acid sequence from amino acid 191 to amino acid 200 of SEQ ID NO:19; and
(d) the amino acid sequence encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 19 or the amino acid sequence of SEQ ID NO:19 from amino acid 61 to amino acid 175.
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:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20 from nucleotide 3746 to nucleotide 4027; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:20 from nucleotide 3815 to nucleotide 4027;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20 from nucleotide 3640 to nucleotide 3940;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fe366_l deposited under accession number ATCC 98364;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fe366_l deposited under accession number
ATCC 98364;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:21;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:21 having biological activity, the fragment comprising the amino acid sequence from amino acid 42 to amino acid 51 of SEQ ID NO:21; (k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:20 from nucleotide 3746 to nucleotide 4027; the nucleotide sequence of SEQ ID NO:20 from nucleotide 3815 to nucleotide 4027; the nucleotide sequence of SEQ ID NO:20 from nucleotide 3640 to nucleotide 3940; the nucleotide sequence of the full-length protein coding sequence of clone fe366_l deposited under accession number ATCC 98364; or the nucleotide sequence of a mature protein coding sequence of clone fe366_l deposited under accession number ATCC 98364. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:21 from amino acid 1 to amino acid 65.
Other embodiments provide the gene corresponding to the cDNA sequence of 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:21; (b) the amino acid sequence of SEQ ID NO:21 from amino acid 1 to amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID NO:21 comprising the amino acid sequence from amino acid 42 to amino acid 51 of SEQ ID NO:21; and (d) the amino acid sequence encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:21 or the amino acid sequence of SEQ ID NO:21 from amino acid 1 to amino acid 65. In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture. The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum. Clone "bd!64 7"
A polynucleotide of the present invention has been identified as clone "bdl64_7". bdl64_7 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bdl64_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bdl64_7 protein").
The nucleotide sequence of bdl64_7 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bdl64_7 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Another potential bdl64_7 reading frame and predicted amino acid sequence is encoded by basepairs 610 to 762 of SEQ ID NO:l and is reported in SEQ ID NO:32. The EcoRI /Notl restriction fragment obtainable from the deposit containing clone bdl64_7 should be approximately 1950 bp.
The nucleotide sequence disclosed herein for bdl64_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bdl64_7 demonstrated at least some similarity with sequences identified as AF001540 (Human clone alphal mRNA, partial sequence), C05823 (similar to none), G22994 (human STS WI-30658), H03651 (yj37el2.sl Homo sapiens cDNA clone 150958 3'), H26492 (EST51a22 Homo sapiens cDNA clone 51a22), H90721 (yv96f02.rl Homo sapiens cDNA clone 250587 5'), N58545 (yv73d07.sl Homo sapiens cDNA clone 2483653'), R10191 (yf35d07.rl Homo sapiens cDNA clone 1288455'), and X17272 (Human heterogenous nuclear RNA W16W). Based upon sequence similarity, bdl64_7 proteins and each similar protein or peptide may share at least some activity.
Clone "bi!29 2"
A polynucleotide of the present invention has been identified as clone "bil29_2". bil29_2 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bil29_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bil29_2 protein").
The nucleotide sequence of bil29_2 as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bil29_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Amino acids 91 to 103 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 104, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone bil29_2 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for bil29_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bil29_2 demonstrated at least some similarity with sequences identified as H88684 (yw23b01.rl Homo sapiens cDNA), R59623 (yh02g07.sl Homo sapiens cDNA clone 42126 3'), T17199 (NIB515 Homo sapiens cDNA 3'end), T24786 (Human gene signature HUMGS06869), T65550 (yc76bl2.sl Homo sapiens cDNA clone 21611 3'), and T65617 (yc76bl2.rl Homo sapiens cDNA clone 21611 5'). The predicted amino acid sequence disclosed herein for bil29_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bil29_2 protein demonstrated at least some similarity to sequences identified as AF016712 (testicular condensing enzyme [Mus musculus]) and U43375 (Similar to sugar transporter (Caenorhabditis elegans cosmid K09C4)). Based upon sequence similarity, bil29_2 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts six potential transmembrane domains within the bil29_2 protein sequence, centered around amino acids 11, 36, 69, 100, 131, and
185 of SEQ ID NO:4, respectively.
Clone "bk95 3"
A polynucleotide of the present invention has been identified as clone "bk95_3". bk95_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bk95_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bk95_3 protein").
The nucleotide sequence of the 5' portion of bk95_3 as presently determined is reported in SEQ ID NO:5. What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO:6. The predicted amino acid sequence of the bk95_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino acids 87 to 99 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 100, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of bk95_3/ including the polyA tail, is reported in SEQ ID NO:7.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone bk95_3 should be approximately 2400 bp.
The nucleotide sequence disclosed herein for bk95_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bk95_3 demonstrated at least some similarity with sequences identified as AA521036 (aa71b06.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:826355 3' similar to SW:SYB2_XENLA P47193 SYNAPTOBREVIN 2), N29686 (yw78a05.sl Homo sapiens cDNA clone 258320 3' similar to SP:SW:SYB2_XENLA P47193 SYNAPTOBREVIN 2), T33715 (Cellubrevin-2 coding sequence), U14567 (***ALU WARNING Human Alu-J subfamily consensus sequence), and U60150 (Mus musculus vesicle-associated membrane protein VAMP-2 mRNA, complete cds). The predicted amino acid sequence disclosed herein for bk95_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bk95_3 protein demonstrated at least some similarity to sequences identified as L14270 (synaptobrevin [Drosophila melanogaster]), M36205 (synaptobrevin 2 (SYB2) [Homo sapiens]), U60961 (cellubrevin [Mus musculus]), U64520 (synaptobrevin-3 [Homo sapiens]), W04181 (Cellubrevin-2), and X76199 (synaptobrevin [Bos taurus]). Based upon sequence similarity, bk95_3 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of bk95_3 indicates that it may contain an Alu repetitive element.
Clone "cel60 6"
A polynucleotide of the present invention has been identified as clone "cgl60_6". cgl60_6 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cgl60_6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cgl60_6 protein").
The nucleotide sequence of cgl60_6 as presently determined is reported in SEQ ID NO:8. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cgl60_6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:9. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone cgl60_6 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for cgl60_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cgl60_6 demonstrated at least some similarity with sequences identified as AA405957 (zu66c07.rl Soares testis NHT Homo sapiens cDNA clone 742956 5') and T19219 (f02011t Testis 1 Homo sapiens cDNA clone f02011 5' end). Based upon sequence similarity, cgl60_6 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts three additional potential transmembrane domains within the cgl60_6 protein sequence, centerd around amino acids 148, 195, and 236 of SEQ ID NO:9, respectively.
Clone "cw775 1" A polynucleotide of the present invention has been identified as clone "cw775_l". cw775_l was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cw775_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as
"cw775_l protein").
The nucleotide sequence of cw775_l as presently determined is reported in SEQ ID NO: 10. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cw775_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:ll.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone cw775_l should be approximately 4200 bp. The nucleotide sequence disclosed herein for cw775_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cw775_l demonstrated at least some similarity with sequences identified as AA104324 (mo50d06.rl Life Tech mouse embryo 10 5dpc 10665016 Mus musculus cDNA clone 557003 5'), AA373350 (EST85423 HSC172 cells I Homo sapiens cDNA 5' end), H30439 (ym58fl0.rl Homo sapiens cDNA clone 52688 5'), N28734 (yx67cl0.rl Homo sapiens cDNA clone 2668025'), and N57005 (yy56h03.sl Homo sapiens cDNA clone 277589 3'). Based upon sequence similarity, cw775_l proteins and each similar protein or peptide may share at least some activity.
Clone "dn740 3"
A polynucleotide of the present invention has been identified as clone "dn740_3". dn740_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. dn740_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dn740_3 protein").
The nucleotide sequence of dn740_3 as presently determined is reported in SEQ ID NO:12. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dn740_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:13. Amino acids 38 to 50 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 51, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone dn740_3 should be approximately 1650 bp.
The nucleotide sequence disclosed herein for dn740_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. dn740_3 demonstrated at least some similarity with sequences identified as AA053844 (zf53h07.rl Soares retina N2b4HR Homo sapiens cDNA clone 380701 5'), AA056525 (zl65g08.rl Stratagene colon (#937204) Homo sapiens cDNA clone 5095345'), H70470 (yr91c07.sl Homo sapiens cDNA clone 2126523'), N53038 (yv53d09.sl Homo sapiens cDNA clone 246449 3'), R56318 (yg90e03.rl Homo sapiens cDNA clone 40653 5'), and W73718 (zd50f06.sl Soares fetal heart NbHH19W Homo sapiens cDNA clone 344099 3'). The predicted amino acid sequence disclosed herein for dn740_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted dn740_3 protein demonstrated at least some similarity to sequences identified as M34651 (ORF-3 protein [Suid herpesvirus 1]), U15306 (NFXl [Homo sapiens]), and Z81103 (M04G12.1 [Caenorhabditis elegans]). Based upon sequence similarity, dn740_3 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts two potential transmembrane domains within the dn740_3 protein sequence, centerd around amino acids 110 and 180 of SEQ ID NO:13, respectively. The nucleotide sequence of dn740_3 indicates that it may contain a simple AT repeat sequence.
Clone "dn904 2"
A polynucleotide of the present invention has been identified as clone "dn904_2". dn904_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. dn904_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dn904_2 protein").
The nucleotide sequence of dn904_2 as presently determined is reported in SEQ ID NO:14. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dn904_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:15.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone dn904_2 should be approximately 2700 bp. The nucleotide sequence disclosed herein for dn904_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. dn904_2 demonstrated at least some similarity with sequences identified as N66026 (za28g05.sl Homo sapiens cDNA clone 2939123' similar to contains Alu repetitive element;contains element MER6 repetitive element) and U67221 (Human clone HS4.14 Alu-Ya5 sequence). The predicted amino acid sequence disclosed herein for dn904_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted dn904_2 protein demonstrated at least some similarity to sequences identified as U79260 (unknown [Homo sapiens]). Based upon sequence similarity, dn904_2 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the dn904_2 protein sequence centered around amino acid 15 of SEQ ID NO:15. The nucleotide sequence of dn904_2 indicates that it may contain an Alu repetitive element.
Clone "do568 11"
A polynucleotide of the present invention has been identified as clone "do568_ll". do568_ll was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. do568_ll is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "do568_ll protein").
The nucleotide sequence of do568_ll as presently determined is reported in SEQ ID NO:16. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the do568_ll protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:17.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone do568_ll should be approximately 2300 bp. The nucleotide sequence disclosed herein for do568_ll was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. do568_ll demonstrated at least some similarity with sequences identified as AA399248 (zt57d07.sl Soares testis NHT Homo sapiens cDNA clone 726445 3'), AA552222 (nk06a07.sl NCI_CGAP_Co2 Homo sapiens cDNA clone AGE:1012692), H41337 (yn91d06.rl Homo sapiens cDNA clone), H56978 (yr07a01.rl Homo sapiens cDNA clone 204552 5'), J05096 (Human Na,K-ATPase subunit alpha 2 (ATP1A2) gene, complete cds), N95160 (zb52c09.sl Soares fetal lung NbHL19W Homo sapiens cDNA clone 307216 3'similar to contains element MER22 repetitive element), R42239 (yf98al0.sl Homo sapiens cDNA clone 30435 3'), T15786 (IB1892 Infant brain, Bento Soares Homo sapiens cDNA 3'end), and T20399 (Human gene signature HUMGS01552). Based upon sequence similarity, do568_ll proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts two potential transmembrane domains within the do568_ll protein sequence, one at the amino terminus and another centered around amino acid 230 of SEQ ID NO:17.
Clone "ek626 3"
A polynucleotide of the present invention has been identified as clone "ek626_3". ek626_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. ek626_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ek626_3 protein"). The nucleotide sequence of ek626_3 as presently determined is reported in SEQ
ID NO:18. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ek626_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:19.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone ek626_3 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for ek626_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ek626_3 demonstrated at least some similarity with sequences identified as AAl 12543 (zm28al2.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526942 5'), AAl 60534 (zo73f06.sl Stratagene pancreas (#937208) Homo sapiens cDNA clone 592547 3'), AA160629 (zo73f06.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 5925475'), AA168779 (ms37g07.rl Stratagene mouse heart (#937316) Mus musculus cDNA clone 613788 5'), AA211632 (zn56b09.rl Stratagene muscle 937209 Homo sapiens cDNA clone 562169 5'), AA224303 (zrl5el0.rl Stratagene NT2 neuronal precursor 937230 Homo sapiens cDNA clone 663498 5'), AA429442 (zw47b06.rl Soares total fetus Nb2HF8 9w Homo sapiens cDNA clone 773171 5'), H22161 (yl38g02.sl Homo sapiens cDNA clone), T52832 (Human gene signature HUMGS08061), U21718 (Rattus norvegicus clone C426 intestinal epithelium proliferating cell-associated mRNA sequence), and W26019 (18b9 Human retina cDNA randomly primed sublibrary Homo sapiens cDNA). The predicted amino acid sequence disclosed herein for dn904_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted dn904_2 protein demonstrated at least some similarity to sequences identified as R99052 (Spider dragline variant, DP-1A.9 monomer) and Z97342 (nuclear antigen homolog [Arabidopsis thaliana]). Based upon sequence similarity, ek626_3 proteins and each similar protein or peptide may share at least some activity.
Clone "fe366 1"
A polynucleotide of the present invention has been identified as clone "fe366_l". fe366_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. fe366_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fe366_l protein").
The nucleotide sequence of fe366_l as presently determined is reported in SEQ ID NO:20. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the fe366_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:21. Amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone fe366_l should be approximately 3100 bp.
The nucleotide sequence disclosed herein for fe366_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fe366_l demonstrated at least some similarity with sequences identified as AA139623 (mq40b07.rl Barstead MPLRB1 Mus musculus cDNA clone 581173 5' similar to WP:F43E2.7 CE07243), AA306766 (EST177699 Jurkat T-cells VI Homo sapiens cDNA 5' end), AA663899 (ae74d05.sl Stratagene schizo brain Sll Homo sapiens cDNA clone 969897 3'), H29956 (yp44b03.rl Homo sapiens cDNA clone 190253 5'), H93431
(ys76dl0.rl Homo sapiens cDNA clone 2207235'), and M61937 (R.norvegicus dihydrodiol dehydrogenase mRNA, complete cds). Based upon sequence similarity, fe366_l proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of fe366_l indicates that it may contain one or more of the following: CAA repeat, Alu repetitive element.
Deposit of Clones Clones bdl64_7, bil29_2, bk95_3, cgl60_6, cw775_l, dn740_3, dn904_2, do568_l 1, ek626_3, and fe366_l were deposited on March 19, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98364, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. § 1.808(b).
Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/Notl digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from pEDόdpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et ah, 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, Notl will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest. Clone Probe Sequence bdl64_7 SEQ ID NO:22 bil29_2 SEQ ID NO:23 bk95_3 SEQ ID NO:24 cgl60_6 SEQ ID NO:25 cw775_l SEQ ID NO:26 dn740_3 SEQ ID NO:27 dn904_2 SEQ ID NO:28 do568_ll SEQ ID NO:29 ek626_3 SEQ ID NO:30 fe366_l SEQ ID NO:31
In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1- dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio /Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention. The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form(s) of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997,
Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1- 39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s) .
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. Preferably, species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustek υison, Canis familiar is, Oryctolagus cuniculus, Bos taunts, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al, 1993, Nature Genetics 3:103-112; Johansson et al, 1995, Genomics 25: 682-690; Lyons et al, 1997, Nature Genetics 15: 47-56; O'Brien et al, 1997, Trends in Genetics 13(10): 393-399; Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein). The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein. The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
Figure imgf000038_0001
* The hybrid length is that anticipated for the hybridized regιon(s) of the hybridizing polynucleotides When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleoUdes and idenhfying the region or regions of optimal sequence complementarity
* SSPE (lxSSPE is 0 ι.5M NaCl, lOmM NaH2P04, and 1 25mM EDTA, pH 74) can be substituted for SSC (lxSSC is 0 15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
*TB - TR The hybndizahon temperature for hybrids anhαpated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G + C bases) For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81 5 + 16 6(log,0[Na+]) + 041(%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC = 0 165 M) Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide /expression control sequence.
A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention. USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al, J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent. Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptoπligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lprfipr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells {e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β2 microglobulin protein or an MHC class II a chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al, J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology
154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation /chemotherapy to stimulate the production of erythroid precursors and /or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes. Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity. A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin- β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al, Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994. Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke). The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
Recepτor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities. The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins. E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by retiirning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and /or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No.4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention. When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Merberg, David Treacy, Maurice Spaulding, Vikki Agostino, Michael
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1800 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
TTTTTTTTTT TACAGACTTC ACAGAGAATG CAGTTGTCTT GACTTCAGGT CTGTCTGTTC 60
TGTTGGCAAG TAAATGCAGT ACTGTTCTGA TCCCGCTGCT ATTAGAATGC ATTGTGAAAC 120
GACTGGAGTA TGATTAAAAG TTGTGTTCCC CAATGCTTGG AGTAGTGATT GTTGAAGGAA 180
AAAATCCAGC TGAGTGATAA AGGCTGAGTG TTGAGGAAAT TTCTGCAGTT TTAAGCAGTC 240
GTATTTGTGA TTGAAGCTGA GTACATTTTG CTGGTGTATT TTTAGGTAAA ATGCTTTTTG 300
TTCATTTCTG GTGGTGGGAG GGGACTGAAG CCTTTAGTCT TTTCCAGATG CAACCTTAAA 360
ATCAGTGACA AGAAACATTC CAAACAAGCA ACAGTCTTCA AGAAATTAAA CTGGCAAGTG 420
GAAATGTTTA AACAGTTCAG TGATCTTTAG TGCATTGTTT ATGTGTGGGT TTCTCTCTCC 480
CCTCCCTTGG TCTTAATTCT TACATGCAGG AACACTCAGC AGACACACGT ATGCGAAGGG 540
CCAGAGAAGC CAGACCCAGT AAGAAAAAAT AGCCTATTTA CTTTAAATAA ACCAAACATT 600
CCATTTTAAA TGTGGGGATT GGGAACCACT AGTTCTTTCA GATGGTATTC TTCAGACTAT 660
AGAAGGAGCT TCCAGTTGAA TTCACCAGTG GACAAAATGA GGAAAACAGG TGAACAAGCT 720
TTTTCTGTAT TTACATACAA AGTCAGATCA GTTATGGGAC AATAGTATTG AATAGATTTC 780
AGCTTTATGC TGGAGTAACT GGCATGTGAG CAAACTGTGT TGGCGTGGGG GTGGAGGGGT 840
GAGGTGGGCG CTAAGCTTTT TTTAAGATTT TTCAGGTACC CTTCACTAAA GGCACCGAAG 900
GCTTAAAGTA GGACAACCAT GGAGCTTCCT GTGGCAGGAG AGACAACAAA GCGCTATTAT 960
CCTAAGGTCA AGAGAAGTGT CAGCCTCACC TGATTTTTAT TAGTAATGAG GACTTGCCTC 1020
AACTCCCTCT TTCTGGAGTG AAGCATCCGA AGGAATGCTT GAAGTACCCC TGGGCTTCTC 1080
TTAACATTTA AGCAAGCTGT TTTTATAGCA GCTCTTAATA ATAAAGCCCA AATCTCAAGC 1140
GGTGCTTGAA GGGGAGGGAA AGGGGGAAAG CGGGCAACCA CTTTTCCCTA GCTTTTCCAG 1200
AAGCCTGTTA AAAGCAAGGT CTCCCCACAA GCAACTTCTC TGCCACATCG CCACCCCGTG 1260
CCTTTTGATC TAGCACAGAC CCTTCACCCC TCACCTCGAT GCAGCCAGTA GCTTGGATCC 1320
TTGTGGGCAT GATCCATAAT CGGTTTCAAG GTAACGATGG TGTCGAGKTC TTTGGTGGGT 1380
TGAACTATGT TAGAAAAGGC CATTAATTTG CCTGCAAATT GTTAACAGAA GGGTATTAAA 1440 ACCACAGCTA AGTAGCTCTA TTATAATACT TATCCAGTGA CTAAAACCAA CTTAAACCAG 1500
TAAGTGGAGA AATAACATGT TCAAGAACTG TAATGCTGGG TGGGAACATG TAACTTGTAG 1560
ACTGGAGAAG A AGGCATTT GAGTGGCTGA GAGGGCTTTT GGGTGGGAAT GCAAAAATTC 1620
TCTGCTAAGA CTTTTTCAGG TGAACATAAC AGACTTGGCC AAGCTAGCAT CTTAGCGGAA 1680
GCTGATCTCC AATGCTCTTC AGTAGGGTCA TGAAGGTTTT TCTTTTCCTG AGAAAACAAC 1740
ACGTATTGTT TTCTCAGGTT TTGCTTTTTG GCCTTTTTCT AGCTTAAAAA AAAAAAAAAA 1800
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Val Trp Val Ser Leu Ser Pro Pro Leu Val Leu lie Leu Thr Cys Arg
1 5 10 15
Asn Thr Gin Gin Thr His Val Cys Glu Gly Pro Glu Lys Pro Asp Pro
20 25 30
Val Arg Lys Asn Ser Leu Phe Thr Leu Asn Lys Pro Asn lie Pro Phe
35 40 45
( 2 ) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1063 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : AAAGTTCCAT CTCTAGAACT GATTTTTATC CGTTCTGTTT TTCAGGTCTT ATCTGTGTTA 60 GTTGTGTGTT ACTATCAGGA GGCCCCCTTT GGACCCAGTG GATACAGATT ACGACTCTTC 120
TTTTATGGTG TATGCAATGT CATTTCTATC ACTTGTGCTT ATACATCATT TTCAATAGTT 180
CCTCCCAGCA ATGGGACCAC TATGTGGAGA GCCACAACTA CAGTCTTCAG TGCCATTTTG 240
GCTTTTTTAC TCGTAGATGA GAAAATGGCT TATGTTGACA TGGCTACAGT TGTTTGCAGC 300
ATCTTAGGTG TTTGTCTTGT CATGATCCCA AACATTGTTG ATGAAGACAA TTCTTTGTTA 360
AATGCCTGGA AAGAAGCCTT TGGGTACACC ATGACTGTGA TGGCTGGACT GACCACTGCT 420
CTCTCAATGA TAGTATACAG ATCCATCAAG GAGAAGATCA GCATGTGGAC TGCACTGTTT 480
ACTTTTGGTT GGACTGGGAC AATTTGGGGA AT TCTACTA TGTTTATTCT TCAAGAACCC 540
ATCATCCCAT TAGATGGAGA AACCTGGAGT TATCTCATTG CTATATGTGT CTGTTCTACT 600
GCAGCATTCT TAGGAGTTTA TTATGCCTTG GACAAATTCC ATCCAGCTTT GGTTAGCACA 660
GTACAACATT TGGAGATTGT GGTAGCTATG GTCTTGCAGC TTCTCGTGCT GCACATATTT 720
CCTAGCATCT ATGATGTTTT TGGAGGGGTA ATCATTATGA TTAGTGTTTT TGTCCTTGCT 780
GGCTATAAAC TTTACTGGAG GAATTTAAGA AGGCAGGACT ACCAGGAAAT ATTAGACTCT 840
CCCATTAAAT GAATACCTGA TTATTATTGT CTCATTAATG TTCAGTTATT AATATGTATA 900
CTGCCATTTT AATGTTTACC TATGAATGTC TTTTGTGTTA TATAACTGAC AGAGTGCTAT 960
AAAATATATA ATATATACAA ATGCAGAAAA TTTATTCTAG TCTAATATAT TCAAATACAA 1020
ATATTAAATA TATGAAATAC GTTAAAAAAA AAAAAAAAAA AAA 1063 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Trp Arg Ala Thr Thr Thr Val Phe Ser Ala lie Leu Ala Phe Leu 1 5 10 15
Leu Val Asp Glu Lys Met Ala Tyr Val Asp Met Ala Thr Val Val Cys 20 25 30 Ser lie Leu Gly Val Cys Leu Val Met lie Pro Asn lie Val Asp Glu 35 40 45
Asp Asn Ser Leu Leu Asn Ala Trp Lys Glu Ala Phe Gly Tyr Thr Met 50 55 60
Thr Val Met Ala Gly Leu Thr Thr Ala Leu Ser Met lie Val Tyr Arg 65 70 75 80
Ser lie Lys Glu Lys lie Ser Met Trp Thr Ala Leu Phe Thr Phe Gly 85 90 95
Trp Thr Gly Thr lie Trp Gly lie Ser Thr Met Phe lie Leu Gin Glu 100 105 110
Pro lie lie Pro Leu Asp Gly Glu Thr Trp Ser Tyr Leu lie Ala lie 115 120 125
Cys Val Cys Ser Thr Ala Ala Phe Leu Gly Val Tyr Tyr Ala Leu Asp 130 135 140
Lys Phe His Pro Ala Leu Val Ser Thr Val Gin His Leu Glu lie Val 145 150 155 160
Val Ala Met Val Leu Gin Leu Leu Val Leu His lie Phe Pro Ser lie 165 170 175
Tyr Asp Val Phe Gly Gly Val lie lie Met lie Ser Val Phe Val Leu 180 185 190
Ala Gly Tyr Lys Leu Tyr Trp Arg Asn Leu Arg Arg Gin Asp Tyr Gin 195 200 205
Glu lie Leu Asp Ser Pro lie Lys 210 215
( 2 ) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 356 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : TGGCCAAAGA GGCCTAGCCG GGAGCGGGCG AGGCGGCGGC GGCAGCAGCG ATGGCAGGAA 60 TAGAGTTGGA GCGGTGCCAG CAGCAGGCGA ACGAGGTGAC GGAAATTATG CGTAACAACT 120 TCGGCAAGGT CCTGGAGCGT GGTGTGAAGC TGGCCGAACT GCAGCAGCGT TCAGACCAAC 180
TCCTGGATAT GAGCTCAACC TTCAACAAGA CTACACAGAA CCTGGCCCAG AAGAAGTGCT 240
GGGAGAACAT CCGTTACCGG ATCTGCGTGG GGCTGGTGGT GGTTGGTGTC CTGCTCATCA 300
TCCTGATTGT GCTGCTGGTC GTCTTTCTCC CTCAGAGCAG TGACAGCAGT AGTGCC 356 (2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Ala Gly lie Glu Leu Glu Arg Cys Gin Gin Gin Ala Asn Glu Val 1 5 10 15
Thr Glu lie Met Arg Asn Asn Phe Gly Lys Val Leu Glu Arg Gly Val 20 25 30
Lys Leu Ala Glu Leu Gin Gin Arg Ser Asp Gin Leu Leu Asp Met Ser 35 40 45
Ser Thr Phe Asn Lys Thr Thr Gin Asn Leu Ala Gin Lys Lys Cys Trp 50 55 60
Glu Asn lie Arg Tyr Arg lie Cys Val Gly Leu Val Val Val Gly Val 65 70 75 80
Leu Leu lie lie Leu lie Val Leu Leu Val Val Phe Leu Pro Gin Ser 85 90 95
Ser Asp Ser Ser Ser Ala 100
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 92
( 2 ) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1131 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
GGGCCTCAAC TTTGGCGTCG TGAGATTCTT GTGAGGCGTC TGCCTGGAAG CCGGCAGCAA 60
TTTTGCTTCT TTAAAGAGAA AAAGAAGGCT AGGGACTCAG ATTCCTGGAT TCTGAGATCC 120
AGACCAGCTC CTCCCAGACC TCTCCAGAAG AAGCCATGGG AACCCCTCGT ATCCAGCATT 180
TGCTGATCCT CCTGGTCCTA GGAGCCTCCC TCCTGACCTC GGGCCTAGAG CTGTATTGTC 240
AAAAGGGTCT GTCCATGACT GTGGAAGCAG ATCCAGCCAA TATGTTTAAC TGGACCACAG 300
AGGAAGTGGA GACTTGTGAC AAAGGGGCAC TTTGCCAGGA AACCATACTA ATAATTAAAG 360
CAGGGACTGA GACAGCCATT TTGGCCACGA AGGGCTGCAT CCCGGAAGGG GAGGAGGCCA 420
TAACAATTGT CCAGCACTCT TCACCTCCCG GCCTGATCGT GACCTCCTAC AGTAACTACT 480
GTGAGGATTC CTTCTGTAAT GACAAAGACA GCCTGTCTCA GTTTTGGGAG TTCAGTGAGA 540
CCACAGCTTC CACTGTGTCA ACAACCCTCC ATTGTCCAAC CTGTGTGGCT TTGGGGACCT 600
GTTTCAGTGC TCCTTCTCTT CCCTGTCCCA ATGGTACAAC TCGATGCTAT CAAGGAAAAC 660
TTGAGATCAC TGGAGGTGGC ATTGAGTCGT CTGTGGAGGT CAAAGGCTGT ACAGCCATGA 720
TTGGCTGCAG GCTGATGTCT GGAATCTTAG CAGTAGGACC CATGTTTGTG AGGGAAGCGT 780
GCCCACATCA GCTGCTCACT CAACCTCGAA AGACTGAAAA TGGGGCCACC TGTCTTCCCA 840
TTCCTGTTTG GGGGTTACAG CTACTGCTGC CATTGCTGCT GCCATCATTT ATTCACTTTT 900
CCTAAGAAGG CACTTCTGGG CCTGGGTCTG AGGACATCTT TTTTGACTGG GAGCCTTCTT 960
ACTGTTGAGG TTCAACAAGC TGAGGAGTAG ATGGGAATTT GAGGGAGAAT ACAGAGATAC 1020 TATGAACGTA TTTGACATTT TTAATACAAT TTCTGCTATA ATTTTTGTAT GCAGTAGGCG 1080 TTACTAATAA ACATTTCTGC TGTGAAAAAA AAAAAAAAAA AAAAAAAAAA A 1131
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met Gly Thr Pro Arg lie Gin His Leu Leu lie Leu Leu Val Leu Gly 1 5 10 15
Ala Ser Leu Leu Thr Ser Gly Leu Glu Leu Tyr Cys Gin Lys Gly Leu 20 25 30
Ser Met Thr Val Glu Ala Asp Pro Ala Asn Met Phe Asn Trp Thr Thr 35 40 45
Glu Glu Val Glu Thr Cys Asp Lys Gly Ala Leu Cys Gin Glu Thr lie 50 55 60
Leu lie lie Lys Ala Gly Thr Glu Thr Ala lie Leu Ala Thr Lys Gly 65 70 75 80
Cys lie Pro Glu Gly Glu Glu Ala He Thr He Val Gin His Ser Ser 85 90 95
Pro Pro Gly Leu He Val Thr Ser Tyr Ser Asn Tyr Cys Glu Asp Ser 100 105 110
Phe Cys Asn Asp Lys Asp Ser Leu Ser Gin Phe Trp Glu Phe Ser Glu 115 120 125
Thr Thr Ala Ser Thr Val Ser Thr Thr Leu His Cys Pro Thr Cys Val 130 135 140
Ala Leu Gly Thr Cys Phe Ser Ala Pro Ser Leu Pro Cys Pro Asn Gly 145 150 155 160
Thr Thr Arg Cys Tyr Gin Gly Lys Leu Glu He Thr Gly Gly Gly He 165 170 175
Glu Ser Ser Val Glu Val Lys Gly Cys Thr Ala Met He Gly Cys Arg 180 185 190 Leu Met Ser Gly He Leu Ala Val Gly Pro Met Phe Val Arg Glu Ala 195 200 205
Cys Pro His Gin Leu Leu Thr Gin Pro Arg Lys Thr Glu Asn Gly Ala 210 215 220
Thr Cys Leu Pro He Pro Val Trp Gly Leu Gin Leu Leu Leu Pro Leu 225 230 235 240
Leu Leu Pro Ser Phe He His Phe Ser 245
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3527 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCTCCGGGCC GGCTGCGGAG CGACTCCCCG CCGCCAAGTG GGCGGCGTGG CTGTCGGGAA 60
AGAAGGGCTG GGGCCTGCCG TTCTTCCTCC CGAGTATCCC CTCCAGCTGG ACGACCCCAC 120
GCTGCAGCAC GGGCTTCCGG CTTCTCTCCT CAGTGGCCAA TTCGAGGGCA CAGCGGGCTC 180
CGGAGGCGCG GCGGCAAGCC TATCCCGCCT CCCAACCACA GCCTCCAGCA CCCGAGAGAA 240
CGGCCGCCCA CAGCACACGT TCTCCGGACA GGAGGGCGAA GGCCCAAGAC CTGGAGAGAT 300
GGTCAGCTCT CAAAAAAGGC ACAAACAATT GAAGGATGGA TACCATGGCA TATGTTAAAA 360
GCGTGTTGAA AGGAAAATAA GAAAGCCAGG AATCTCAGGA TGAATCAGTC TAGATCGAGA 420
TCAGATGGTG GCAGTGAAGA AACCTTACCT CAAGACCATA ATCATCATGA AAATGAGAGA 480
AGATGGCAGC AAGAGCGTCT CCACAGAGAA GAGGCCTATT ATCAGTTTAT TAATGAACTC 540
AATGATGAAG ATTATCGGCT TATGAGAGAC CATAATCTTT TAGGCACCCC TGGAGAAATA 600
ACATCAGAAG AACTGCAACA GCGGTTAGAT GGCGTCAAGG AACAACTAGC ATCTCAGCCT 660
GACTTGAGAG ATGGAACGAA TTACAGAGAC TCAGAAGTCC CTAGAGAAAG TTCACATGAA 720
GATTCTCTTC TAGAATGGTT GAACACCTTT CGGCGCACAG GAAATGCAAC TCGAAGTGGA 780
CAAAATGGGA ACCAAACTTG GAGAGCTGTG AGTCGAACAA ACCCGAACAA TGGAGAGTTT 840 CGGTTTAGTT TGGAAATCCA CGTAAATCAT GAAAATAGAG GATTTGAAAT TCATGGAGAA 900
GATTATACAG ACATTCCACT TTCAGATAGT AACAGAGATC ATACTGCAAA TAGGCAACAA 960
AGGTCAACTA GTCCTGTGGC TAGGCGAACA AGAAGCCAAA CCTCAGTGAA TTTCAATGGT 1020
AGTAGTTCCA ACATTCCAAG GACTAGGCTT GCTTCAAGGG GGCAAAATCC AGCTGAAGGA 1080
TCTTTCTCAA CATTGGGAAG GTTAAGAAAT GGAATTGGGG GAGCAGCTGG CATTCCTCGA 1140
GCTAACGCTT CACGCACTAA TTTCAGTAGT CACACAAACC AATCAGGTGG TAGTGAACTC 1200
AGGCAAAGGG AGGGGCAACG GTTTGGAGCA GCACATGTTT GGGAAAATGG GGCTAGAAGT 1260
AATGTTACAG TGAGGAATAC AAACCAAAGA TTAGAGCCAA TAAGATTACG ATCTACTTCC 1320
AATAGTCGAA GCCGTTCACC AATTCAGAGA CAGAGTGGCA CTGTTTATCA TAATTCCCAA 1380
AGGGAAAGTA GACCAGTACA GCAAACCACT AGAAGATCTG TTAGGAGGAG AGGTAGAACT 1440
CGAGTCTTTT TAGAGCAAGA TAGAGAACGA GAACGCAGAG GTACTGCATA TACCCCATTC 1500
TCTAATTCAA GGCTTGTGTC AAGAATAACA GTAGAAGAAG GAGAAGAATC CAGCAGATCC 1560
TCAACTGCTG TACGACGACA TCCAACAATC ACACTGGACC TTCAAGTGAG AAGGATCCGT 1620
CCTGGAGAAA ATAGAGATCG GGATAGTATT GCAAATAGAA CTCGATCCAG AGTAGGGCTA 1680
GCAGAAAATA CAGTCACTAT TGAAAGCAAT AGTGGGGGCT TTCGCCGAAC CATTTCTCGT 1740
TTAGAGCGGT CAGGTATTCG AACCTATGTT AGTACCATAA CAGTTCCCCT TCGTAGGATT 1800
TCTGAGAATG AGCTTGTTGA GCCATCATCA GTGGCTCTTC GGTCAATTTT AAGGCAGATC 1860
ATGACTGGGT TTGGAGAACT GAGTTCTCTA ATGGAGGCCG ATTCTGAGTC AGAACTTCAA 1920
AGAAATGGCC AGCATTTACC AGACATGCAC TCAGAACTGA GTAACTTAGG TACAGATAAC 1980
AACAGGAGCC AGCACAGGGA AGGTTCCTCT CAAGACAGGC AGGCCCAAGG AGACAGCACT 2040
GAAATGCATG GTGAAAACGA GACCACCCAG CCTCATACTC GAAACAGTGA CAGTAGGGGT 2100
GGCAGGCAGT TGCGAAATCC AAACAATTTA GTTGAAACTG GAACACTACC CATTCTTCGC 2160
CTTGCTCACT TTTTTTTACT AAATGAAAGT GATGATGATG ATCGAATACG TGGTTTAACC 2220
AAAGAGCAGA TTGACAATCT TTCCACCAGG CACTATGAGC ATAACAGTAT TGATAGTGAA 2280
CTAGGTAAAA TCTGTAGTGT TTGTATTAGT GACTATGTAA CTGGAAACAA GCTCAGGCAA 2340
TTACCTTGCA TGCATGAATT TCACATTCAT TGTATTGACC GATGGCTCTC AGAGAATTGC 2400
ACTTGTCCGA TCTGTCGGCA GCCTGTTTTA GGGTCTAACA TAGCAAACAA TGGGTAAGGT 2460
GATGGGATCT ACTCAAATAC TGTTTTTTAG TAGAACTGAA TGTTCAAGCA TTGTTTTGCT 2520 GAGTTATTTG TGATTAGCTA ACCAGGATGA AAAATAACAG ATTATATATA GTTTGAACTA 2580
TTTTTCGTGT GCTTTTTTAA ACTTGTTAAA AAGAAATTTA TATAAAATTT AAAATACAAA 2640
TGTTAAATTA TCCAGAAATA CAGAATAGTT AATATTGCTA GAACCAAATA ACCTCTAAAA 2700
TGTTTTTATT TTGGTAATTT TGTCATGCTA AGCACTTTTG TATCTGCACA ATTCAGTAGG 2760
TTAAGAATCA ATCTTCTTTT TCTTAATAGT ACAGCAGACT TTAGCTTCAA GTTTCATAGG 2820
CTTAGTACTT ATATCTAGAC ATTTGTGTCT AAATAAGCTT TTCATTAACT TTTTATTTTA 2880
AGGACAGTAT CTTTTCATGA AAGAGTATTT GGCTGAATGT TTGCTATATA TATGTTACTT 2940
GAAATGTTAA ATTTAATATG CAGCATACCA TAGGTGTATA TATAGGTATA TAATTTTAAG 3000
GTTAAAATAT TCAGTCTAAC AAGTTTGGTT CTTATTTAAG CTTTTGGGCT AATACTGCAT 3060
ATGGCACAAT GTTTAATATT GGCAAGTTCA TCTCAGAGAA AGGGGATTCA GATATAATTT 3120
TAAAGTAGAG ATAATTTACT GAAGCGTCTC TGACAATCTA ACTTATTAGA CAGCAAGCAA 3180
TATATAATAC TGAAAAAGTA TTCAGAAATG GAAAATTTAC ATCATATAGG TTATTTAACT 3240
TGTGTTCAGC CTTTTTGTAA CTTTTTTGAA AGTGCAAACA ATTCTTTGGA TTATTAAATA 3300
AGGTATACAG TATGCATGGT TTCTCAAATT TAGCTTTAAA ATCTAAAAGT CTATAAAGAA 3360
TCAGATGCAT AGGCAATATG TTAAGTTCAC TTGGAGGCTA AAAATCTCCA GTGAAAACAA 3420
AACGAAAACC TTTAAGAGAA TGTAGAGTTT ATATAAACAC AAAGTATGCA TTGAAGATCT 3480
GTTTCTACCA ATAAACATTA AAACAAAAAA AAAAAAAAAA AAAAAAA 3527 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 685 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Asn Gin Ser Arg Ser Arg Ser Asp Gly Gly Ser Glu Glu Thr Leu 1 5 10 15
Pro Gin Asp His Asn His His Glu Asn Glu Arg Arg Trp Gin Gin Glu 20 25 30 Arg Leu His Arg Glu Glu Ala Tyr Tyr Gin Phe He Asn Glu Leu Asn 35 40 45
Asp Glu Asp Tyr Arg Leu Met Arg Asp His Asn Leu Leu Gly Thr Pro 50 55 60
Gly Glu He Thr Ser Glu Glu Leu Gin Gin Arg Leu Asp Gly Val Lys 65 70 75 80
Glu Gin Leu Ala Ser Gin Pro Asp Leu Arg Asp Gly Thr Asn Tyr Arg 85 90 95
Asp Ser Glu Val Pro Arg Glu Ser Ser His Glu Asp Ser Leu Leu Glu 100 105 110
Trp Leu Asn Thr Phe Arg Arg Thr Gly Asn Ala Thr Arg Ser Gly Gin 115 120 125
Asn Gly Asn Gin Thr Trp Arg Ala Val Ser Arg Thr Asn Pro Asn Asn 130 135 140
Gly Glu Phe Arg Phe Ser Leu Glu He His Val Asn His Glu Asn Arg 145 150 155 160
Gly Phe Glu He His Gly Glu Asp Tyr Thr Asp He Pro Leu Ser Asp 165 170 175
Ser Asn Arg Asp His Thr Ala Asn Arg Gin Gin Arg Ser Thr Ser Pro 180 185 190
Val Ala Arg Arg Thr Arg Ser Gin Thr Ser Val Asn Phe Asn Gly Ser 195 200 205
Ser Ser Asn He Pro Arg Thr Arg Leu Ala Ser Arg Gly Gin Asn Pro 210 215 220
Ala Glu Gly Ser Phe Ser Thr Leu Gly Arg Leu Arg Asn Gly He Gly 225 230 235 240
Gly Ala Ala Gly He Pro Arg Ala Asn Ala Ser Arg Thr Asn Phe Ser 245 250 255
Ser His Thr Asn Gin Ser Gly Gly Ser Glu Leu Arg Gin Arg Glu Gly 260 265 270
Gin Arg Phe Gly Ala Ala His Val Trp Glu Asn Gly Ala Arg Ser Asn 275 280 285
Val Thr Val Arg Asn Thr Asn Gin Arg Leu Glu Pro He Arg Leu Arg 290 295 300
Ser Thr Ser Asn Ser Arg Ser Arg Ser Pro He Gin Arg Gin Ser Gly 305 310 315 320
Thr Val Tyr His Asn Ser Gin Arg Glu Ser Arg Pro Val Gin Gin Thr 325 330 335
Thr Arg Arg Ser Val Arg Arg Arg Gly Arg Thr Arg Val Phe Leu Glu 340 345 350
Gin Asp Arg Glu Arg Glu Arg Arg Gly Thr Ala Tyr Thr Pro Phe Ser 355 360 365
Asn Ser Arg Leu Val Ser Arg He Thr Val Glu Glu Gly Glu Glu Ser 370 375 380
Ser Arg Ser Ser Thr Ala Val Arg Arg His Pro Thr He Thr Leu Asp 385 390 395 400
Leu Gin Val Arg Arg He Arg Pro Gly Glu Asn Arg Asp Arg Asp Ser 405 410 415
He Ala Asn Arg Thr Arg Ser Arg Val Gly Leu Ala Glu Asn Thr Val 420 425 430
Thr He Glu Ser Asn Ser Gly Gly Phe Arg Arg Thr He Ser Arg Leu 435 440 445
Glu Arg Ser Gly He Arg Thr Tyr Val Ser Thr He Thr Val Pro Leu 450 455 460
Arg Arg He Ser Glu Asn Glu Leu Val Glu Pro Ser Ser Val Ala Leu 465 470 475 480
Arg Ser He Leu Arg Gin He Met Thr Gly Phe Gly Glu Leu Ser Ser 485 490 495
Leu Met Glu Ala Asp Ser Glu Ser Glu Leu Gin Arg Asn Gly Gin His 500 505 510
Leu Pro Asp Met His Ser Glu Leu Ser Asn Leu Gly Thr Asp Asn Asn 515 520 525
Arg Ser Gin His Arg Glu Gly Ser Ser Gin Asp Arg Gin Ala Gin Gly 530 535 540
Asp Ser Thr Glu Met His Gly Glu Asn Glu Thr Thr Gin Pro His Thr 545 550 555 560
Arg Asn Ser Asp Ser Arg Gly Gly Arg Gin Leu Arg Asn Pro Asn Asn 565 570 575
Leu Val Glu Thr Gly Thr Leu Pro He Leu Arg Leu Ala His Phe Phe 580 585 590
Leu Leu Asn Glu Ser Asp Asp Asp Asp Arg He Arg Gly Leu Thr Lys 595 600 605
Glu Gin He Asp Asn Leu Ser Thr Arg His Tyr Glu His Asn Ser He 610 615 620 Asp Ser Glu Leu Gly Lys He Cys Ser Val Cys He Ser Asp Tyr Val 625 630 635 640
Thr Gly Asn Lys Leu Arg Gin Leu Pro Cys Met His Glu Phe His He 645 650 655
His Cys He Asp Arg Trp Leu Ser Glu Asn Cys Thr Cys Pro He Cys 660 665 670
Arg Gin Pro Val Leu Gly Ser Asn He Ala Asn Asn Gly 675 680 685
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1463 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAGCCTGGGC TCCGCGCAGC CCACCGATCT GGGCGCCCAC AAGCGGCCGG CATCCGTGTC 60
GAGCAGCGCT GCCGTGGAGC ACGAGCAGCG TGAGGCGGCA GCCAAGGAGA AACAACCGCC 120
GCCGCCTGCG CACCGGGGCC CGGCCGACAG CCTGTCCACC GCGGCCGGGG CCGCCGAGCT 180
GAGCGCGGAA GGTGCGGGCA AGAGCCGCGG GTCTGGAGAG CAGGACTGGG TCAACAGGCC 240
CAAGACCGTG CGCGACACGC TGCTGGCGCT GCACCAGCAC GGCCACTCGG GGCCCTTCGA 300
GAGCAAGTTT AAGAAGGAGC CGGCCCTGAC TGCAGGCAGG TTGTTGGGTT TCGAGGCCAA 360
CGGGGCCAAC GGGTCTAAAG CAGTTGCAAG AACAGCAAGG AAAAGGAAGC CCTCTCCAGA 420
ACCAGAAGGT GAAGTCGGGC CCCCTAAGAT CAACGGAGAG GCCCAGCCGT GGCTGTCCAC 480
ATCCACAGAG GGGCTCAAGA TCCCCATGAC TCCTACATCC TCTTTTGTGT CTCCGCCACC 540
ACCCACTGCC TCACCTCATT CCAACCGGAC CACACCGCCT GAAGCGGCCC AGAATGGCCA 600
GTCCCCCATG GCAGCCCTGA TCTTAGTAGC AGACAATGCA GGGGGCAGTC ATGCCTCAAA 660
AGATGCCAAC CAGGTTCACT CCACTACCAG GAGGAATAGC AACAGTCCGC CCTCTCCGTC 720
CTCTATGAAC CAAAGAAGGC TGGGCCCCAG AGAGGTGGGG GGCCAGGGAG CAGGCAACAC 780
AGGAGGACTG GAGCCAGTGC ACCCTGCCAG CCTCCCGGAC TCCTCTCTGG CAACCAGTGC 840 CCCGCTGTGC TGCACCCTCT GCCACGAGCG GCTGGAGGAC ACCCATTTTG TGCAGTGCCC 900
GTCCGTCCCT TCGCACAAGT TCTGCTTCCC TTGCTCCAGA CAAAGCATCA AACAGCAGGG 960
AGCTAGTGGA GAGGTCTATT GTCCCAGTGG GGAAAAATGC CCTCTTGTGG GCTCCAATGT 1020
CCCCTGGGCC TTTATGCAAG GGGAAATTGC AACCATCCTT GCTGGAGATG TGAAAGTGAA 1080
AAAAGAGAGA GACTCGTGAC TTTTCCGGTT TCAGAAAAAC CCAATGATTA CCCTTAATTA 1140
AAACTGCTTG AATTGTATAT ATATCTCCAT ATATATATAT ATCCAAGACA AGGGAAATGT 1200
AGACTTCATA AACATGGCTG TATAATTTTG ATTTTTTTTG AATACATTGT GTTTCTATAT 1260
TTTTTTTGAC GACAAAAGGT ATGTACTTAT AAAGACATTT TTTTCTTTTG TTAACGTTAT 1320
TAGCATATCT TTGTGCTTTA TTATCCTGGT GACAGTTACC GTTCTATGTA GGCTGTGACT 1380
TGCGCTGCTT TTTTAGAGCA CTTGGCAAAT CAGAAATGCT TCTAGCTGTA TTTGTATGCA 1440
CTTATTTTAA AAAAAAAAAA AAA 1463 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Thr Pro Thr Ser Ser Phe Val Ser Pro Pro Pro Pro Thr Ala Ser 1 5 10 15
Pro His Ser Asn Arg Thr Thr Pro Pro Glu Ala Ala Gin Asn Gly Gin 20 25 30
Ser Pro Met Ala Ala Leu He Leu Val Ala Asp Asn Ala Gly Gly Ser 35 40 45
His Ala Ser Lys Asp Ala Asn Gin Val His Ser Thr Thr Arg Arg Asn 50 55 60
Ser Asn Ser Pro Pro Ser Pro Ser Ser Met Asn Gin Arg Arg Leu Gly 65 70 75 80
Pro Arg Glu Val Gly Gly Gin Gly Ala Gly Asn Thr Gly Gly Leu Glu 85 90 95 Pro Val His Pro Ala Ser Leu Pro Asp Ser Ser Leu Ala Thr Ser Ala 100 105 110
Pro Leu Cys Cys Thr Leu Cys His Glu Arg Leu Glu Asp Thr His Phe 115 120 125
Val Gin Cys Pro Ser Val Pro Ser His Lys Phe Cys Phe Pro Cys Ser 130 135 140
Arg Gin Ser He Lys Gin Gin Gly Ala Ser Gly Glu Val Tyr Cys Pro 145 150 155 160
Ser Gly Glu Lys Cys Pro Leu Val Gly Ser Asn Val Pro Trp Ala Phe 165 170 175
Met Gin Gly Glu He Ala Thr He Leu Ala Gly Asp Val Lys Val Lys 180 185 190
Lys Glu Arg Asp Ser 195
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2547 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CATTTTTCTG GTCCTTCTTA AAAGTAATCA CTCTTAAATT TTGTGCTTAT TCTGTTGTTT 60
TAAAAAATAG TTTAAACAAA TATGTGTGTA CTCATAAACA TAGGTTACTT TTGCTTCTTT 120
TTGAGATATA TTTAAATTTT ATTGTGGTCT ACATATTCTT CAGCAGTTTG TTTTTTTACC 180
CAATATTATG TTTCATCTGT ATTACTGCAT TTACTATCCC TAGTTGATTC ACTTCCCTGA 240
AGTACAATAT TCAGTTGTGT GGCTATACCA TAATTTAGTT ATTCATTTTG TTGTCAGTAA 300
AATTTGGGTG ATTATCAGAT TTTTTTCTAG CATGAAAAAT GCTACTARGA ACATTCSTGT 360
ATGTGTCTAA TGGTATACAC TTTCAAGTGT TTTTTTATAT ATGTGAGAGT AGATTACTTG 420
GACCTTGAAG ATGAACATGC TATCTTTTCC AGATACTGCC AATTATTTCA GCAAGATATG 480
AGTTCCCATC ATTTTATATT TGTCAGCATT TGATATTTCC AGGCCTAGTG ATTTCCAGTC 540
ATTTACTGGA TATAATATGA TTATCTCTGT AGGGAGTTGA TTTCCATCTC CTCAATTACT 600 AATAAAGTTA AAAATCTTTT CATATGTTTT ATTGCCATTT TTATTTCTTC TGTAAAGTAC 660
CTACTCATGG CTTTTTCTCA TTTTTTGTTT GTCATCATTG AATTATAGGA GTTTTGAGAG 720
AGTGAGCAAG CTAGTCTGTG TGTGTGTGTG TGTGCGTGTG TGTGTATCTC CTTAATGTGT 780
TATATGTGAT TGGAACTTCT TCTCCCACCT TGATGCTTCC TTTCTTCCCC ACTTGTTTTA 840
GGTATCTTCT GATGAAGTGG AGTTATTTAT GGTATGTTCT CAGGAGCTAC AATTTTTAAT 900
TTCAATATAA TCAGTGTTTT TAATTATCTT ATGTTTAGCT CTTTTGGGTC ATGCTTAGGA 960
AATTCTTCTT AAATTTCATT GATAACAGTC TTCCATACTT TCTTCTAAAG TCTTATATTT 1020
TGGCCTTTCA TATTTATTCC TTTAATCCAM CTGGAGTAGA TTTTTTTTTT CCCTCTGTAG 1080
AGTTTGGAGT AGAGATTTTA TTTCCTTTTT TTTTTTTTTT TTTTTTTCTT TTTTTTTGAG 1140
ACAGAGTCTT GCTCTGTCGC CCAGGCTGGA GTGCAGTGGC ACTATCTCAG CTCACTGCAA 1200
CCTCCACCTC CTGGGTTCAA GCGATTCTCC TGCCTCCGCC TCCCGAGTAG CTGGGACTAC 1260
AGGCATGTGC CACCACGCCC AGCTAATTTT TTGTATTTTT TTTAGTAGAG ATGGGGTTCC 1320
ACCATGTTAG CTAGGATGAT TTCGATTTCC TGACCTTGTG ATCCGCCCGC CTCGGCCTCC 1380
CAAAATGCTG GGATTATAGG TGTGAGCCAC CACGTGGCCT CATTTCATTC TTTCATGTGG 1440
ATAGGCAGTT GTTCCAGAAG TATATAGTGA GGAGCTTCTT CTTTCTCTAA TGATCTGCAA 1500
TGTCACCTTC ATCATTTATG AAGGTTGCAC ATATACATGG GAATTTTTTA GTCTGGCATT 1560
AAATGTTCTT CAAAAGAGTT CCTGCAAACG TTTTTGTTTT TATTTCCTAC TGTTCCCTTC 1620
ACGTACTCTC TACTGAACTA AACTCTGTAA TGTGTCTCGA AACTGTCCCA CAATTTTCCT 1680
TGTCTTAAGA GTTTAATGCT TTCATACACC TCTCACATTC AGCCTTGTGC TATTGTCTTA 1740
GGTATATTTA TTTCTCTTTT GCTCCCAATT ATGTTGTAAA CTTTTGGAAG CAGGAAGGAT 1800
ATATTGTTCA TCTTTGGTAG CATTAAACAA TGAATACAGT GTTTTTTACT TAATAGATAT 1860
TTGGTAAATC ATTGAACTAA ATTGGGGTTT GGAATTGAAG GTCTTAGAAA TTACCTGACC 1920
ACTCCCATTA TATTTGCCCA TCCATGATCA CTGAGATTTA TAGAGATTAG ATGCAATGCC 1980
CAGTTTCACA TATGTTTTTG CATCACTGTC TCTTTTTTTC TTGAGCTTAT TCCAGAGTGT 2040
CTTTTAATAT CCATTCCATG ATCAAATGGC TGAACTATTA AAATGCTGTC CAGAAGTGTA 2100
AAGCAATATG AAGATGCTAG AAAAGTTGAA GAGACACATA TATGGTAGGT CCAAGACCAT 2160
TACACTTACT GAGTCCATTA CTAAAAATGA TGTTCACTTA ACATCAAAAC ACTCAGGATT 2220
ACCCAAGCAC AATATACTGA TTTGCACCTC TGCCTTTGTT CATGCCCCTT GTTCAGGAGA 2280 ACTGCTTTCA TGTGCTACTG TCCATAGATC TTCTCTATCC TTACAGATTA ATTTCTTCCT 2340
TTTGAATGCT ATGTTTCCAT ACTTTGACAT TCCTTCTGCA CCATTCAGAC CATATTTTAG 2400
TTCTTTTTTA TGGTATCTCT CACTTTTGAT TGTCACCCCT TAAGTCAAAG ACAATTTTTT 2460
CATCTGTGTC TTCTCAACAC CCAGCACAGG GCTATGTTTG GTAAAAATTA GGTATCCAAG 2520
ATGTACTAAA TGAAAAAAAA AAAAAAA 2547 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Phe Phe Lys Arg Val Pro Ala Asn Val Phe Val Phe He Ser Tyr 1 5 10 15
Cys Ser Leu His Val Leu Ser Thr Glu Leu Asn Ser Val Met Cys Leu 20 25 30
Glu Thr Val Pro Gin Phe Ser Leu Ser 35 40
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2245 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GCTCAACGGC CTCTTCTGGT TGCTGTCTTC CTCGTCCCTC CGGCCCTTCT TCCTACTCAG 60
CGTCTCACTT TTGGCCTATT TTCTGCTGGA TCTCTGGCAG CCTCGCTTTC TCCCTGACGT 120
TTCAGCATCA TCCCCAGAGG AGCCACACTC TGACAGTGAG GGTGCGGGGT CAGGCGCCCG 180 GCCGCACCTG CTGAGTGTGC CCGAGTTGTG CAGATACCTG GCTGAGAGCT GGCTCACCTT 240
CCAGATTCAC CTGCAGGAGC TGCTGCAGTA CAAGAGGCAG AATCCAGCTC AGTTCTGCGT 300
TCGARTCTGC TCTGGCTGTG CTGTGTTGGC TGTGTTGGGA CACTATGTTC CAGGGATTAT 360
GATTTCCTAC ATTGTCTTGT TGAGTATCCT GCTGTGGCCC CTGGTGGTTT ATCATGARCT 420
GATCCAGAGG ATGTWCACTC GCCTGGAGCC CCTGCTCATG CAGCTGGACT ACAGCATGAA 480
GGCAGAAKCC AATGCCCTGC ATCACAAACA CGACAAGAGG AAGCGTCAGG GGAAGAATGC 540
ACCCCCAGGA GGTGATGAGC CACTGGCAGA GACAGAGAGT GAAAGCGAGG CAGAGCTGGC 600
TGGCTTCTCC CCAGTGGTGG ATGTGAAGAA AACAGCATTG GCCTTGGCCA TTACAGACTC 660
AGAGCTGTCA GATGAGGAGG CTTCTATCTT GGAGAGTGGT GGCTTCTCCG TATCCCGGGC 720
CACAACTCCG CAGCTGACTG ATGTCTCCGA GGATTTGGAC CAGCAGAGCC TGCCAAGTGA 780
ACCAGAGGAG ACCCTAAGCC GGGACCTAGG GGAGGGAGAG GAGGGAGAGC TGGCCCCTCC 840
CGAAGACCTA CTAGGCCGTC CTCAAGCTCT GTCAAGGCAA GCCCTGGACT TGGAGGAAGA 900
GGAAGAGGAT GTGGCAGCTA AGGAAACCTT GTTGCGGCTC TCATCCCCCC TCCACTTTGT 960
GAACACGCAC TTCAATGGGG CAGGGTCCCC CCCAGATGGA GTGAAATGCT CCCCTGGAGG 1020
ACCAGTGGAG ACACTGAGCC CCGAGACAGT GAGTGGTGGC CTCACTGCTC TGCCCGGCAC 1080
CCTGTCACCT CCACTTTGCC TTGTTGGAAG TGACCCAGCC CCCTCCCCTT CCATTCTCCC 1140
ACCTGTTCCC CAGGACTCAC CCCAGCCCCT GCCTGCCCCT GAGGAAGAAG AGGCACTCAC 1200
CACTGAGGAC TTTGAGTTGC TGGATCAGGG GGAGCTGGAG CAGCTGAATG CAGAGCTGGG 1260
CTTGGAGCCA GAGACACCGC CAAAACCCCC TGATGCTCCA CCCCTGGGGC CCGACATCCA 1320
TTCTCTGGTA CAGTCAGACC AAGAAGCTCA GGCCGTGGCA GAGCCATGAG CCAGCCGTTG 1380
AGGAAGGAGC TGCAGGCACA GTAGGGCTTC CTGGCTAGGA GTGTTGCTGT TTCCTCCTTT 1440
GCCTACCACT CTGGGGTGGG GCAGTGTGTG GGGAAGCTGG CTGTCGGATG GTAGCTATTC 1500
CACCYTCTGC CTGCCTGCCT GCCTGCTGTC CTGGGCATGG TGCAGTACCT GTGCCTAGGA 1560
TTGGTTTTAA ATTTGTAAAT AATTTTCCAT TTGGGTTAGT GGATGTGAAC AGGGCTAGGG 1620
AAGTCCTTCC CACAGCCTGC GCTTGCCTCC CTGCCTCATC TCTATTCTCA TTCCACTATG 1680
CCCCAAGCCC TGGTGGTCTG GCCCTTTCTT TTTCCTCCTA TCCTCAGGGA CCTGTGCTGC 1740
TCTGCCCTCA TGTCCCACTT GGTTGTTTAG TTGAGGCACT TTATAATTTT TCTCTTGTCT 1800
TGTGTTCCTT TCTGCTTTAT TTCCCTGCTG TGTCCTGTCC TTAGCAGCTC AACCCCATCC 1860 TTTGCCAGCT CCTCCTATCC CGTGGGCACT GGCCAAGCTT TAGGGAGGCT CCTGGTCTGG 1920
GAAGTAAAGA GTAAACCTGG GGCAGTGGGT CAGGCCAGTA GTTACACTCT TAGGTCACTG 1980
TAGTCTGTGT AACCTTCACT GCATCCTTGC CCCATTCAGC CCGGCCTTTC ATGATGCAGG 2040
AGAGCAGGGA TCCCGCAGTA CATGGCGCCA GCACTGGAGT TGGTGAGCAT GTGCTCTYTY 2100
TTGAGATTAG GAGCTTCCTT ACTGCTCCTC TGGGTGATCC AAGTGTAGTG GGACCCCCTA 2160
CTAGGGTCAG GAAGTGGACA CTAACATCTG TGCAGGTGTT GACTTGAAAA ATAAAGTGTT 2220
GATTGGCTAG AAAAAAAAAA AAAAA 2245 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met He Ser Tyr He Val Leu Leu Ser He Leu Leu Trp Pro Leu Val 1 5 10 15
Val Tyr His Glu Leu He Gin Arg Met Xaa Thr Arg Leu Glu Pro Leu 20 25 30
Leu Met Gin Leu Asp Tyr Ser Met Lys Ala Glu Xaa Asn Ala Leu His 35 40 45
His Lys His Asp Lys Arg Lys Arg Gin Gly Lys Asn Ala Pro Pro Gly 50 55 60
Gly Asp Glu Pro Leu Ala Glu Thr Glu Ser Glu Ser Glu Ala Glu Leu 65 70 75 80
Ala Gly Phe Ser Pro Val Val Asp Val Lys Lys Thr Ala Leu Ala Leu 85 90 95
Ala He Thr Asp Ser Glu Leu Ser Asp Glu Glu Ala Ser He Leu Glu 100 105 110
Ser Gly Gly Phe Ser Val Ser Arg Ala Thr Thr Pro Gin Leu Thr Asp 115 120 125
Val Ser Glu Asp Leu Asp Gin Gin Ser Leu Pro Ser Glu Pro Glu Glu 130 135 140 Thr Leu Ser Arg Asp Leu Gly Glu Gly Glu Glu Gly Glu Leu Ala Pro 145 150 155 160
Pro Glu Asp Leu Leu Gly Arg Pro Gin Ala Leu Ser Arg Gin Ala Leu 165 170 175
Asp Leu Glu Glu Glu Glu Glu Asp Val Ala Ala Lys Glu Thr Leu Leu 180 185 190
Arg Leu Ser Ser Pro Leu His Phe Val Asn Thr His Phe Asn Gly Ala 195 200 205
Gly Ser Pro Pro Asp Gly Val Lys Cys Ser Pro Gly Gly Pro Val Glu 210 215 220
Thr Leu Ser Pro Glu Thr Val Ser Gly Gly Leu Thr Ala Leu Pro Gly 225 230 235 240
Thr Leu Ser Pro Pro Leu Cys Leu Val Gly Ser Asp Pro Ala Pro Ser 245 250 255
Pro Ser He Leu Pro Pro Val Pro Gin Asp Ser Pro Gin Pro Leu Pro 260 265 270
Ala Pro Glu Glu Glu Glu Ala Leu Thr Thr Glu Asp Phe Glu Leu Leu 275 280 285
Asp Gin Gly Glu Leu Glu Gin Leu Asn Ala Glu Leu Gly Leu Glu Pro 290 295 300
Glu Thr Pro Pro Lys Pro Pro Asp Ala Pro Pro Leu Gly Pro Asp He 305 310 315 320
His Ser Leu Val Gin Ser Asp Gin Glu Ala Gin Ala Val Ala Glu Pro 325 330 335
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1406 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CTTGTGGGAA GAGCTGAAGC AGGCGCTCTT GGCTCGGCGC GGCCCGCTGC AATCCGTGGA 60 GGAACGCGCC GCCGAGCCAC CATCATGCCT GGGCACTTAC AGGAAGGCTT CGGCTGCGTG 120 GTCACCAACC GATTCGACCA GTTATTTGAC GACGAATCGG ACCCCTTCGA GGTGCTGAAG 180
GCAGCAGAGA ACAAGAAAAA AGAAGCCGGC GGGGGCGGCG TTGGGGGCCC TGGGGCCAAG 240
AGCGCAGCTC AGGCCGCGGC CCAGACCAAC TCCAACGCGG CAGGCAAACA GCTGCGCAAG 300
GAGTCCCAGA AAGACCGCAA GAACCCGCTG CCCCCCAGCG TTGGCGTGGT TGACAAGAAA 360
GAGGAGACGC AGCCGCCCGT GGCGCTTAAG AAAGAAGGAA TAAGACGAGT TGGAAGAAGA 420
CCTGATCAAC AACTTCAGGG TGAAGGGAAA ATAATTGATA GAAGACCAGA AAGGCGACCA 480
CCTCGTGAAC GAAGATTCGA AAAGCCACTT GAAGAAAAGG GTGAAGGAGG CGAATTTTCA 540
GTTGATAGAC CGATTATTGA CCGACCTATT CGAGGTCGTG GTGGTCTTGG AAGAGGTCGA 600
GGGGGCCGTG GACGTGGAAT GGGCCGAGGA GATGGATTTG ATTCTCGTGG CAAACGTGAA 660
TTTGATAGGC ATAGTGGAAG TGATAGATCT TCTTTTTCAC ATTACAGTGG CCTGAAGCAC 720
GAGGACAAAC GTGGAGGTAG CGGATCTCAC AACTGGGGAA CTGTCAAAGA CGAATTAACT 780
GACTTGGATC AATCAAATGT GACTGAGGAA ACACCTGAAG GTGAAGAACA TCATCCAGTG 840
GCAGACACTG AAAATAAGGA GAATGAAGTT GAAGAGGTAA AAGAGGAGGG TCCAAAAGAG 900
ATGACTTTGG ATGAGTGGAA GGCTATTCAA AATAAGGACC GGGCAAAAGT AGAATTTAAT 960
ATCCGAAAAC CAAATGAAGG TGCTGATGGG CAGTGGAAGA AGGGATTTGT TCTTCATAAA 1020
TCAAAGAGTG AAGAGGCTCA TGCTGAAGAT TCGGTTATGG ACCATCATTT CCGGAAGCCA 1080
GCAAATGATA TAACGTTTCA GCTGGAGATC AATTTTGGAG ACCTTGGCCG CCCAGGACGT 1140
GGCGGCAGGG GAGGACGAGG TGGACGTGGG CGTGGTGGGC GCCCAAACCG TGGCAGCAGG 1200
ACCGACAAGT CAAGTGCTTT TGCTCCTGAT GTGGATGACC CAGAGGCATT CCCAGTTTTG 1260
GCTTAAMTGG ATGCCATAAG ACAACCCTGG TTCCTTTGTG AACCCTTTTG TTCAAAGCTT 1320
TTGCATGCTT AAGGATTCCA AACGACTAAG AAATTAAAAA AAAAAAAAAA AAAAAAAAAA 1380
AAAAAAAAAA AAAAAAAAAA AAAAAA 1406 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Pro Gly His Leu Gin Glu Gly Phe Gly Cys Val Val Thr Asn Arg 1 5 10 15
Phe Asp Gin Leu Phe Asp Asp Glu Ser Asp Pro Phe Glu Val Leu Lys 20 25 30
Ala Ala Glu Asn Lys Lys Lys Glu Ala Gly Gly Gly Gly Val Gly Gly 35 40 45
Pro Gly Ala Lys Ser Ala Ala Gin Ala Ala Ala Gin Thr Asn Ser Asn 50 55 60
Ala Ala Gly Lys Gin Leu Arg Lys Glu Ser Gin Lys Asp Arg Lys Asn 65 70 75 80
Pro Leu Pro Pro Ser Val Gly Val Val Asp Lys Lys Glu Glu Thr Gin 85 90 95
Pro Pro Val Ala Leu Lys Lys Glu Gly He Arg Arg Val Gly Arg Arg 100 105 110
Pro Asp Gin Gin Leu Gin Gly Glu Gly Lys He He Asp Arg Arg Pro 115 120 125
Glu Arg Arg Pro Pro Arg Glu Arg Arg Phe Glu Lys Pro Leu Glu Glu 130 135 140
Lys Gly Glu Gly Gly Glu Phe Ser Val Asp Arg Pro He He Asp Arg 145 150 155 160
Pro He Arg Gly Arg Gly Gly Leu Gly Arg Gly Arg Gly Gly Arg Gly 165 170 175
Arg Gly Met Gly Arg Gly Asp Gly Phe Asp Ser Arg Gly Lys Arg Glu 180 185 190
Phe Asp Arg His Ser Gly Ser Asp Arg Ser Ser Phe Ser His Tyr Ser 195 200 205
Gly Leu Lys His Glu Asp Lys Arg Gly Gly Ser Gly Ser His Asn Trp 210 215 220
Gly Thr Val Lys Asp Glu Leu Thr Asp Leu Asp Gin Ser Asn Val Thr 225 230 235 240
Glu Glu Thr Pro Glu Gly Glu Glu His His Pro Val Ala Asp Thr Glu 245 250 255
Asn Lys Glu Asn Glu Val Glu Glu Val Lys Glu Glu Gly Pro Lys Glu 260 265 270
Met Thr Leu Asp Glu Trp Lys Ala He Gin Asn Lys Asp Arg Ala Lys 275 280 285
Val Glu Phe Asn He Arg Lys Pro Asn Glu Gly Ala Asp Gly Gin Trp 290 295 300
Lys Lys Gly Phe Val Leu His Lys Ser Lys Ser Glu Glu Ala His Ala 305 310 315 320
Glu Asp Ser Val Met Asp His His Phe Arg Lys Pro Ala Asn Asp He 325 330 335
Thr Phe Gin Leu Glu He Asn Phe Gly Asp Leu Gly Arg Pro Gly Arg 340 345 350
Gly Gly Arg Gly Gly Arg Gly Gly Arg Gly Arg Gly Gly Arg Pro Asn 355 360 365
Arg Gly Ser Arg Thr Asp Lys Ser Ser Ala Phe Ala Pro Asp Val Asp 370 375 380
Asp Pro Glu Ala Phe Pro Val Leu Ala 385 390
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4237 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GCGGACGCGG CCAGTCAGGT GCTCCTGGGC TCCGGTCTCA CCATCCTGTC CCAGCCGCTC 60
ATGTACGTGA AAGTGCTCAT CCAGGTGGGA TATGAGCCTC TTCCTCCAAC AATAGGACGA 120
AATATTTTTG GGCGGCAAGT GTGTCAGCTT CCTGGTCTCT TTAGTTATGC TCAGCACATT 180
GCCAGTATCG ATGGGAGGCG CGGGTTGTTC ACAGGCTTAA CTCCAAGACT GTGTTCGGGA 240
GTCCTTGGAA CTGTGGTCCA TGGTAAAGTT TTACAGCATT ACCAGGAGAG TGACAAGGGT 300
GAGGAGTTAG GAMCTGGAAA TGTACARAAA GAAGTCTCAT CTTCCTTTGA MCACGTTATC 360
AAGGAGACAA CTCGAGAGAT GATCGCTCGT TCTGCTGCTA CCCTCATCAC ACATCCCTTC 420
CATGTTGATC ACTCTGAGAT CTATGGTACA RTTCATTGGC AGAGAATCCA AGTACTGTGG 480
ACTTTGTGAT TCCATAATAA CCATCTATCG GGAAGAGGGC ATTCTAGGAT TTTTCGCGGG 540 TCTTGTTCCT CGCCTTCTAG GTGACATCCT TTCTTTGTGG CTGTGTAACT CACTGGCCTA 600
CCTCGTCAAT ACCTATGCAC TGGACAGTGG GGTTTCTACC ATGAATGAAA TGAAGAGTTA 660
TTCTCAAGCT GTCACAGGAT TTTTTGCGAG TATGTTGACC TATCCCTTTG TGCTTGTCTC 720
CAATCTTATG GCTGTCAACA ACTGTGGTCT TGCTGGTGGA TGCCCTCCTT ACTCCCCAAT 780
ATATACGTCT TGGATAGACT GTTGGTGCAT GCTACAAAAA GAGGGGAATA TGAGCCGAGG 840
AAATAGCTTA TTTTTCCGGA AGGTCCCCTT TGGGAAGACT TATTGTTGTG ACCTGAAAAT 900
GTTAATTTGA AGATGTGGGG CAGGGACAGT GACATTTCTG TAGTCCCAGA TGCACAGAAT 960
TATGGGAGAG AATGTTGATT TCTATAC GT GTGGCGCGCT TTTTTAATAA TCATTTAATC 1020
TTGGGAAAAT TCAGGTGTTT GGTGTCTGCC TTTTTTGTTC TTTTTTCCAG CACAACATAA 1080
CTTACCACTG ATACTCCCCC TTTAGTTATT CTGAATTAGG ATATTTTTGC TCCAAATTCT 1140
TATTTTACTT AACCAGAAGG GAAAAAAAGT TGTATTTTCC TGAAGCTACA GGCACTTTGT 1200
CATGTGATTT TTGAGTCTCA ATTTAAGGCT TTGTAAAATG AAGAGTAGAA TTCCAAGAAA 1260
AATGAGAAAT AATTTTGTAA AACTTAACAA AATCACTAAA TTAAACTATA TGGGAGGTTA 1320
TGAATTACTT TTTCTTGGGT AGACCCTAAA ATGTCAGTAG CATGCACCAG AATCTGACTC 1380
CCATTATGCT TCTAAGCACA TTTCATTGAC CTTGTCTCTC ATACTTCAAG AAAAGGACAG 1440
TACATTGCTA CATTACCCTA GAAAGTCTGT GTGAGGATCT GCCCCTTCAG TCTGTTATTG 1500
CAAAGTAATA AAATGTCACC TACAGGGAGC CTCTGAGCCT ACTCTAGTTC AAGAGGCTAC 1560
CTGAAAAAAA ATAAATAAGA TAAAGGGTCA GCAACAACAA AGAAAAAGAC AATTACAGAA 1620
AATAAGCAAG ATTTGGAAAG GAAGTATAAT GGCACTTTTT TCCTCAAAGG AAGTTCTTGT 1680
TTTCACATAA AATATGAAAA GCAGATCCTG CAGGAGTAAC CCCCTTCTTT AAGAGCCAAG 1740
TATTTGCCAG TGCTTAAATT ACACCATACC GTTCTAATTA TATATAATCT TTTGTTCTTC 1800
AGTTTTTTGT TTTGTTTCCT TTTTGTTATT GTTGCCGAAG GTGAGTAGTT TTGCATTTCT 1860
GATGACAGCC TTGGAAAGTA TATTTGTAAC TCCATGTCTG GTAATGCCAA CCCAAGTCGA 1920
CATGGGTCTT AGGACACTGA CCACCTCACA TGCCATACCC TCAGTTAAGC ATGTTAACAT 1980
TTATAGGAGG AAAAAAATCA CTTTGGGAGA AAATAAAATT CAACTCAAGC ATAAAGCTTC 2040
TGTTTACTCA GGCCTTCTAA AAAGCAGGTT AAAATGCTCT AAAATGAGAA AGCCTGTGGT 2100
TTCACTTATT TATATAACTC ACTGGGACAT TGCCAAATGA GTAAGCACTT AATTCGCTGC 2160
TTCTGAGACT TCTCTGTCAA AACAGCCCCA CTGATAATAT TAGACAGAAC GAGAATGCAG 2220 GGGTCTCTTC CCTCCCCTGG GGTTTAGGAA GCTCATGAGG AGCTCGGCTT AAAATGTCTT 2280
TGATGTCTCT TCCTTTGTCT CAAAAAGTAA TGTCAATTTT ATATACTATT TCAATATTAC 2340
TATCTGCATT TGTTTTAATA TAAAAATGTT TGCTGCCTAC CTTTTTCTCC CAAAAAATCT 2400
TTAAGTAAAG ATGATCTGGG AAAATGAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2460
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2520
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2580
AAAAAAAAAA AAAAAAAAGC GGCCGCAGGT CTAGAATTCA ATCGGAAGGT ATATAGCTTA 2640
TTTGTTGCTT TTCATTGTAA TTTAACATGG TTAATGGTTA ATTACTATTT AACACACATT 2700
TCAAATGAAT ATTATTTGGG GGATTAGATT GAGTGAAATT AACCTGCTAT TAAATAGTAA 2760
ACTTTTCCTC TGGAGTCACT TTTTTCCCCC TTCAAAGTAT GTTACTGAGG AAGTAAACTT 2820 ττττττττττ TTTTGGTTTT TGTTTTTTGA GACACAGTCT CGCTCTGTTG CCCAGGCTGC 2880
TGGAGTGCCG TGGCGCAATC TCGGCTCACT GCAACCTCCG CCTCCTGGAT TCAAACAATT 2940
CTCCTGCTTC AGCCTCCTGA GTAGCTGGGA TTACAGGCAC ATGCCACCAC GCCCGGCTAA 3000
TTTTTGTATT TTTAGTAGAG ACTGGGTTTC ACCATGTTGG TCAGGCTGGT CTCAAACTCC 3060
TGACCTCGTG ATCCACCCGC CTCGGCCTCC CAAAATCCTG GGATTACAGG CGCGAGCCAC 3120
CACACCCGGC TGGAAGTAAA CATTTTTAAA GCTACTTTTA CTCATTCTAG CCTTGTAGAA 3180
TGACCATTGC AGCTTGAGGG ACCTAGTTCT TACCTTTTCT TGCAACCAAC ACACTTGCAA 3240
TTGTGTCTGG TATGCTTGTT CCTGCTGCTA ATAAAGTAAG GCCCATTACT GTATCGGGAA 3300
TTTCTAGTGT TTCCCCTGTA ATAAACAGAT ATTTCAAGTT ACAAATCTTA AAGATTCACT 3360
AACCATCCTT TGCAGTTATT TTGGATATTT CCTTCGTGAA CAAAAATAAA ATAGGCACAT 3420
TTAGAATTCA GAGCCAATAT GTGCTTGCTT ATTAGTTTTT TAGCTAGCAA CATATTTGAA 3480
TCAGGCTGGT AATTCGGGTA ACCCAGGTAG CACAGATTTT TAATGACATA TYTAAAGATA 3540
CGTAACAGCT AAAATTTCTG CCAGTGAGAA ATTTTCCTGT TTGATATTTC TTACAAAAGA 3600
TGTTTATGTC CACCATTATY TTCATTCAGG GGCTGTGCTG AATATTTGAT AATGAGACTG 3660
ATCATTCCGC TTTTTCTTTC TTAAAAATAT TAGGCAGAGT TAAGCAAATT AATTATAGCT 3720
ATCTTTAAGC TATAAATGTG TTAACATGTA TATATACCAT TTATTATGTT CTACTTTAGT 3780
GATATACCTT AATTTAGTGG GCTTTGGCAG GGCGGGGGAG GGGGAACGTT CATTAATCTC 3840
TGAGGAAAAC AAAACCTGTT TTCTACTTGA GTCTAACATA TGGTCCCAAT TTATTAATAC 3900 TTCTGTTAAA TTTGATGTCA GGTCAACATT TTTCAGAAAT GTATTTATTC TCAGAAACAG 3960
AACCAGAGAG AAGTTAAACA AAAGGTTATG TAACTGTTCC TTTAATGTTG TAATTGAAAA 4020
CTTGGTTTAG CGTCTTTTTT TTCTTTCTCT TTTTTTTTCT TAAAATGCCA ACTAAAATAA 4080
TTAGAAAGTA GCTTATTTAT TGCATGCTTA TACATTGATA TTGGAATTGG AATTGGTTGT 4140
TAATTTCTGT TACTGGCTTT GCTAGAATTC ATATGTGCAT AAATAACACT AATATTTATC 4200
ATCTTGGAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 4237 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Met Tyr He Tyr His Leu Leu Cys Ser Thr Leu Val He Tyr Leu Asn 1 5 10 15
Leu Val Gly Phe Gly Arg Ala Gly Glu Gly Glu Arg Ser Leu He Ser 20 25 30
Glu Glu Asn Lys Thr Cys Phe Leu Leu Glu Ser Asn He Trp Ser Gin 35 40 45
Phe He Asn Thr Ser Val Lys Phe Asp Val Arg Ser Thr Phe Phe Arg 50 55 60
Asn Val Phe He Leu Arg Asn Arg Thr Arg Glu Lys Leu Asn Lys Arg 65 70 75 80
Leu Cys Asn Cys Ser Phe Asn Val Val He Glu Asn Leu Val 85 90
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TNTTGAAGACT GTTGCTTGTT TGGAATGT 29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CNCCATCTAAT GGGATGATGG GTTCTTGA 29
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ANTTTCCGTCA CCTCGTTCGC CTGCTGCT 29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: GNATACGAGGG GTTCCCATGG CTTCTTCT 29
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TNTACGACGAC ATCCAACAAT CACACTGG 29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: TNGTCCGGTTG GAATGAGGTG AGGCAGTG 29
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "olgionucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GNTCCTCACTA TATACTTCTG GAACAACT 29
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GNCCTAAGAGT GTAACTACTG GCCTGACC 29
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "olgionucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TNTCCTCGTGC TTCAGGCCAC TGTAATGT 29
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ANGCCCACTAA ATTAAGGTAT ATCACTAA 29
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met Trp Gly Leu Gly Thr Thr Ser Ser Phe Arg Trp Tyr Ser Ser Asp 1 5 10 15
Tyr Arg Arg Ser Phe Gin Leu Asn Ser Pro Val Asp Lys Met Arg Lys 20 25 30
Thr Gly Glu Gin Ala Phe Ser Val Phe Thr Tyr Lys Val Arg Ser Val 35 40 45
Met Gly Gin 50

Claims

What is claimed is:
1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 463 to nucleotide 606;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 501;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bdl64_7 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 19 to amino acid 28 of SEQ ID NO:2;
(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).
2. The polynucleotide of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with the polynucleotide of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein encoded by the polynucleotide of claim 2, which process comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and
(b) purifying said protein from the culture.
6. A protein produced according to the process of claim 5.
7. The protein of claim 6 comprising a mature protein.
8. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 19 to amino acid 28 of SEQ ID NO:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone bdl64_7 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
9. The protein of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
10. A composition comprising the protein of claim 8 and a pharmaceutically acceptable carrier.
11. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition of claim 10.
12. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:l.
13. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 202 to nucleotide 849;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 511 to nucleotide 849;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone bil29_2 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 103 to amino acid 112 of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
14. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4; (b) the amino acid sequence of SEQ ID NO:4 from amino acid 88 to amino acid 209;
(c) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 103 to amino acid 112 of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone bil29_2 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
15. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
16. 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 156 to nucleotide 902;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 225 to nucleotide 902;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 237 to nucleotide 654;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cgl60_6 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:9;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:9 having biological activity, the fragment comprising the amino acid sequence from amino acid 119 to amino acid 128 of SEQ ID NO:9;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
17. A protein comprising 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 28 to amino acid 166;
(c) fragments of the amino acid sequence of SEQ ID NO:9 comprising the amino acid sequence from amino acid 119 to amino acid 128 of SEQ ID NO:9; and
(d) the amino acid sequence encoded by the cDNA insert of clone cgl60_6 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
18. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:8.
19. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 10 from nucleotide 400 to nucleotide 2454;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 10 from nucleotide 1454 to nucleotide 1787;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cw775_l deposited under accession number ATCC 98364; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cw775_l deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:ll;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:ll having biological activity, the fragment comprising the amino acid sequence from amino acid 337 to amino acid 346 of SEQ ID NO:ll;
(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).
20. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:ll;
(b) fragments of the amino acid sequence of SEQ ID NO: 11 comprising the amino acid sequence from amino acid 337 to amino acid 346 of SEQ ID NO:ll; and
(c) the amino acid sequence encoded by the cDNA insert of clone cw775_l deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
21. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:10.
22. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 506 to nucleotide 1096;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 656 to nucleotide 1096;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:12 from nucleotide 2 to nucleotide 1078;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dn740_3 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:13;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:13 having biological activity, the fragment comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:13;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
23. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:13; (b) the amino acid sequence of SEQ ID NO: 13 from amino acid 1 to amino acid 191;
(c) fragments of the amino acid sequence of SEQ ID NO:13 comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:13; and
(d) the amino acid sequence encoded by the cDNA insert of clone dn740_3 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
24. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:12.
25. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:14 from nucleotide 1563 to nucleotide 1685;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:14 from nucleotide 1100 to nucleotide 1646;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone dn904_2 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:15;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:15 having biological activity, the fragment comprising the amino acid sequence from amino acid 15 to amino acid 24 of SEQ ID NO:15; (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).
26. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:15;
(b) the amino acid sequence of SEQ ID NO:15 from amino acid 1 to amino acid 28;
(c) fragments of the amino acid sequence of SEQ ID NO:15 comprising the amino acid sequence from amino acid 15 to amino acid 24 of SEQ ID NO:15; and
(d) the amino acid sequence encoded by the cDNA insert of clone dn904_2 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
27. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:14.
28. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16 from nucleotide 359 to nucleotide 1369;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:16 from nucleotide 1547 to nucleotide 1868;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone do568_ll deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364; (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone do568_ll deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:17;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 17 having biological activity, the fragment comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:17;
(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).
29. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 17;
(b) fragments of the amino acid sequence of SEQ ID NO: 17 comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO: 17; and
(c) the amino acid sequence encoded by the cDNA insert of clone do568_ll deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
30. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:16.
31. 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 85 to nucleotide 1263;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 from nucleotide 265 to nucleotide 608;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ek626_3 deposited under accession number ATCC 98364;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364;
(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, the fragment comprising the amino acid sequence from amino acid 191 to amino acid 200 of SEQ ID NO:19;
(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).
32. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:19;
(b) the amino acid sequence of SEQ ID NO:19 from amino acid 61 to amino acid 175; (c) fragments of the amino acid sequence of SEQ ID NO:19 comprising the amino acid sequence from amino acid 191 to amino acid 200 of SEQ ID NO:19; and
(d) the amino acid sequence encoded by the cDNA insert of clone ek626_3 deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
33. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:18.
34. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20 from nucleotide 3746 to nucleotide 4027;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20 from nucleotide 3815 to nucleotide 4027;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20 from nucleotide 3640 to nucleotide 3940;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fe366_l deposited under accession number ATCC 98364;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fe366_l deposited under accession number ATCC 98364;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:21;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:21 having biological activity, the fragment comprising the amino acid sequence from amino acid 42 to amino acid 51 of SEQ ID NO:21; (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
35. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:21;
(b) the amino acid sequence of SEQ ID NO:21 from amino acid 1 to amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID NO:21 comprising the amino acid sequence from amino acid 42 to amino acid 51 of SEQ ID NO:21; and
(d) the amino acid sequence encoded by the cDNA insert of clone fe366_l deposited under accession number ATCC 98364; the protein being substantially free from other mammalian proteins.
36. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:20.
PCT/US1998/005474 1997-03-19 1998-03-19 Secreted proteins and polynucleotides encoding them WO1998041539A2 (en)

Priority Applications (4)

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CA002284109A CA2284109A1 (en) 1997-03-19 1998-03-19 Secreted proteins and polynucleotides encoding them
EP98912986A EP0971954A2 (en) 1997-03-19 1998-03-19 Secreted proteins and polynucleotides encoding them
JP54082198A JP2001517941A (en) 1997-03-19 1998-03-19 Secreted proteins and polynucleotides encoding them
AU67650/98A AU6765098A (en) 1997-03-19 1998-03-19 Secreted proteins and polynucleotides encoding them

Applications Claiming Priority (4)

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US82049397A 1997-03-19 1997-03-19
US4096398A 1998-03-18 1998-03-18
US09/040,963 1998-03-18
US08/820,493 1998-03-18

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JP (1) JP2001517941A (en)
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19929887A1 (en) * 1999-06-29 2001-01-11 Multigene Biotech Gmbh cDNA sequences of two interacors FANCIP2 and FANCIP3 of the Fanconi anemia protein of complementation group A.
CN102770441A (en) * 2009-12-14 2012-11-07 肿瘤疗法科学股份有限公司 TMEM22 peptides and vaccines including the same
US8921056B2 (en) 2003-10-10 2014-12-30 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt
US8926970B2 (en) 2006-10-20 2015-01-06 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Rspondin antibodies as inhibiting factors of angiogenesis and vaculogenesis

Citations (2)

* 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
WO1997007198A2 (en) * 1995-08-11 1997-02-27 Genetics Institute, Inc. Dna sequences and secreted proteins encoded thereby

Patent Citations (2)

* 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
WO1997007198A2 (en) * 1995-08-11 1997-02-27 Genetics Institute, Inc. Dna sequences and secreted proteins encoded thereby

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ADAMS D. ET AL.: "3,400 new expressed sequence tags identify diversity of transcripts in human brain" NATURE GENETICS, vol. 4, no. 3, July 1993, pages 256-267, XP000674622 *
Database EMBL, entry HS139309, Accession number N78139, 4 April 1996 95% identity with Seq.ID:1 nt.901-1546 XP002068326 *
Database EMBL, entry HS19181, Accession number R10191, 21 April 1995 97% identity with Seq.ID:1 nt.1-401 XP002068324 cited in the application *
Database EMBL, entry HS492173, Accession number H26492, 15 July 1995 99% identity with Seq.ID:1 nt.170-828 XP002068325 cited in the application *
Database EMBL, entry HSC8235, Accession number C05823, 25 August 1996 98% identity with Seq.ID:1 nt.1262-1792 reverse orientation XP002068327 cited in the application *
Database EMBL, entry HSHBC4826, Accession number D83821, 14 April 1996 97% identity with Seq.ID:1 nt.419-961 XP002068382 *
GURU S.C. ET AL.: "A transcript map for the 2.8-Mb region containing the multiple endocrine neoplasia type 1 locus" GENOME RESEARCH, vol. 7, no. 7, July 1997, pages 725-735, XP002068381 *
JONG HWA KIM ET AL.: "Nucleotide sequence of a novel transcript confined in HeLa cell nucleus" NUCLEIC ACIDS RESEARCH, vol. 18, no. 3, 1990, page 669 XP002068323 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19929887A1 (en) * 1999-06-29 2001-01-11 Multigene Biotech Gmbh cDNA sequences of two interacors FANCIP2 and FANCIP3 of the Fanconi anemia protein of complementation group A.
US8921056B2 (en) 2003-10-10 2014-12-30 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt
US8951745B2 (en) 2003-10-10 2015-02-10 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt
US9081011B2 (en) 2003-10-10 2015-07-14 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-spondins) and/or Wnt
US8926970B2 (en) 2006-10-20 2015-01-06 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Rspondin antibodies as inhibiting factors of angiogenesis and vaculogenesis
US9226963B2 (en) 2006-10-20 2016-01-05 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Antagonist anti-Rspondin3 antibodies
US10273276B2 (en) 2006-10-20 2019-04-30 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Rspondins as modulators of angiogenesis and vasculogenesis
US10538563B2 (en) 2006-10-20 2020-01-21 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Rspondins as modulators of angiogenesis and vasculogenesis
CN102770441A (en) * 2009-12-14 2012-11-07 肿瘤疗法科学股份有限公司 TMEM22 peptides and vaccines including the same
US8697631B2 (en) 2009-12-14 2014-04-15 Oncotherapy Science, Inc. TMEM22 peptides and vaccines including the same

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WO1998041539A3 (en) 1998-11-26
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JP2001517941A (en) 2001-10-09
EP0971954A2 (en) 2000-01-19

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