WO1998014470A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998014470A2
WO1998014470A2 PCT/US1997/018032 US9718032W WO9814470A2 WO 1998014470 A2 WO1998014470 A2 WO 1998014470A2 US 9718032 W US9718032 W US 9718032W WO 9814470 A2 WO9814470 A2 WO 9814470A2
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WIPO (PCT)
Prior art keywords
polynucleotide
protein
seq
amino acid
clone
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PCT/US1997/018032
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French (fr)
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WO1998014470A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Vikki Spaulding
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Genetics Institute, Inc.
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Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to AU48094/97A priority Critical patent/AU4809497A/en
Publication of WO1998014470A2 publication Critical patent/WO1998014470A2/en
Publication of WO1998014470A3 publication Critical patent/WO1998014470A3/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

Definitions

  • the present invention provides novel proteins , along with therapeutic, diagnostic and research utilities for these proteins.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 28 to nucleotide 276; the nucleotide sequence of the full length protein coding sequence of clone AE402_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AE402_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:4 from nucleotide 61 to nucleotide 513; the nucleotide sequence of SEQ ID NO:4 from nucleotide 322 to nucleotide 513; the nucleotide sequence of the full length protein coding sequence of clone AE610_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AE610_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a 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:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 20 to nucleotide 523; the nucleotide sequence of the full length protein coding sequence of clone AH106_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AH106_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 130 to nucleotide 309; the nucleotide sequence of the full length protein coding sequence of clone AH196_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AH196_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 12 from nucleotide 69 to nucleotide 467; the nucleotide sequence of the full length protein coding sequence of clone AI6_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AI6_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AI6_li deposited under accession number ATCC 98190. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 69 to amino acid 133.
  • 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: 13 or the amino acid sequence of SEQ ID NO: 13 from amino acid 69 to amino acid 133.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 from nucleotide 55 to nucleotide 337; the nucleotide sequence of the full length protein coding sequence of clone AJ 13_1 i deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AJ13_1 i deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190.
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94.
  • 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: 17 or the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19 from nucleotide 33 to nucleotide 422; the nucleotide sequence of SEQ ID NO:19 from nucleotide 1 14 to nucleotide 422; the nucleotide sequence of the full length protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:22 from nucleotide 47 to nucleotide 517; the nucleotide sequence of SEQ ID NO:22 from nucleotide 116 to nucleotide 517; the nucleotide sequence of the full length protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AI142_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24 from nucleotide 312 to nucleotide 417; the nucleotide sequence of the full length protein coding sequence of clone AK604_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK604_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:27 from nucleotide 76 to nucleotide 372; the nucleotide sequence of the full length protein coding sequence of clone AK620_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK620_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:29 from nucleotide 367 to nucleotide 552; the nucleotide sequence of the full length protein coding sequence of clone AK650_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK650_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 from nucleotide 116 to nucleotide 310; the nucleotide sequence of SEQ ID NO:32 from nucleotide 173 to nucleotide 310; the nucleotide sequence of the full length protein coding sequence of clone AM226_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AM226_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:35 from nucleotide 281 to nucleotide 418; the nucleotide sequence of SEQ ID NO:35 from nucleotide 353 to nucleotide 418; the nucleotide sequence of the full length protein coding sequence of clone AR417_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AR4 I li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a 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:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 496 to nucleotide 583; the nucleotide sequence of SEQ LD NO:38 from nucleotide 565 to nucleotide 583; the nucleotide sequence of the full length protein coding sequence of clone AU43_1 i deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AU43_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:39;
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:41 from nucleotide 55 to nucleotide 405; the nucleotide sequence of SEQ ID NO:41 from nucleotide 148 to nucleotide 405; the nucleotide sequence of the full length protein coding sequence of clone AW60_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AW60_1 i deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190.
  • 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:42;
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:44 from nucleotide 337 to nucleotide 525; the nucleotide sequence of SEQ ID NO:44 from nucleotide 406 to nucleotide 525; the nucleotide sequence of the full length protein coding sequence of clone BA176_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BA176_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47 from nucleotide 536 to nucleotide 628; the nucleotide sequence of the full length protein coding sequence of clone BD140_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BD140_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:50 from nucleotide 303 to nucleotide 617; the nucleotide sequence of SEQ ID NO:50 from nucleotide 345 to nucleotide 617; the nucleotide sequence of the full length protein coding sequence of clone BD407_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BD407_li deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ D NO: 51 from amino acid 1 to amino acid 32.
  • 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:51 ;
  • protein comprises the amino acid sequence of SEQ ID NO:51 or the amino acid sequence of SEQ ID NO:51 from amino acid 1 to amino acid 32.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:52 from nucleotide 178 to nucleotide 534; the nucleotide sequence of the full length protein coding sequence of clone BF290_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BF290_1 i deposited under accession number ATCC 98190.
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • 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 Fig. 1 is a schematic representation of the pED6 and pNotS vectors used for deposit of clones disclosed herein.
  • Fig. 2 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AE610_li.
  • Fig. 3 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AH106_li, AM226_li.
  • Fig. 4 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AH196_li.
  • Fig. 5 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AI6_li.
  • Fig. 6 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AR417_li.
  • Fig. 7 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AW60_li.
  • Fig. 8 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: BD140_li.
  • Fig. 9 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: BF290_li.
  • Nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application. In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids.
  • the actual nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full length and mature) can then be determined from such nucleotide sequence.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence.
  • reported protein sequences include "Xaa” designators. These "Xaa” designators indicate either (1) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined more accurately).
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences 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 endoplpasmic reticulum.
  • AE402_li One protein of the present invention has been identified as protein "AE402_li”.
  • a partial cDNA clone encoding AE402_li was first isolated from a murine adult spleen cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • I.M.A.G.E. Consoritum library The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AE402_li”.
  • Applicants' methods identified clone AE402_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AE402_li as presently determined is reported in SEQ ID NO:l . What applicants believe is the proper reading frame and the predicted amino acid sequence of the AE402_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Additional nucleotide sequence from the 3' portion of AE402_li, including the polyA tail, is reported in SEQ ID NO:3.
  • Protein "AE610 li” One protein of the present invention has been identified as protein "AE610_li”.
  • a partial cDNA clone encoding AE610_li was first isolated from a murine adult spleen cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AE610_li”.
  • Applicants' methods identified clone AE610_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AE610_li as presently determined is reported in SEQ ID NO:4. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AE610_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:5.
  • Amino acids 1 to 87 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 88. Additional nucleotide sequence from the 3' portion of AE610_li, including the polyA tail, is reported in SEQ ID NO:6.
  • AH106_li One protein of the present invention has been identified as protein "AH106_li”.
  • a partial cDNA clone encoding AH106_li was first isolated from a murine fetal thymus cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified at GenBank accession number T81127.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AH106_li”.
  • Applicants' methods identified clone AH106_li as encoding a secreted protein.
  • the nucleotide sequence of AH106_li as presently determined is reported in SEQ ID NO: 1
  • AH196_li One protein of the present invention has been identified as protein "AH196_li”.
  • a partial cDNA clone encoding AH196_li was first isolated from a murine fetal thymus cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone AH196_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AH196_li as presently determined is reported in SEQ ID NO:9. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AH196_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 10. Additional nucleotide sequence from the
  • AI6_li One protein of the present invention has been identified as protein "AI6_li”.
  • a partial cDNA clone encoding AI6_li was first isolated from a human blood cell (Thl or Th2) cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • clone AI6_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AI6_1 i as presently determined is reported in SEQ ID NO: 12. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AI6_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:13. Additional nucleotide sequence from the 3' portion of AI6_li, including the polyA tail, is reported in SEQ ID NO: 14.
  • a partial cDNA clone encoding AJ13_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AJ13_li”.
  • Applicants' methods identified clone AJ13_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AJ13_li as presently determined is reported in SEQ ID NO:15.
  • An additional internal nucleotide sequence from AJ13_li as presently determined is reported in SEQ ID NO: 16. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO:17.
  • Additional nucleotide sequence from the 3' portion of AJ13_li, including the polyA tail, is reported in SEQ ID NO: 18. Protein "AJ27 li"
  • AJ27_li A partial cDNA clone encoding AJ27_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone AJ27_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AJ27_li as presently determined is reported in SEQ ID NO: 19. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AJ27_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
  • Amino acids 1 to 27 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28. Additional nucleotide sequence from the 3' portion of AJ27_li, including the polyA tail, is reported in SEQ ID NO:21.
  • AJ142_li One protein of the present invention has been identified as protein "AJ142_li”.
  • a partial cDNA clone encoding AJ142_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • AK604_li A partial cDNA clone encoding AK604_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified as "yc80gl l.rl Homo sapiens cDNA clone 22157 5'" (T64857, BlastN).
  • the sequence also showed at least some identity with a partial cDNA sequence identified as "H. sapiens partial cDNA sequence; clone c-lpgl 1 " (Z40033, BlastN).
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK604_li”.
  • AK604_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AK604_li as presently determined is reported in SEQ ID NO:24. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK604_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:25. Additional nucleotide sequence from the 3' portion of AK604_li, including the polyA tail, is reported in SEQ ID NO:26.
  • Protein "AK620 li” One protein of the present invention has been identified as protein "AK620_li”.
  • a partial cDNA clone encoding AK620_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone AK620_li as encoding a secreted protein.
  • AK620_li The nucleotide sequence of AK620_li as presently determined is reported in SEQ ID NO:27. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK620_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:28..
  • AK650_li A partial cDNA clone encoding AK650_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK650_li”.
  • Applicants' methods identified clone AK650_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AK650_ li as presently determined is reported in SEQ ID NO: 29. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK650_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:30. Additional nucleotide sequence from the
  • AK650_li 3' portion of AK650_li, including the polyA tail, is reported in SEQ ID NO:31.
  • AM226_li A partial cDNA clone encoding AM226_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM226_li”.
  • AM226_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AM226_li as presently determined is reported in SEQ ID NO:32. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AM226_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:33. Amino acids 1 to 19 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20. Additional nucleotide sequence from the 3' portion of AM226_li, including the polyA tail, is reported in SEQ ID NO:34.
  • AR417_li A partial cDNA clone encoding AR417_li was first isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • nucleotide sequence of the 5' portion of AR417_li as presently determined is reported in SEQ ID NO:35. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AR417_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:36. Amino acids 1 to 24 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 25. Additional nucleotide sequence from the 3' portion of AR417_li, including the polyA tail, is reported in SEQ ID NO:37.
  • AU43_li A partial cDNA clone encoding AU43_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone AU43_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AU43_li as presently determined is reported in SEQ ID NO:38. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AU43_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:39.
  • Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3' portion of AU43_li, including the polyA tail, is reported in SEQ ID NO:40.
  • AW60_li A partial cDNA clone encoding AW60_li was first isolated from a human ovary (PA-1 teratocarcinoma) cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AW60_li”.
  • Applicants' methods identified clone AW60_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AW60_li as presently determined is reported in SEQ ID NO:41. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AW60_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:42. Amino acids 1 to 31 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 32. Additional nucleotide sequence from the 3' portion of AW60_li, including the polyA tail, is reported in SEQ ID NO:43.
  • BA176_li A partial cDNA clone encoding BA176_li was first isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "BA176_li”.
  • Applicants' methods identified clone BA176_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of BA176_li as presently determined is reported in SEQ ID NO:44. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BA176_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:45.
  • Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3' portion of BA176_li, including the polyA tail, is reported in SEQ ID NO:46.
  • Protein "BD 140 li” One protein of the present invention has been identified as protein "BD140_li”.
  • a partial cDNA clone encoding BD140_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yn98c02.rl
  • Homo sapiens cDNA (H43507, Fasta), "yn67g04.rl Homo sapiens cDNA” (H22693, Fasta) and "yn82e07.sl Homo sapiens cDNA clone 174948 3'" (H38408, BlastN).
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3'
  • Applicants' methods identified clone BD140_li as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of BD140_li as presently determined is reported in SEQ ID NO:47. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BD140_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ED NO:48. Additional nucleotide sequence from the
  • BD140_li 3' portion of BD140_li, including the polyA tail, is reported in SEQ ID NO:49.
  • Protein "BD407 li” One protein of the present invention has been identified as protein "BD407_li”.
  • a partial cDNA clone encoding BD407_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone BD407_li as encoding a secreted protein.
  • Protein "BF290 li” One protein of the present invention has been identified as protein "BF290_li”.
  • a partial cDNA clone encoding BF290_li was first isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E.
  • Applicants' methods identified clone BF290_li as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of BF290_li as presently determined is reported in SEQ ID NO:52. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BF290_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:53. Additional nucleotide sequence from the 3' portion of BF290_li, including the polyA tail, is reported in SEQ ID NO:54. Deposit of Clones
  • Clones AE402_li, AE610_li, AH106_li, AH196_li, AI6_li, AJ13_li, AJ27_li, AJ142_li, AK604_li, AK620_li, AK650_li, AM226_li, AR417_li, AU43_li, AW60_li, BA176_li, BD140_li, BD407_li and BF290_li were deposited on October 2, 1996 with the American Type Culture Collection under accession number ATCC 98190, from which each clone comprising a particular polynucleotide is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit.
  • Each clone can be removed from the vector in which it was deposited by performing an EcoRI NotI digestion (5' cite, EcoRI; 3' cite, NotI) to produce the appropriate fragment for such clone.
  • Each clone was deposited in either the pED6 or pNotS vector depicted in Fig. 1. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' cite and EcoRI will produce the 3' cite 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 design of the oligonucleotide probe should preferably follow these parameters:
  • the oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g 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
  • SDS 100 ⁇ g/ml of yeast RNA
  • 10 mM EDTA approximately 10 mL per 150 mm filter.
  • the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1 % SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio/Technology JO, 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 of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • the sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides 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.
  • Species homologs of the disclosed proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed proteins; that is, naturally-occurring alternative forms of the isolated proteins which are identical, homologous or related to that encoded by the polynucleotides disclosed herein.
  • the isolated polynucleotide endcoing the protein of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman etal., Nucleic Acids Res. 1_9, 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 etal., Nucleic Acids Res. 1_9, 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.
  • 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.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo205 cells human Colo205 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 eukaryotes
  • prokaryotes such as bacteria.
  • 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.
  • 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.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • Kits for expression and purification of such fusion proteins 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 Kod
  • 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.
  • 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.
  • 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 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.
  • 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 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 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.
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (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.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen- specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1 , B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 59:11102- 11105 (1992).
  • murine models of GVHD can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr 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.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • 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.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981 ; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
  • Assays for T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631- 640, 1990.
  • Assays for lymphocyte survival apoptosis include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991 ; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1 :639-648, 1992.
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551 , 1991.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders
  • Assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81 :2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, LK. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. 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.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • 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. W091 /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 include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71 :382-84 (1978).
  • a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • FSH follicle stimulating hormone
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Nature 321 :779-782, 1986; Vale et al., Nature 321 -.116-119, 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 (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.
  • 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 may themselves be useful as inhibitors of receptor/ligand interactions.
  • Suitable assays for receptor-ligand activity include without limitation those described i 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.
  • 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 BL-l . 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 septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia- reperfusion injury such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS
  • 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, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • 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 When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • 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.
  • compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about O.l ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the 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).
  • KLH keyhole limpet hemocyanin
  • 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.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • matrices may be formed of materials presently in use for other implanted medical applications.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and TGF- ⁇ ), and insulinlike growth factor (IGF).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulinlike growth factor
  • the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF I may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 1 :
  • AAAAAA 246 INFORMATION FOR SEQ ID NO: 4:
  • Ser lie Ala Ala Gly Val Lys Pro Thr Lys Ser Leu lie lie Ser Ser 20 25 30
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 :
  • NTACTGGGCA TAATTATATN TTAATCATAT ATGGAAATGT GCAACATATG GTATTTGTTA 300
  • GAGTCCCATC GTGGWGGTAC TCAGTGGCAG TATGGAGCCG GCCTTCCACA GAGGAGATCT 180
  • CAAAGAAGGC CAGAACTGGC TGGAAAAGAA GGACGTGGTG GGAAGAGCAA GANGGTTTTT 420
  • MOLECULE TYPE protein
  • Lys Lys Tyr lie Thr Gly Thr Asp lie Leu Asp Xaa Xaa Leu Glu Asp 50 55 60 lie Leu Glu Ser lie Asn Ser lie Lys Ser Arg Leu Ser Lys Ser Gly 65 70 75 80
  • GGTAGCTTCA AACAGCTCAC AGTTAAGGAA GCTGTGCTGA ATGGTTTGGT GGCCACTGAG 300 GTGTTGATGT GGTTTTATGT CGGAGAGATT ATAGGCA 337
  • AGTACCAAGC CTTTCTGCTC CAGGTCAAGG AACCAGCAGC ACAAACACTG TTGGGGCAAC 420
  • AAGAGACTCT TTGCTGAAAT TGAATTGCAC TTATACATGT AAATTGTCAA CATGTAATTT 300
  • AAAAAAAAAA AAAAAAAA 318 (2) INFORMATION FOR SEQ ID NO: 32:
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 32:
  • GACTCTGCAG TCTACACTTC TCCTGTTACT GCTTGTGCCT CTGATAAAGC CAGCACCACC 180

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Abstract

Novel proteins are disclosed.

Description

SECRETED PROTEINS
FIELD OF THE INVENTION
The present invention provides novel proteins , along with therapeutic, diagnostic and research utilities for these proteins.
BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins that the present invention is directed.
SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a 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 : 1 from nucleotide 28 to nucleotide 276;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AE402_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AE402_1 i deposited under accession number ATCC 98190; (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AE402_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AE402_1 i deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 28 to nucleotide 276; the nucleotide sequence of the full length protein coding sequence of clone AE402_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AE402_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190.
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; and
(c) the amino acid sequence encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190; 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 protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 61 to nucleotide 513; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 322 to nucleotide 513;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AE610_1 i deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AE610_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:5; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:5 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:4 from nucleotide 61 to nucleotide 513; the nucleotide sequence of SEQ ID NO:4 from nucleotide 322 to nucleotide 513; the nucleotide sequence of the full length protein coding sequence of clone AE610_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AE610_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190.
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:5;
(b) fragments of the amino acid sequence of SEQ ID NO:5; and
(c) the amino acid sequence encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:5.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 20 to nucleotide 523;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AH 106_1 i deposited under accession number ATCC
98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AH 106_1 i deposited under accession number ATCC
98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 20 to nucleotide 523; the nucleotide sequence of the full length protein coding sequence of clone AH106_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AH106_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8; (b) fragments of the amino acid sequence of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 130 to nucleotide 309;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AH 196_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AH196_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 130 to nucleotide 309; the nucleotide sequence of the full length protein coding sequence of clone AH196_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AH196_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10; (b) fragments of the amino acid sequence of SEQ ID NO: 10; and
(c) the amino acid sequence encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a 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 69 to nucleotide 467;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AI6_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AI6_1 i deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AI6_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AI6_1 i deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 13;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 12 from nucleotide 69 to nucleotide 467; the nucleotide sequence of the full length protein coding sequence of clone AI6_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AI6_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AI6_li deposited under accession number ATCC 98190. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 69 to amino acid 133.
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 69 to amino acid 133; (c) fragments of the amino acid sequence of SEQ ID NO : 13 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone AI6_1 i deposited under accession number ATCC 98190; 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 69 to amino acid 133.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a 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 55 to nucleotide 337;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ 13_ 1 i deposited under accession number ATCC 98190; (d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ13_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:17; (h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 17 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16 from nucleotide 55 to nucleotide 337; the nucleotide sequence of the full length protein coding sequence of clone AJ 13_1 i deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AJ13_1 i deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94. 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) the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94;
(c) fragments of the amino acid sequence of SEQ ID NO: 17; and
(d) the amino acid sequence encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 33 to nucleotide 422; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 19 from nucleotide 114 to nucleotide 422;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190; (e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19 from nucleotide 33 to nucleotide 422; the nucleotide sequence of SEQ ID NO:19 from nucleotide 1 14 to nucleotide 422; the nucleotide sequence of the full length protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 20;
(b) fragments of the amino acid sequence of SEQ ID NO:20; and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:20.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:22;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22 from nucleotide 47 to nucleotide 517;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:22 from nucleotide 116 to nucleotide 517; (d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:23;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:23 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)- (g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:22 from nucleotide 47 to nucleotide 517; the nucleotide sequence of SEQ ID NO:22 from nucleotide 116 to nucleotide 517; the nucleotide sequence of the full length protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AI142_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190.
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:23; (b) fragments of the amino acid sequence of SEQ ID NO: 23; and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:23. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24 from nucleotide 312 to nucleotide 417;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK604_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK604_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK604_1 i deposited under accession number ATCC 98190; (g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:25;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:25 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24 from nucleotide 312 to nucleotide 417; the nucleotide sequence of the full length protein coding sequence of clone AK604_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK604_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:25; (b) fragments of the amino acid sequence of SEQ ID NO: 25; and
(c) the amino acid sequence encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:25. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:27;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:27 from nucleotide 76 to nucleotide 372;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK620_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190; (e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK620_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK620_1 i deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:28;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:28 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:27 from nucleotide 76 to nucleotide 372; the nucleotide sequence of the full length protein coding sequence of clone AK620_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK620_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190.
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:28; (b) fragments of the amino acid sequence of SEQ ID NO:28; and
(c) the amino acid sequence encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:28. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29 from nucleotide 367 to nucleotide 552; (c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK650_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK650_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:30;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:30 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:29 from nucleotide 367 to nucleotide 552; the nucleotide sequence of the full length protein coding sequence of clone AK650_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AK650_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190.
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:30; (b) fragments of the amino acid sequence of SEQ ID NO:30; and
(c) the amino acid sequence encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:30. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:32; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:32 from nucleotide 116 to nucleotide 310;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:32 from nucleotide 173 to nucleotide 310;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AM226_li deposited under accession number
ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AM226_li deposited under accession number
ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:33;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:33 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 from nucleotide 116 to nucleotide 310; the nucleotide sequence of SEQ ID NO:32 from nucleotide 173 to nucleotide 310; the nucleotide sequence of the full length protein coding sequence of clone AM226_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AM226_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190. 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:33; (b) fragments of the amino acid sequence of SEQ ID NO:33; and
(c) the amino acid sequence encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:33. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35 from nucleotide 281 to nucleotide 418;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 35 from nucleotide 353 to nucleotide 418;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AR417_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AR417_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:36; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:36 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:35 from nucleotide 281 to nucleotide 418; the nucleotide sequence of SEQ ID NO:35 from nucleotide 353 to nucleotide 418; the nucleotide sequence of the full length protein coding sequence of clone AR417_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AR4 I li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190.
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:36;
(b) fragments of the amino acid sequence of SEQ ID NO:36; and
(c) the amino acid sequence encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:36.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:38;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 38 from nucleotide 496 to nucleotide 583;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 565 to nucleotide 583;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AU43_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AU43_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AU43_1 i deposited under accession number ATCC 98190; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:39;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:39 having biological activity; (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 496 to nucleotide 583; the nucleotide sequence of SEQ LD NO:38 from nucleotide 565 to nucleotide 583; the nucleotide sequence of the full length protein coding sequence of clone AU43_1 i deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AU43_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:39;
(b) fragments of the amino acid sequence of SEQ ID NO:39; and
(c) the amino acid sequence encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:39.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:41 from nucleotide 55 to nucleotide 405;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 148 to nucleotide 405; (d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AW60_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AW60_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:42;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:42 having biological activity; (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:41 from nucleotide 55 to nucleotide 405; the nucleotide sequence of SEQ ID NO:41 from nucleotide 148 to nucleotide 405; the nucleotide sequence of the full length protein coding sequence of clone AW60_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone AW60_1 i deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190.
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:42;
(b) fragments of the amino acid sequence of SEQ ID NO:42; and
(c) the amino acid sequence encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:42. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:44; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:44 from nucleotide 337 to nucleotide 525;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:44 from nucleotide 406 to nucleotide 525;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone B A 176_ 1 i deposited under accession number ATCC
98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BA176_1 i deposited under accession number ATCC
98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:45;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:45 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:44 from nucleotide 337 to nucleotide 525; the nucleotide sequence of SEQ ID NO:44 from nucleotide 406 to nucleotide 525; the nucleotide sequence of the full length protein coding sequence of clone BA176_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BA176_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190. 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:45; (b) fragments of the amino acid sequence of SEQ ID NO:45; and
(c) the amino acid sequence encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:45. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:47;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:47 from nucleotide 536 to nucleotide 628;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BD140_1 i deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BD140_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BD 140_1 i deposited under accession number ATCC 98190 ;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:48;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:48 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47 from nucleotide 536 to nucleotide 628; the nucleotide sequence of the full length protein coding sequence of clone BD140_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BD140_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190.
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:48; (b) fragments of the amino acid sequence of SEQ ID NO:48; and
(c) the amino acid sequence encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:48. In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:50;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO : 50 from nucleotide 303 to nucleotide 617;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:50 from nucleotide 345 to nucleotide 617;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BD407_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BD407_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:51 ; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:51 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:50 from nucleotide 303 to nucleotide 617; the nucleotide sequence of SEQ ID NO:50 from nucleotide 345 to nucleotide 617; the nucleotide sequence of the full length protein coding sequence of clone BD407_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BD407_li deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ D NO: 51 from amino acid 1 to amino acid 32.
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:51 ;
(b) the amino acid sequence of SEQ ID NO:51 from amino acid 1 to amino acid 32;
(c) fragments of the amino acid sequence of SEQ ID NO:51 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:51 or the amino acid sequence of SEQ ID NO:51 from amino acid 1 to amino acid 32.
In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:52;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:52 from nucleotide 178 to nucleotide 534; (c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BF290_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BF290_li deposited under accession number ATCC 98190;
(0 a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BF290_1 i deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:53;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:53 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:52 from nucleotide 178 to nucleotide 534; the nucleotide sequence of the full length protein coding sequence of clone BF290_li deposited under accession number ATCC 98190; or the nucleotide sequence of the mature protein coding sequence of clone BF290_1 i deposited under accession number ATCC 98190. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190.
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:53; (b) fragments of the amino acid sequence of SEQ ID NO:53; and
(c) the amino acid sequence encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:53. 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 FIGURES Fig. 1 is a schematic representation of the pED6 and pNotS vectors used for deposit of clones disclosed herein.
Fig. 2 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AE610_li.
Fig. 3 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AH106_li, AM226_li.
Fig. 4 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AH196_li.
Fig. 5 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AI6_li. Fig. 6 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AR417_li.
Fig. 7 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: AW60_li.
Fig. 8 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: BD140_li.
Fig. 9 is an autoradiograph evidencing the expression of the following clone(s) disclosed herein: BF290_li.
DETAILED DESCRIPTION ISOLATED PROTEINS
Nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application. In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids. The actual nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence.
For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing. Because of the partial ambiguity in reported sequence information, reported protein sequences include "Xaa" designators. These "Xaa" designators indicate either (1) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined more accurately).
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences 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 endoplpasmic reticulum.
Protein "AE402 li"
One protein of the present invention has been identified as protein "AE402_li". A partial cDNA clone encoding AE402_li was first isolated from a murine adult spleen cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yh02hl2.rl Homo sapiens cDNA clone 42238 5'" (R60758, BlastN) and "yh02hl2.sl Homo sapiens cDNA clone 42238 3'" (R60759, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the
I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AE402_li".
Applicants' methods identified clone AE402_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AE402_li as presently determined is reported in SEQ ID NO:l . What applicants believe is the proper reading frame and the predicted amino acid sequence of the AE402_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Additional nucleotide sequence from the 3' portion of AE402_li, including the polyA tail, is reported in SEQ ID NO:3.
Protein "AE610 li" One protein of the present invention has been identified as protein "AE610_li". A partial cDNA clone encoding AE610_li was first isolated from a murine adult spleen cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yfl9g02.rl Homo sapiens cDNA" (R08399, Fasta), "yw68d09.sl Homo sapiens cDNA clone 257393 3'" (N27174, BlastN), "yil0a04.rl Homo sapiens cDNA" (R62698, Fasta) and "yh78el0.sl Homo sapiens cDNA clone 135882 3"' (R33815, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AE610_li".
Applicants' methods identified clone AE610_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AE610_li as presently determined is reported in SEQ ID NO:4. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AE610_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:5. Amino acids 1 to 87 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 88. Additional nucleotide sequence from the 3' portion of AE610_li, including the polyA tail, is reported in SEQ ID NO:6.
Protein "AH 106 li"
One protein of the present invention has been identified as protein "AH106_li". A partial cDNA clone encoding AH106_li was first isolated from a murine fetal thymus cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified at GenBank accession number T81127. The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AH106_li".
Applicants' methods identified clone AH106_li as encoding a secreted protein. The nucleotide sequence of AH106_li as presently determined is reported in SEQ ID
NO:7. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AH106_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8..
Protein "AH 196 li"
One protein of the present invention has been identified as protein "AH196_li". A partial cDNA clone encoding AH196_li was first isolated from a murine fetal thymus cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yjl2f04.rl Homo sapiens cDNA clone 148543 5'" (H12523, BlastN) and "yjl2f04.sl Homo sapiens cDNA clone 148543 3'" (H12470, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AH196_li".
Applicants' methods identified clone AH196_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AH196_li as presently determined is reported in SEQ ID NO:9. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AH196_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 10. Additional nucleotide sequence from the
3' portion of AH196_li, including the polyA tail, is reported in SEQ ID NO:l 1.
Protein "AI6 li"
One protein of the present invention has been identified as protein "AI6_li". A partial cDNA clone encoding AI6_li was first isolated from a human blood cell (Thl or Th2) cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yj42h04.rl Homo sapiens cDNA" (H03613, Fasta) and "yx60fl0.sl Homo sapiens cDNA clone 266155 3'" (N21637, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AI6_li".
Applicants' methods identified clone AI6_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AI6_1 i as presently determined is reported in SEQ ID NO: 12. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AI6_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:13. Additional nucleotide sequence from the 3' portion of AI6_li, including the polyA tail, is reported in SEQ ID NO: 14.
Protein "AJ13 li"
One protein of the present invention has been identified as protein "AJ13_li". A partial cDNA clone encoding AJ13_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yo61h02.rl Homo sapiens cDNA clone 182451 5'" (H42116, BlastN), "yr84a08.rl Homo sapiens cDNA clone 211958 5'" (H75363, BlastN) and "yg83h03.sl Homo sapiens cDNA clone 40148 3'" (R53978, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AJ13_li".
Applicants' methods identified clone AJ13_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AJ13_li as presently determined is reported in SEQ ID NO:15. An additional internal nucleotide sequence from AJ13_li as presently determined is reported in SEQ ID NO: 16. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ID NO:17. Additional nucleotide sequence from the 3' portion of AJ13_li, including the polyA tail, is reported in SEQ ID NO: 18. Protein "AJ27 li"
One protein of the present invention has been identified as protein "AJ27_li". A partial cDNA clone encoding AJ27_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yx25h01.rl Homo sapiens cDNA clone 262897 5'" (N28373, BlastN) and "yx62d05.rl Homo sapiens cDNA clone 266313 5'" (N35654, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AJ27_li".
Applicants' methods identified clone AJ27_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AJ27_li as presently determined is reported in SEQ ID NO: 19. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AJ27_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 1 to 27 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28. Additional nucleotide sequence from the 3' portion of AJ27_li, including the polyA tail, is reported in SEQ ID NO:21.
Protein "AJ 142 li"
One protein of the present invention has been identified as protein "AJ142_li". A partial cDNA clone encoding AJ142_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yq85bl2.rl Homo sapiens cDNA clone 202559 5'" (H53268, BlastN) and "yq85bl2.sl Homo sapiens cDNA clone 2025593'" (H53269, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AJ142_li". Applicants' methods identified clone AJ142_li as encoding a secreted protein.
The nucleotide sequence of AJ142_li as presently determined is reported in SEQ ID
NO:22. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AJ142_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:23. Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24.
Protein "AK604 li"
One protein of the present invention has been identified as protein "AK604_li". A partial cDNA clone encoding AK604_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified as "yc80gl l.rl Homo sapiens cDNA clone 22157 5'" (T64857, BlastN). The sequence also showed at least some identity with a partial cDNA sequence identified as "H. sapiens partial cDNA sequence; clone c-lpgl 1 " (Z40033, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK604_li".
Applicants' methods identified clone AK604_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AK604_li as presently determined is reported in SEQ ID NO:24. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK604_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:25. Additional nucleotide sequence from the 3' portion of AK604_li, including the polyA tail, is reported in SEQ ID NO:26.
Protein "AK620 li" One protein of the present invention has been identified as protein "AK620_li". A partial cDNA clone encoding AK620_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "ye7607.rl Homo sapiens cDNA clone 123684 5'" (R02637, BlastN) and "yx90e05.sl Homo sapiens cDNA clone 2690243'" (N26101 , BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK620_li".
Applicants' methods identified clone AK620_li as encoding a secreted protein.
The nucleotide sequence of AK620_li as presently determined is reported in SEQ ID NO:27. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK620_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:28..
Protein "AK650 li"
One protein of the present invention has been identified as protein "AK650_li". A partial cDNA clone encoding AK650_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yp60g06.rl Homo sapiens cDNA clone 191866 5'" (H40407, BlastN) and "yp60g06.sl Homo sapiens cDNA clone 191866 3'" (H40350, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the
I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK650_li".
Applicants' methods identified clone AK650_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AK650_ li as presently determined is reported in SEQ ID NO: 29. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK650_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:30. Additional nucleotide sequence from the
3' portion of AK650_li, including the polyA tail, is reported in SEQ ID NO:31.
Protein "AM226 li"
One protein of the present invention has been identified as protein "AM226_li". A partial cDNA clone encoding AM226_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yf09a01.rl Homo sapiens cDNA clone 126312 5'" (R06469, BlastN) and "yy49b06.sl Homo sapiens cDNA clone 276851 3'" (N39415, BlastN). The sequence also showed some similarity with bovine osteoinductuve factor (OIF) (M37974, BlastN), with which it may share some activity. The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM226_li".
Applicants' methods identified clone AM226_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AM226_li as presently determined is reported in SEQ ID NO:32. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AM226_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:33. Amino acids 1 to 19 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20. Additional nucleotide sequence from the 3' portion of AM226_li, including the polyA tail, is reported in SEQ ID NO:34.
Protein "AR417 li"
One protein of the present invention has been identified as protein "AR417_li". A partial cDNA clone encoding AR417_li was first isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified at GenBank accession numbers R18973, R42209 ("yf89g09.sl Homo sapiens cDNA clone 29781 3'"), R12416 ("yf56a02.rl Homo sapiens cDNA clone 26106 5'") and R15309 ("yf89g09.rl Homo sapiens cDNA"). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AR417_li". Applicants' methods identified clone AR417_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AR417_li as presently determined is reported in SEQ ID NO:35. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AR417_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:36. Amino acids 1 to 24 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 25. Additional nucleotide sequence from the 3' portion of AR417_li, including the polyA tail, is reported in SEQ ID NO:37.
Protein "AU43 li"
One protein of the present invention has been identified as protein "AU43_li". A partial cDNA clone encoding AU43_li was first isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yi49f07.rl Homo sapiens cDNA clone 142597 5'" (R70850, BlastN) and "yd68e02.sl Homo sapiens cDNA clone 1134023'" (T78464, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AU43_li".
Applicants' methods identified clone AU43_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of AU43_li as presently determined is reported in SEQ ID NO:38. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AU43_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:39. Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3' portion of AU43_li, including the polyA tail, is reported in SEQ ID NO:40.
Protein "AW60 li"
One protein of the present invention has been identified as protein "AW60_li". A partial cDNA clone encoding AW60_li was first isolated from a human ovary (PA-1 teratocarcinoma) cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "ym57fl l.rl Homo sapiens cDNA clone 52343 5'" (H23492, BlastN), "ym57f08.rl Homo sapiens cDNA" (H23390, Fasta) and "ym57f 11.sl Homo sapiens cDNA clone 52343 3'" (H23494, BlastN). The sequence also showed at least some identity with a sequence identified as "Homo sapiens clone S31il25" (L40397, Fasta)The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AW60_li".
Applicants' methods identified clone AW60_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of AW60_li as presently determined is reported in SEQ ID NO:41. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AW60_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:42. Amino acids 1 to 31 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 32. Additional nucleotide sequence from the 3' portion of AW60_li, including the polyA tail, is reported in SEQ ID NO:43.
Protein "B A 176 li"
One protein of the present invention has been identified as protein "BA176_li". A partial cDNA clone encoding BA176_li was first isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yi75gl l.rl Homo sapiens cDNA" (R77409, Fasta), "yj50M2.rl Homo sapiens cDNA" (H03089, Fasta) and "yi75gl l.sl Homo sapiens cDNA clone 1451243'" (R77410, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "BA176_li".
Applicants' methods identified clone BA176_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of BA176_li as presently determined is reported in SEQ ID NO:44. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BA176_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:45. Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3' portion of BA176_li, including the polyA tail, is reported in SEQ ID NO:46.
Protein "BD 140 li" One protein of the present invention has been identified as protein "BD140_li". A partial cDNA clone encoding BD140_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yn98c02.rl
Homo sapiens cDNA" (H43507, Fasta), "yn67g04.rl Homo sapiens cDNA" (H22693, Fasta) and "yn82e07.sl Homo sapiens cDNA clone 174948 3'" (H38408, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3'
UTR (including a polyA tail). This full-length clone is also referred to herein as "BD140_li".
Applicants' methods identified clone BD140_li as encoding a secreted protein.
The nucleotide sequence of the 5' portion of BD140_li as presently determined is reported in SEQ ID NO:47. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BD140_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ED NO:48. Additional nucleotide sequence from the
3' portion of BD140_li, including the polyA tail, is reported in SEQ ID NO:49.
Protein "BD407 li" One protein of the present invention has been identified as protein "BD407_li". A partial cDNA clone encoding BD407_li was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "ys65a05.rl Homo sapiens cDNA" (H84524, Fasta) and "yzl5h02.sl Homo sapiens cDNA clone 283155 3'" (N51349, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "BD407_li".
Applicants' methods identified clone BD407_li as encoding a secreted protein.
The nucleotide sequence of BD407_li as presently determined is reported in SEQ ID
NO: 50. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BD407_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ LD NO:51. Amino acids 1 to 14 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 15.
Protein "BF290 li" One protein of the present invention has been identified as protein "BF290_li". A partial cDNA clone encoding BF290_li was first isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with ESTs reported by the I.M.A.G.E. Consortium identified as "yhl0f04.rl Homo sapiens cDNA" (R61165, Fasta) and "yy35dl2.sl Homo sapiens cDNA clone 273239 3'" (N33175, BlastN). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consoritum library. The clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "BF290_li".
Applicants' methods identified clone BF290_li as encoding a secreted protein. The nucleotide sequence of the 5' portion of BF290_li as presently determined is reported in SEQ ID NO:52. What applicants believe is the proper reading frame and the predicted amino acid sequence of the BF290_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:53. Additional nucleotide sequence from the 3' portion of BF290_li, including the polyA tail, is reported in SEQ ID NO:54. Deposit of Clones
Clones AE402_li, AE610_li, AH106_li, AH196_li, AI6_li, AJ13_li, AJ27_li, AJ142_li, AK604_li, AK620_li, AK650_li, AM226_li, AR417_li, AU43_li, AW60_li, BA176_li, BD140_li, BD407_li and BF290_li were deposited on October 2, 1996 with the American Type Culture Collection under accession number ATCC 98190, from which each clone comprising a particular polynucleotide is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit.
Each clone can be removed from the vector in which it was deposited by performing an EcoRI NotI digestion (5' cite, EcoRI; 3' cite, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNotS vector depicted in Fig. 1. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' cite and EcoRI will produce the 3' cite 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.
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 JO, 773-778 (1992) and in R.S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention. The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
Species homologs of the disclosed proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed proteins; that is, naturally-occurring alternative forms of the isolated proteins which are identical, homologous or related to that encoded by the polynucleotides disclosed herein. The isolated polynucleotide endcoing the protein of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman etal., Nucleic Acids Res. 1_9, 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 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 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
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 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 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 fnterleukin 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, 59:11102- 11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr 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 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, LK. 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. W091 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71 :382-84 (1978). Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Nature 321 :779-782, 1986; Vale et al., Nature 321 -.116-119, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
Receptor Li gand 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 i 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.
An ti -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 BL-l . Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
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, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in 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.lμg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the 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 insulinlike 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 LaVallie, Edward Racie, Lisa Merberg, David Treacy, Maurice Spaulding, Vikki
(ii) TITLE OF INVENTION: SECRETED PROTEINS (iii) NUMBER OF SEQUENCES: 54
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Brown, Scott A.
(B) REGISTRATION NUMBER: 32,724
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8224
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
AAGCTTGGGG TTTTCTGGGC TACTACGATG GCGATGAGTT TCGAGTGGCC GTGGCAGTAC 60
CGCTTCCCGC CCTTCTTTAC GTTACAGCCG AACGTGGACA CCCGGCAGAA GCAGCTGGCC 120
GCCTGGTGCT CTCTGGTTCT GTCCTTCTGC CGCCTGCACA AACAGTCCAG CATGACGGTG 180
ATGGAAGCCC AGGAGAGCCC GCTTTTCAAC AACGTCAAGC TACAGCGGAA ACTTCCTGTG 240
GAGTCAATTC AGATTGTATT AGAAGAACTG AGAAAG 276 ( 2 ) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Ala Met Ser Phe Glu Trp Pro Trp Gin Tyr Arg Phe Pro Pro Phe 1 5 10 15
Phe Thr Leu Gin Pro Asn Val Asp Thr Arg Gin Lys Gin Leu Ala Ala 20 25 30
Trp Cys Ser Leu Val Leu Ser Phe Cys Arg Leu His Lys Gin Ser Ser 35 40 45
Met Thr Val Met Glu Ala Gin Glu Ser Pro Leu Phe Asn Asn Val Lys 50 55 60
Leu Gin Arg Lys Leu Pro Val Glu Ser lie Gin lie Val Leu Glu Glu 65 70 75 80
Leu Arg Lys
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
GTGAGTACAT ACACACANGC GCNTGCAGCA CANGATTNTG TCTCATCGTC NTCCCACCCN 60
NNNNGGNGNN GNTGCCTCCC TTAGTCAGGN GANGATGNAT CCTTTCCNAG GGGNTGGGGG 120
GNANCATTGG ATGCGGGCAG CNTTCCAGGC AANATGAAGA TNGGAGGCCC ACGGGCATGG 180
CAGTGAGAGG NGTGGCCCCA CACNGATTTA TGATNTTGAA ATCTCAACTC CCAAAAAAGA 240
AAAAAA 246 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 632 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
AGCTTCGGAA TAATAATTTT GGCAAATCTA TCTTCTGAAC CACTCATTTC TGTGGTCTTA 60
ATGGCTCCAA TTTGGGGACC AATAATGTTC ATTGTCTCAG GATCCCTGTC AATTGCAGCA 120
GGAGTGAAAC CTACAAAAAG CCTGATCATC AGCAGTCTAA CTCTGAACAC TATCACCTCT 180
GTGTTGGCTG CAACTGCAAG CATAATGGGT GTAGTCAGTG TGGCTGTGGG TTCACAGTTT 240
CCGTTTCGGT ATAATTATAC AATCACCAAG GGTTTGGATA TTTTGATGTT AATTTTAAAT 300
ATGCTAGAAT TCTGCATTGC TGTGTCCATC TCTGCTTTTG GATGTAAAGC TTCCTGTTGT 360
AACTCCAGCG AGGTTCTTGT AGTGCTACCA TCAAATCCTG CTGTGACTGT GATGGCACCC 420
CCCACACCAC TTAATGAAGG TTTGAGGCCA CCAAAAGATC AACAGACAAA TGCTCCAGAA 480
ATCTATGCTG ACTGTGACAC AAGAAGCCTC ACATGAAGAA ATTACCAGTA TCCAACTTCG 540
ATACTGATAG ACTTGTTGAT ATTATTATTA TATGTAATCC AATTATGAAC TGTGTGTGTA 600
TAGAGAGATA ATAAATTCAA AATTATGTTC TC 632 (2) INFORMATION FOR SEQ ID NO : 5 : ( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 151 amino acids
( B ) TYPE : amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
Met Ala Pro lie Trp Gly Pro lie Met Phe lie Val Ser Gly Ser Leu 1 5 10 15
Ser lie Ala Ala Gly Val Lys Pro Thr Lys Ser Leu lie lie Ser Ser 20 25 30
Leu Thr Leu Asn Thr lie Thr Ser Val Leu Ala Ala Thr Ala Ser lie 35 40 45
Met Gly Val Val Ser Val Ala Val Gly Ser Gin Phe Pro Phe Arg Tyr 50 55 60
Asn Tyr Thr lie Thr Lys Gly Leu Asp lie Leu Met Leu lie Leu Asn 65 70 75 80
Met Leu Glu Phe Cys lie Ala Val Ser lie Ser Ala Phe Gly Cys Lys 85 90 95
Ala Ser Cys Cys Asn Ser Ser Glu Val Leu Val Val Leu Pro Ser Asn 100 105 110
Pro Ala Val Thr Val Met Ala Pro Pro Thr Pro Leu Asn Glu Gly Leu 115 120 125
Arg Pro Pro Lys Asp Gin Gin Thr Asn Ala Pro Glu lie Tyr Ala Asp 130 135 140
Cys Asp Thr Arg Ser Leu Thr 145 150
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 365 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
CTATGGGGAC CAAAGTGNTT TTTCNTTCAG GAAGTGGAGA TGCATGGCCA TCTCCCCCTC 60
CCTTTTTCCT TCTCNTGNTT TTCTTTCCCC ATAGAAAGTA CCTTGAAGTA GCACAGTCCG 120
TCCTTGCATG TGCNCGNGCT NTCNTTTGAG TAAAAGTATA CATGGAGTAA AAATCATATT 180
AAGCATCAGA TTCAACTTAT ATTTTNTATT TCATNTTCTT CCTTTCCCTT CTCCCACNTT 240
NTACTGGGCA TAATTATATN TTAATCATAT ATGGAAATGT GCAACATATG GTATTTGTTA 300
AATACGTTTG TTTTTATTGC AGAGCAAAAA TAAATCAAAT TAGAAGCAAA AAAAAAAAAA 360
AAAAA 365 (2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 689 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
CCCANAGAGN CCTAGGAAGA TGAACAAACG ACAGCTCTAC TACCAGGTTT TAAACTTTGC 60
CATGATCGTG TCTTCTGCGC TCATGATCTG GAAAGGCCTG ATTGTTCTCA CGGGCAGCGA 120
GAGTCCCATC GTGGWGGTAC TCAGTGGCAG TATGGAGCCG GCCTTCCACA GAGGAGATCT 180
BCTGTTCCTC ACGAATTTCC GGGAGGACCC CATCAGAGCT GGTGAAATAG TTGTTTTTAA 240
GGTTGAAGGA AGAGACATTC CGATAGTTCA CAGAGTAATC AAGGTTCATG AAAAAGATAA 300
TGGTGACATC AARTTTCTGA CTAAAGGAGA TAATAATGAA GTYGATGATA GAGGCTTGTA 360
CAAAGAAGGC CAGAACTGGC TGGAAAAGAA GGACGTGGTG GGAAGAGCAA GANGGTTTTT 420
ACCATATGTT GGTATGGTCA CCATAATAAT GAATGACTAT CCAAAATTCA AKTATGCTCT 480
TTTGGCTGTA ATGGGTGCAT ATGTGTTACT AAAACGTGAA TCCTAAAATG AGAAGCAGTT 540
CCTGGGACCA GATTGAAATG AATTCTGTTG AAAAAGAGAA AAACTAATAT ATTTGAGATG 600
TTCCATTTTC TGTATAAAAG GGAACAGTGT GGAGATGTTT TTGTCTTGTC CAAATAAAAG 660
ATTCACCAGT AAAAAAAAAA AAAAAAAAA 689 ( 2 ) INFORMATION FOR SEQ ID NO : 8 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 168 amino acids
( B ) TYPE : amino acid
( C ) STRANDEDNESS :
( D) TOPOLOGY : l inear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Asn Lys Arg Gin Leu Tyr Tyr Gin Val Leu Asn Phe Ala Met lie 1 5 10 15
Val Ser Ser Ala Leu Met lie Trp Lys Gly Leu lie Val Leu Thr Gly 20 25 30
Ser Glu Ser Pro lie Val Xaa Val Leu Ser Gly Ser Met Glu Pro Ala 35 40 45
Phe His Arg Gly Asp Leu Leu Phe Leu Thr Asn Phe Arg Glu Asp Pro 50 55 60 lie Arg Ala Gly Glu lie Val Val Phe Lys Val Glu Gly Arg Asp lie 65 70 75 80
Pro lie Val His Arg Val lie Lys Val His Glu Lys Asp Asn Gly Asp 85 90 95 lie Lys Phe Leu Thr Lys Gly Asp Asn Asn Glu Val Asp Asp Arg Gly 100 105 110
Leu Tyr Lys Glu Gly Gin Asn Trp Leu Glu Lys Lys Asp Val Val Gly 115 120 125
Arg Ala Arg Xaa Phe Leu Pro Tyr Val Gly Met Val Thr lie lie Met 130 135 140
Asn Asp Tyr Pro Lys Phe Xaa Tyr Ala Leu Leu Ala Val Met Gly Ala 145 150 155 160
Tyr Val Leu Leu Lys Arg Glu Ser 165
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
CTCTCCCCCC CCCCTCTCTC TCTCTCTCGC ATACTAACTA GGTTTGACTG TATTACTCGT 60
ACCAGATTTA AAATTAGACT AGCCTTGCCA CAACGCCCTA CTGAGAGGTA CTGTCGAACT 120
GTAGACAGCA TGATGTTCTT TGATGGTGAA AGTCTAAATC TGGACCGTGT TCAGAGATAC 180
CAAATGATGA GGCTGAAAAG GGGAAAGGGG GTTCTTCAGT CTCTTCTTCT TCTTCTTTTT 240
ATTTTTTTTT CCATGATGTT TTCTCTATGG CCAGTGCAAA TGGTGTTGTC ACCCTTGCAT 300
GTTGCCAAC 309 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Met Phe Phe Asp Gly Glu Ser Leu Asn Leu Asp Arg Val Gin Arg 1 5 10 15
Tyr Gin Met Met Arg Leu Lys Arg Gly Lys Gly Val Leu Gin Ser Leu 20 25 30
Leu Leu Leu Leu Phe lie Phe Phe Ser Met Met Phe Ser Leu Trp Pro 35 40 45
Val Gin Met Val Leu Ser Pro Leu His Val Ala Asn 50 55 60
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 257 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AGGTCTCTCT GGTTCTTTCT ATATCATCAT TTTATTATTA TGTCCTAATA TAAAGTACTG 60
GCTCATAGGG CCAGGGTATT ATTATAGAAT ATTATTNTCG CATGTAAACA AAGATATCTT 120
TGCTTTAAGA TGTGAGAAGA AATGAATTTA CTTTGTTTGC ATTAAGTTAN GGAAGAGTTG 180
TAATATATAC TTTAAGAAAG AAGAGAAGAA AACTAGTATC TN AAGCGGT AAAAAAAAAA 240
AAAAAAAAAA AAAAAAA 257 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CACGAGGATT GATTTCCATC TTGCCTCTCC ANAAGGCAAA ACCTTAGTTT TTGAACAAAG 60
AAAATCAGAT GGAGTTCACA CTGNTANANA CTGAANTTGG TGATTACATG TTCTGCTTTG 120
ACAATACATT CAGCACCATT TCTGAGAANG TGATTTTCTT TGAATTAATC CTGGATAATA 180
TGGGAGAACA GGCACAAGAA CAAGAAGATT GGAAGAAATA TATTACTGGC ACAGATATAT 240
TGGATNTNAN NCTGGAAGAC ATCCTGGAAT CCATCAACAG CATCAAGTCC AGACTAAGCA 300
AAAGTGGGCA CATACAAACT CTGCTTAGAG CATTTGAAGC TCGTGATCGA AACATACAAG 360
AAAGCAACTT TGATAGAGTC AATTTCTGGT CTATGGTTAA TTTAGTGGTC ATGGTGGTGG 420
TGTCAGCCAT TCAAGTTTAT ATGCTGAAGA GTCTGTTTGA AGATAAG 467 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Glu Phe Thr Leu Xaa Xaa Thr Glu Xaa Gly Asp Tyr Met Phe Cys 1 5 10 15
Phe Asp Asn Thr Phe Ser Thr lie Ser Glu Xaa Val lie Phe Phe Glu 20 25 30
Leu lie Leu Asp Asn Met Gly Glu Gin Ala Gin Glu Gin Glu Asp Trp 35 40 45
Lys Lys Tyr lie Thr Gly Thr Asp lie Leu Asp Xaa Xaa Leu Glu Asp 50 55 60 lie Leu Glu Ser lie Asn Ser lie Lys Ser Arg Leu Ser Lys Ser Gly 65 70 75 80
His lie Gin Thr Leu Leu Arg Ala Phe Glu Ala Arg Asp Arg Asn lie 85 90 95
Gin Glu Ser Asn Phe Asp Arg Val Asn Phe Trp Ser Met Val Asn Leu 100 105 110
Val Val Met Val Val Val Ser Ala He Gin Val Tyr Met Leu Lys Ser 115 120 125
Leu Phe Glu Asp Lys 130
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
TGTTTGAAGA TAAGAGGAAA AGTAGAACTT AAAACTCCAA ACTAGAGNAC GTAACATTGA 60
AAAATGAGGC ATAAAAATGC AATAAACTGT TACAGTCAAG ACCATTAATG GTNTTNTCCA 120
AAATATTTTG AGATATAAAA GTAGGAAACA GGTATAATTT TAATGTGAAA ATTAAGTNTT 180
CACTTTCTGT GCAAGTAATC CTGCTGATCC AGTTGTACTT AAGTGTGTAA CAGGAATATT 240 TTGCAGAATA TAGGTTTAAC TGAATGAAGC CATATTAATA ACTGCATTTT CCTAACTTTG 300 AAAAATTTTG CAAATGTCTT AGGTGATTTA AATAAATGAG TATTGGGCCT AATTGCAAAA 360 AAAAAAAAAA AAAAAAAAAA AAAAAAA 387
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GAATTCTCTT GAAGNTGGGG GGTGCNGGNN GGGGAAANCG NNTCTCCNNT CCANAAGCGG 60
GGGCCNTTTT GTCCGTNNNC TTGTGNAAAA AANCCCGGNG NTGGTGAACG CTGNTNTTAN 120
TTACTCCAAA CCTCGANTGG NCNNTTNGTG GTNCNNCGCC GAGGNTGANN TGGNTCCCCC 180
CCCCCCTGNT NNAATNCCNA AAACTNTTCN GAACCCGAAA ANAATTNTCC ATTCTGCCNN 240
NANTGGTTTC NTCCNNCNNC TCCTNATTAA AGAAGCNNT 279 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGCGGGTGAC ATTCAGCCGG CGGTTCGGGG GGACGGANTC TCCATTCCAG AACCATGGCC 60
CAATTTGTCC GTAACCTTGT GGAGAAGACC CCGGCGCTGG TGAACGCTGC TGTGACTTAC 120
TCGAAGCCTC GATTGGCCAC ATTTTGGTAC TACGCCAAGG TTGAGCTGGT TCCTCCCACC 180
CCTGCTGAGA TCCCTAGAGC TATTCAGAGC CTGAAAAAAA TAGTCAATAG TGCTCAGACT 240
GGTAGCTTCA AACAGCTCAC AGTTAAGGAA GCTGTGCTGA ATGGTTTGGT GGCCACTGAG 300 GTGTTGATGT GGTTTTATGT CGGAGAGATT ATAGGCA 337
(2) INFORMATION FOR SEQ ID NO: 17:
(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: 17:
Met Ala Gin Phe Val Arg Asn Leu Val Glu Lys Thr Pro Ala Leu Val 1 5 10 15
Asn Ala Ala Val Thr Tyr Ser Lys Pro Arg Leu Ala Thr Phe Trp Tyr 20 25 30
Tyr Ala Lys Val Glu Leu Val Pro Pro Thr Pro Ala Glu He Pro Arg 35 40 45
Ala He Gin Ser Leu Lys Lys He Val Asn Ser Ala Gin Thr Gly Ser 50 55 60
Phe Lys Gin Leu Thr Val Lys Glu Ala Val Leu Asn Gly Leu Val Ala 65 70 75 80
Thr Glu Val Leu Met Trp Phe Tyr Val Gly Glu He He Gly 85 90
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AAATTANAGG AAGANCCTNT TGAAAAAATT TNTGTTTGTN AAAAAGNTAG GGNAATTGTT 60
ATTTTGGAAA TAGCCTNCCC NAGNGNGGAN AGGGGGGNAT TTTAAGNANG NTTTTTTGNA 120
AAATTTTTNG NCGNNGGNNA GAANCNAAAA AGNGGAATTT GNNTTTTAAG GGGGNTANTT 180 GNTTGTTTGG GTTTAANACC CTTGCCAAAA NNAAANACCC CCAAGNNANT TNAANNAGGG 240
TATAANTTAG NATTTTTCCC TGGANTTAAA NAGNANATTA TATNCTGGAA NAAANGNAAN 300 GGTTGGTATN AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 345
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 456 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
AGAGATTCAG GACCTGCAGA GTCGCCAGAA GCATGAAATT GAATCTTTGT ATACTAAACT 60
GGGCAAGGTT CCCCCTGCTG TCATTATTCC CCCAGCTGCT CCTCTGTCGG GGAGAAGAAG 120
GAGACCCACT AAAAGCAAAG GCAGCAAGTC TAGTCGCAGC AGCTCATTGG GCAATAAAAG 180
CCCACAGCTT TCAGGCAACC TGTCTGGTCA GAGTGGAACT TCAGTCTTAC ACCCCCAACA 240
GACCCTCCAC CCTCCTGGCA ACATCCCANA NTCCGGGCAG AATCAGCTGT TACAGCCCCT 300
TAAGCCATCT CCCTCCAGTG ACAACCTCTA TTCAGCCTTC ACCAGTGATG GTGCCATTTC 360
AGTACCAAGC CTTTCTGCTC CAGGTCAAGG AACCAGCAGC ACAAACACTG TTGGGGCAAC 420
AGTGAACAGC CAAGCCGCCC AAGCTCAGCC TCCTGC 456 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Lys Leu Asn Leu Cys He Leu Asn Trp Ala Arg Phe Pro Leu Leu 1 5 10 15 Ser Leu Phe Pro Gin Leu Leu Leu Cys Arg Gly Glu Glu Gly Asp Pro 20 25 30
Leu Lys Ala Lys Ala Ala Ser Leu Val Ala Ala Ala His Trp Ala He 35 40 45
Lys Ala His Ser Phe Gin Ala Thr Cys Leu Val Arg Val Glu Leu Gin 50 55 60
Ser Tyr Thr Pro Asn Arg Pro Ser Thr Leu Leu Ala Thr Ser Xaa Xaa 65 70 75 80
Pro Gly Arg He Ser Cys Tyr Ser Pro Leu Ser His Leu Pro Pro Val 85 90 95
Thr Thr Ser He Gin Pro Ser Pro Val Met Val Pro Phe Gin Tyr Gin 100 105 110
Ala Phe Leu Leu Gin Val Lys Glu Pro Ala Ala Gin Thr Leu Leu Gly 115 120 125
Gin Gin 130
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 188 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TACCCTGCCC TCCTCCCTTT TTTNNACCCC TCTCTTTTTT ATTTTTTCTT TGCTCTTTAG 60
AACCCAGTGA AAAATACCAG GGTACTGGGG TGCAACTCTT TCTTATGATA GGTCATTAGT 120
GCTTTAAGCA AAAGATATTA GCAGCTTTGA CTGCAGCATT AGCAATTAGG NAAAAAAAAA 180
AAAAAAAA 188 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 752 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CCTTATGGCC TACTTTAAAA AAAAACCAAT ACCAAAGAAG CCTACAATGT TGGCCTTAGC 60
CAAAATTCTG TTGATTTCAA CGTTGTTTTA TTCACTTCTA TCGGGGAGCC ATGGAAAAGA 120
AAATCAAGAC ATAAACACAA CACAGAACAT NGCAGAAGTT TTTAAAACAA TGGAAAATAA 180
ACCTATTTCT TTGGAAAGTG AAGCAAACTT AAACTCAGAT AAAGAAAATA TAACCACCTC 240
AAATCTCAAG GCGAGTCATT CCCCTCCTTT GAATCTACCC AACAACAGCC ACGGAATAAC 300
AGATTTCTCC AGTAACTCAT CAGCAGAGCA TTCTTTGGGC AGTCTAAAAC CCACATCTAC 360
CATTTCCACA AGCCCTCCCT TGATCCATAG CTTTGTTTCT AAAGTGCCTT GGAATGCACC 420
TATAGCAGAT GAAGATCTTT TGCCCATCTC AGCACATCCC AATGSTACAC CTGCTCTGTY 480
TTCARAAAAC TTCACTTGGT CTTTGTCAAT GACACCGTGA AAACTCCTGA TAACAGTTCC 540
ATTACAGTTA GCATCCTCTY TTCARAACCA ACTTCTCCAT CTGTGACCCC CTTGATAGTG 600
GAACCAAGTG GATGGNTTAC CACAAACAGT GATAGNTTCA CTGGGTTTAC CCCTTATCAA 660
GNAAAAACAA CTTTACAGCC TACCTTAAAA TTCACCAATA ATTCAAAACT NTTTCCAAAT 720
ANGTCAGATC CCCCAAAAAA AAAAAAAAAA AA 752 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Met Leu Ala Leu Ala Lys He Leu Leu He Ser Thr Leu Phe Tyr Ser 1 5 10 15
Leu Leu Ser Gly Ser His Gly Lys Glu Asn Gin Asp He Asn Thr Thr 20 25 30
Gin Asn Xaa Ala Glu Val Phe Lys Thr Met Glu Asn Lys Pro He Ser 35 40 45
Leu Glu Ser Glu Ala Asn Leu Asn Ser Asp Lys Glu Asn He Thr Thr 50 55 60
Ser Asn Leu Lys Ala Ser His Ser Pro Pro Leu Asn Leu Pro Asn Asn 65 70 75 80
Ser His Gly He Thr Asp Phe Ser Ser Asn Ser Ser Ala Glu His Ser 85 90 95
Leu Gly Ser Leu Lys Pro Thr Ser Thr He Ser Thr Ser Pro Pro Leu 100 105 110
He His Ser Phe Val Ser Lys Val Pro Trp Asn Ala Pro He Ala Asp 115 120 125
Glu Asp Leu Leu Pro He Ser Ala His Pro Asn Xaa Thr Pro Ala Leu 130 135 140
Xaa Ser Xaa Asn Phe Thr Trp Ser Leu Ser Met Thr Pro 145 150 155
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
AAGCTTGGCA CGAGGTCTTT AGAAGAACTA CAAAACCTGA ATGGAAAACT TCGAAGTGAA 60
GGACAAGGNA ATATGGGCTT TACTAGGCAG AATCACAGGG CAGAAGTTGA ATATACCGGC 120
AATTTTGAGA GCACCCAAGG AGAGAAAACC AAGTAAAAAA AGAAGGAGGC ACACAAAAGA 180
CATCTACTCT TCCTGCAGTA CTTTATAGTT GTGGGATTTG TAAGAAGAAC CATGATCAGC 240
ATCTTCTTTT ATTGTGTGAT ACCTGTAAAC TACATTACCA TTTTGGATGT CTGGATCCTC 300
CTCTAACAAG GATGCCAAGA AAGACCCAAA ACAGTTATTG GCAGTGCTCG GAATGTGACC 360
AGGCAGGGAG CAGTGACATG GAAGCAGATA TGGCCATGGA AACCCTACCA GATGGAA 417 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Met Pro Arg Lys Thr Gin Asn Ser Tyr Trp Gin Cys Ser Glu Cys Asp
1 5 10 15
Gin Ala Gly Ser Ser Asp Met Glu Ala Asp Met Ala Met Glu Thr Leu 20 25 30
Pro Asp Gly 35
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
TCTGTGTTCA GTATAATTTT ATTTTTCTCA ACCTTAAATA TGAACTTAGG AAATAAGGAG 60
GGAAGTACAA AGATTATTGA CTATACAACN TACCAGCTGA AAGAAAGATC TTCATCAACA 120
TCTGTATCTT TCCAGAGGTA TACAGAATTA AAATTNNATN TTCAAGCTTT AATGATCCAG 180
TTTTAAGTCA ACGGCAGAAG TATGTTGAAT ATTTCATCAC TCAATCTTGA ACTGATTTAG 240
AAGAGACTCT TTGCTGAAAT TGAATTGCAC TTATACATGT AAATTGTCAA CATGTAATTT 300
GGAATTTTCT GATTAATAAA TGTGGTTTTG GACATCTAAA AAAAAAAAAA AAAAAAAAA 359 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CTCNCAAATC GGCNCGNGCA ACGAACGGCT TGGGCGCGGA CTGGTATCCG GGGACTGTGA 60
CTTGCAGGGT CCGCCATGGA GCCAGAGCAG ATGCTGGAGG GACAAACGCA GGTTGCAGAA 120
AATCCTCACT CTGAGTACGG TCTCACAGAC AACGTTGAGA GAATAGTAGA AAATGAGAAG 180
ATTAATGCAG AAAAGTCATC AAAGCAGAAG GTAGATCTCC AGTCTTTGCC AACTCGTGCC 240
TACCTGGATC AGACACTTGT GCCTATCTTA TTACAGGGAC TTGCTGTGCT TGCCAAGGAA 300
AGACCACCAC ATCCCATTGA ATTTCTAGCA TCTTATCTTT TAAAAAACAA GGCACAGTTT 360
GAAGATYGAA ACTGAMTTAA TGGGRAGAAC AGAAAAATTT AGTTGSTACT GTAGATTTAC 420
ATGATTAAGA RGCAGCTTTA ATTGCCATGA TCATTCCCTT TTTTTGGAAG GATAAGNACC 480
TTNCGGANAA CAGNACCTAT TTTTGGGATT GCAGNAGNTA AAATATTTCC CNTATTTTGA 540
NTTAATNACC ATAAACCNTA CCTATTTAAT GNGNGTATTT TGTGCAATTT TTTTTTNAGN 600
TTGTTTTTAA ATTTGTTTTT AAAATGACCT TNAAAATNAA NTGTNNAAAC ACCNTTTAAA 660
AAAAAAAAAA AAAAA 675 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Met Glu Pro Glu Gin Met Leu Glu Gly Gin Thr Gin Val Ala Glu Asn 1 5 10 15
Pro His Ser Glu Tyr Gly Leu Thr Asp Asn Val Glu Arg He Val Glu 20 25 30
Asn Glu Lys He Asn Ala Glu Lys Ser Ser Lys Gin Lys Val Asp Leu 35 40 45 Gin Ser Leu Pro Thr Arg Ala Tyr Leu Asp Gin Thr Leu Val Pro He 50 55 60
Leu Leu Gin Gly Leu Ala Val Leu Ala Lys Glu Arg Pro Pro His Pro 65 70 75 80
He Glu Phe Leu Ala Ser Tyr Leu Leu Lys Asn Lys Ala Gin Phe Glu 85 90 95
Asp Xaa Asn
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 552 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CACGAGGGTT TGGTGAGGAA ATTACCAGAG AACTATTAAA GACTTGGATG CTCTTCTCGG 60
CTTTGCTATT AAGTAAGTTG GACAAGTTGT TTGGCTTCTT TGAGCCTCTG TTTTCTCCAT 120
TCTAAAATTC TAAAATGGGA GTGTTGAATT AGATCAGTGG CTTTCGAACT TTCTGCTCCT 180
AGTAGTGAGA AATACATTTT ACTCCACTCC CTGGTATGTA CACGCATTCC TGTGTTTTGT 240
GAAAACCTGA CACCATGCTC CTCCCTCACT ACATGTAAAA CACTTTTATT CATTAAAAAG 300
AAAACTGACT GGCTTGGACC TACAAATTAG TTTCATTATT TGTTAATGTT TGAAAGCCAT 360
TAAAAGATGA ATATTAAGGT TTCTTTATAC TCAATACTTG TAGTTTTGTT TGGGGGAATG 420
AGAGGATGCC CTTGGTACCT TTGTGAGGCC TCTCCACTGA GGGTCAATCA TGACTTCTGT 480
TTTAAACCAG CCCATCCCAT CTTCTCCAGC TGCTCTCCTT ATGTCTTGCT TCTCTCCCCT 540
CCAACCTTCT CA 552 (2) INFORMATION FOR SEQ ID NO : 30 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Met Asn He Lys Val Ser Leu Tyr Ser He Leu Val Val Leu Phe Gly 1 5 10 15
Gly Met Arg Gly Cys Pro Trp Tyr Leu Cys Glu Ala Ser Pro Leu Arg 20 25 30
Val Asn His Asp Phe Cys Phe Lys Pro Ala His Pro He Phe Ser Ser 35 40 45
Cys Ser Pro Tyr Val Leu Leu Leu Ser Pro Pro Thr Phe Ser 50 55 60
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CAGGGCCCCA TCCTTCAGTG CATTGCACAC TTTGCATGNT GGGTCAGGGA AGATTGTGGA 60
GAGAGGACAG TGCACATGGT TTCCCCCACN TNGNCTGCGT GGGGGTATGT CCTGCTTCCG 120
CCACTTCCAA CTGTGGCANT TGGGCACGCC CCTNTCAGGG CACCTTCCCT TTTTGTTTCC 180
GCAAAATGAG GTTGTAATAG TGCCTGCCGC ACTGTNTGGC ACACAGTAAG NTCTCAAGAA 240
ATGTTAGCTG TTGTTGCCGT TAGAACACCA TAGNTAGAAT ACCATACNTG GCATTCACTT 300
AAAAAAAAAA AAAAAAAA 318 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 310 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
ATTGAGGAAA ACCACAAAAA ACTTCAAAAC AGCTACAACG GGAAAAAGAG AGTTTTGTCC 60
CACAGTCAGC AGGCCACTAG TTTATTAACT TCCAGTCACC TTGATTTTTG CTAAAATGAA 120
GACTCTGCAG TCTACACTTC TCCTGTTACT GCTTGTGCCT CTGATAAAGC CAGCACCACC 180
AACCCAGCAG GACTCACGCA TTATCTATGA TTATGGAACA GATAATTTTG AAGAATCCAT 240
ATTTAGCCAA GATTATGAGG ATAAATACCT GGATGGAAAA AATATTAAGG AAAAAGAAAC 300
TGTGATAATA 310 (2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Lys Thr Leu Gin Ser Thr Leu Leu Leu Leu Leu Leu Val Pro Leu 1 5 10 15
He Lys Pro Ala Pro Pro Thr Gin Gin Asp Ser Arg He He Tyr Asp 20 25 30
Tyr Gly Thr Asp Asn Phe Glu Glu Ser He Phe Ser Gin Asp Tyr Glu 35 40 45
Asp Lys Tyr Leu Asp Gly Lys Asn He Lys Glu Lys Glu Thr Val He 50 55 60
He 65
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 303 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CCCAAGNAAN TTTCAANTTT TTGCCTTTNC TGGCCTTTAN TGGATCCCNA AAGCATTTAA 60
GGNANATGTT CCNAAAANTT TGNAAAGNTA AANGTTTCCC ATGATCGCTC ATTTTTTTTT 120
TATGATTCAN ANGTTATTCC TTATAAAGTA AGNANTTTGT TTTCCTCCTA TCAAGGCAGN 180
TATTTTATTA AATTTTTCAN TTAGTTTGAG NAATAGCAGA TAGTTTCATA TTTAGGGAAA 240
NTTTCCAAAT AAAATAAATG TTATTNTTTG ATAAAGAGNT AAAAAAAAAA AAAAAAAAAA 300
AAA 303 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 418 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
AAGCTTGNGC ACGNGGCACA AGTAGCTACG NCTGCAAGCA CCTGCCACCA TAAAGGGGNT 60
GCATTTTGCC ACCATAAANG GGNTGCATTT TTTTAAAAAG CCTAGGCNGC TCTAACATCA 120
TCTGATATGG ACACAANGCN AACAGTTTCC NTATNTACAT CCNTACCTCT AAAAGATACT 180
TCAAAGTGAC AAAAACGTGT TCCTTCCCCA CTTAGAGACA ATGATTAACA GGGCCCTATA 240
TGTTCTTACC ACATACAGAG GATGCATTTA TTTTTGCTCT ATGACACTTG CAAAAATCTC 300
TACTGTAATT AATTTGGGTC TATTATTAAC TCTCTGTTCC ATCATAGAAT GTGGCCAGGC 360
CTTACAATGG AGAGCCAGAG TTAAAACTTC AAGTTGCATC TGTTTTTGGG CTGAGTCA 418 (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
( C ) STRANDEDNESS :
( D) TOPOLOGY : l inear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 36 :
Met Thr Leu Ala Lys He Ser Thr Val He Asn Leu Gly Leu Leu Leu
1 5 10 15
Thr Leu Cys Ser He He Glu Cys Gly Gin Ala Leu Gin Trp Arg Ala 20 25 30
Arg Val Lys Thr Ser Ser Cys He Cys Phe Trp Ala Glu Ser 35 40 45
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 331 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
AGTTTGTTCT GTAAATATTT NGAAAAGTGA CAGCTNTCAA CTTCAGGGTA ACTATTTCTA 60
AAAATGTAAA TANGTATTAA TCCTTGTATC TTTTATGGTA ATTTNGCATA TTGATATGAA 120
TTANATAAAA TTGTTTAAAA TAAAAGGTGT CCTTGAATTA CTGACCACCC ATAGATGTNT 180
ACTGTTACCA GGTTTTACAA TGCAAATTTT CACTAATACC TGGGTTTAAT ACAGCTCACA 240
TCACTGAATG TTACACATGA GTTTAAATGG GTTAATATAC AGGTTTTGTT ATAATAAAGT 300
TACTGATTAA ATTAAAAAAA AAAAAAAAAA A 331 (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 583 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
CACGNGGGTG AGGCCGACTG CTGAAGACAG CTCGCCACCC TCCTTGCCTC CACTCCAATC 60
CAGGGGCTGG GGCCACATTC TTTGCCTTCA TTTATCCTCA GATCAGGTGA GATCGACAGG 120
AGGTGTTGAT GGCAGTGCCA GCAATTATTG CTAATCCGTT TGCATCCTTA TGCATAGATC 180
TGAATTCAGA CTTTGTGAAT TTCCAGAGGT GTGGGTNATA TAATAGAATT CAGTGAGTGG 240
GCATGGCTGA TCTTGTGCAA ATTAAAAGTT ATGGGGCATA AGAATAGCAA AAGTTGAACT 300
TCTTTTAAAA AGGAAAGTAC CCTGAGAGCC AGTATTGGTT GAGGCTCTTC AGTATGCCCA 360
GGTTGGCAGC ACTGAGAACC GCAGGAACGG CCTGTTGTTA CAAAAAGGAG ATTGACTCAG 420
CTGCCCTTGG TGCATCTGAC TGACTATGAC TGCTGAGAGA TTCCAAGGAC CCTTAATGCC 480
AGGGCTAACC TCTCCATGTG CAGTGAGACC TCTGGAGGAA GTGTCATCCT CTGGCTTTGT 540
GTGGTACTCA TTATGGTGCA GTGCGGGCAT GAAATGAAGA CAC 583 (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Met Cys Ser Glu Thr Ser Gly Gly Ser Val He Leu Trp Leu Cys Val
1 5 10 15
Val Leu He Met Val Gin Cys Gly His Glu Met Lys Thr 20 25
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
CCCAAATAGG CTTACAGATA CGATATGTTT TAAATGTTTN GTATTTAACA AAAACATACT 60
GACACTGTTT GGAAATGGCA ACAGGAAGAT AGCAAAATGA ATACTAACAT TACGAAAAGA 120
TGAACAGGTA CATGTTCCAA GGCAGGTGGC TGTGAACTTC CTCTGAGTGA AGGCATCCCC 180
TCCAGCACCT TTCAGCCTGC TAGTTAGGAC GACCCGCCGC CACCCTCCAG GACNTCCAGC 240
CCTGCANTGC NTTTCTTTTN TTTTAAATAA TTCTTCATTG AGTTCTAATA TGTAAAAAAA 300
AAAAAAAAAA A 311 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
AAGCTTGGCA CGAGGGCGGT TGAGGCCTTC GGTGGTGAAC GAGTCTCCAG CACCATGTCT 60
GGTTTGTCTG GCCCACCAGC CCGGCGCGGC CCTTTTCCGT TAGCGTTGCT GCTTTTGTTC 120
CTGCTCGGCC CCAGATTGGT CCTTGCCATC TCCTTCCATC TGCCCATTAA CTCTCGCAAG 180
TGCCTCCGTG AGGAGATTCA CAAGGACCTG CTAGTGACTG GCGCGTACGA GATCTCCGAC 240
CAGTCTGGGG GCGCTGGCGG CCTGCGCAGC CACCTCRAGA TCACAGATTC TGCTGGCCAT 300
ATTCTCTACT CCAAAGAGGA TGCAACCAAG GGGAAATTTG CCTTTACCAC TGAAGATTAT 360
GACATGTTTG AAGTGTGTTT TGAGAGCAAG GGAACAGGGC GGATA 405 (2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 42 :
Met Ser Gly Leu Ser Gly Pro Pro Ala Arg Arg Gly Pro Phe Pro Leu
1 5 10 15
Ala Leu Leu Leu Leu Phe Leu Leu Gly Pro Arg Leu Val Leu Ala He 20 25 30
Ser Phe His Leu Pro He Asn Ser Arg Lys Cys Leu Arg Glu Glu He 35 40 45
His Lys Asp Leu Leu Val Thr Gly Ala Tyr Glu He Ser Asp Gin Ser 50 55 60
Gly Gly Ala Gly Gly Leu Arg Ser His Leu Xaa He Thr Asp Ser Ala 65 70 75 80
Gly His He Leu Tyr Ser Lys Glu Asp Ala Thr Lys Gly Lys Phe Ala 85 90 95
Phe Thr Thr Glu Asp Tyr Asp Met Phe Glu Val Cys Phe Glu Ser Lys 100 105 110
Gly Thr Gly Arg He 115
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
TCTTTCAATT TACCTTGTGA AAACACCCTT AACTTTTTCT TNACCCTTAG CTGAAATGTT 60
NACATAGCTT NTGGTGATAT CTTTTCATGA TTTTATATNT CTTAAAATGG TGATGGATGT 120
GACACCTCAT AAAAGTGAGC TTTGAACTGT AGATAACTCT TAAAGAAAAT GTCATTTTAG 180
ACAATTAAAA TATTTGTGCT CAAAAAAAAA AAAAAAAAAA AAAAA 225 (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
CGAGGGCAGG TCAGTCAGGT TCCTGGGCGC TCTGTTACAC AAGCAAGATA CAGCCAGCCC 60
CACCTAATTT TGTTTCCCTG GCACCCTCCT GCTCAGTGCG ACATTGTCAC ACTTAACCCA 120
TCTGTTTTCT CTAATGCACG ACAGATTCCT TTCAGACAGG ACAACTGTGA TATTTCAGTT 180
CCTGATTGTA AATACCTCCT AAGCCTGAAG CTTCTGTTAC TAGCCATTGT GAGCTTCAGT 240
TTCTTCATCT GCAAAATGGG CATAATACAA TCTATTCTTG CCACATCAAG GGATTGTTAT 300
TCCTTTAAAA AAAAACCAAT ACCAAAGAAG CCTACAATGT TGGCCTTAGC CAAAATTCTG 360
TTGATTTCAA CGTTGTTTTA TTCACTTCTA TCGGGGAGCC ATGGAAAAGA AAATCAAGAC 420
ATACACACAA CACAGAACAT TGCAGAAGTT TTTAANACAA TGGAAAATAA ACCTATTTCT 480
TTGGAAAGTG AAGCAAACTT AAACTCAGAT AAAGNAAATA TAACC 525 (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Met Leu Ala Leu Ala Lys He Leu Leu He Ser Thr Leu Phe Tyr Ser 1 5 10 15
Leu Leu Ser Gly Ser His Gly Lys Glu Asn Gin Asp He His Thr Thr 20 25 30
Gin Asn He Ala Glu Val Phe Xaa Thr Met Glu Asn Lys Pro He Ser 35 40 45
Leu Glu Ser Glu Ala Asn Leu Asn Ser Asp Lys Xaa Asn He Thr 50 55 60
(2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 302 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
TCAAAAGGTN ACACAAAATT ACTGTCACGT GGATTTTGTC AAGGAGAATC ATAAAAGCAG 60
GAGACCAGTA GCAGAAATGT AGACAGGATG TATCATCCAA AGGTTTTCTT TCTTACAATT 120
TTTGGCCATC CTGAGGCATT TACTAAGTAG CCTTAATTTG TATTTTAGTA GTATTTTCTT 180
AGTAGAAAAT ATTTGTGGAA TCAGATAAAA CTAAAAGATT TCACCATTAC AGCCCTGCCT 240
CATAACTAAA TAATAAAAAT TATTCCACCA AAAAATTNTA AAACAAAGNA AAAAAAAAAA 300
AA 302 (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 628 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
CACGAGGTTT CAGACCAGCT TGTGTCAATA GGGTCCTACA GAGCAGCTGA TATCAGCAGT 60
TTTACTAGTA TGCAGGACCT GAAAGAATAT CTCAAAGGGA AAACAATGTT TCATAATGTT 120
CAGGAAGTTA TCTATAGAGC AGCTAAGGAG CTATAATCTT GTAACAGAGT CTACGTGATT 180
GTAGGACAAT AGGCACCACA CAAATATGAG GAAGCAGGTC AGAGAGCGGG CTGACTTAAT 240
GATTAATGCT GAATGTGCTA CAAGCTTGTT TCATTTTCAT TTCTCCTCCT CCCTTTTTTC 300
CTGATTAATT TAATAAAGTT CATAGGGGAG GCTTCAAACA CATGAGAAAT TAAAACCTTT 360
ATTACCAGAG TCAGAGCCTG ACTATATTGA TTGAGTGAAG CTTTCCTTTA TAAAATGCAA 420
AGCATGTAAA CAATTCCAAC ACAGTAACAT ATTCATGAGT TTTTAAATTC ATGAGTTTTA 480 GAGAAAATAT TTTACTTAAA ACCAGCACTT GATGATCTCT GACAATGTTA TGTAGCCTGA 540 ACCTGGAGTT TTGGCTGATG GGTTGTCTCA GCCTGTGACA GGTTTTAGCT GGCTTTGGTT 600 CATCTTGTAT CACACCCCCA CACTCACA 628
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Pro Glu Pro Gly Val Leu Ala Asp Gly Leu Ser Gin Pro Val Thr Gly
1 5 10 15
Phe Ser Trp Leu Trp Phe He Leu Tyr His Thr Pro Thr Leu Thr 20 25 30
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 436 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
AGCAGCTAAG GGGAAATAAT CTTGTAACAG GGTCTGGGTG ATTNTGAGGT AATAGGCCCC 60
AAACAACCAT GGGGAAGCAG GTCAGAGGGC AAGCTGGCNT AGTGTTTAAC ATTGAATGGG 120
CTGAAAGTTT GGTTNATTTT TGTTTCTTGT TTCTCCCCCT CCCTTCTNAC CTGAATAATT 180
TTATGAAGTT TATAGGGATG GTTTCAGGAC CTCCATTCTA TCTGTTCCTG AAATATTACA 240
AAAAGATTAT TATTGTAGCA CTNATNTAAT TGGGGTTTTA TTTCGTTGTT NGCATGTCTG 300
TTTCTTCCCC AGTGAGTTGT AAATTGCTTA AGGGCAAACA GACGCATCCT ATTTATCTGT 360
CTGTCACTAA CATTAAGCAC AGCATTTGGT ATACAGTCAT CACTCTAATA AAGTTTGAAA 420 AAAAAAAAAA AAAAAA 436
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CACGAGGGAA AAAAAGAGTT TTTTTTTTAG ATCATCAGCT ATTGTTAGTG TTTGTGTATG 60
TTATGTGTGG CTCAAGACAA CTTTGCTTCT TTTAATATAG GCAGGGAAGT CAAAAGATTG 120
GATATCCCTG CTTTATACCA AGAAAGACAA CACCCCACAT TTGCAGTGCC TGAAAACACT 180
ACCAGCCATC TGAAAAACAT GTGACTTCTA ACTTCTGTTC TTTTTTGTAG CAGTGGAATC 240
CCACGGTGAT ATCTGAGGGA TGTGGTTACC TTTTGGAGGA GGTTGACGGT TTCTAAGGAT 300
GATTCTTTCT GAGTGAAATA TTGTCAGTGT CATTGACCTT TTCATTATTT CAACTATTAT 360
TATTCCAGGT TATCAATACT CTGGCTGACC ATCATCATCG TGAGACTGAC TTTGGTGTAG 420
GAGTTCGAGA CCACCCTGGC CAACATGGCA AAACCCCATC TCCACAAAAA TTGGATAATT 480
TGATAATTAT CATTATTGGG TTTCTGAGAC GTTACACATT TAACATTNTN TTCTGCACAA 540
GTTGCCTTTG TGTGAGTATA CTAACTTTCT GTAGAGGTAN ACTTGTAATC ACAAATAAGA 600
ATAAATTATA TAAAACAAAA AAAAAAAAAA AAAAAA 636 (2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: Phe Phe Leu Ser Glu He Leu Ser Val Ser Leu Thr Phe Ser Leu Phe 1 5 10 15
Gin Leu Leu Leu Phe Gin Val He Asn Thr Leu Ala Asp His His His 20 25 30
Arg Glu Thr Asp Phe Gly Val Gly Val Arg Asp His Pro Gly Gin His 35 40 45
Gly Lys Thr Pro Ser Pro Gin Lys Leu Asp Asn Leu He He He He 50 55 60
He Gly Phe Leu Arg Arg Tyr Thr Phe Asn He Xaa Phe Cys Thr Ser 65 70 75 80
Cys Leu Cys Val Ser He Leu Thr Phe Cys Arg Gly Xaa Leu Val He 85 90 95
Thr Asn Lys Asn Lys Leu Tyr Lys Thr 100 105
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 536 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
GGCACGAGGA GCGGGAGCTG GTGCCTTCCC GGAAGGGCTC AGAGGCGGGC TCGGGCAAGC 60
ACTTTAACCT TTTAAGCCCA ACCAGATGAG TTGCCTGCAG TTTTGGAGGC CTTCAGAGCA 120
TTTCACTAGA CCTCTGTCTG TGTCGGTCCA ATGTCTTTAG CCAAGCTTTG ATTAAAGATG 180
ACTTCCTTGT TTGCTCAAGA AATTCGCCTT TCTAAAAGAC ATGAAGAAAT AGTATCACAA 240
AGATTAATGT TACTTCAACA AATGGAGAAT AAATTGGGTG ATCAACACAC AGAAAAGGCA 300
TCTCAACTCC AAACTGTTGA GACTGCTTTT AAAAGGAACC TTAGTCTTTT AAAGGATATA 360
GAAGCAGCAG AAAAGTCACT ACAGACCAGG ATTCACCCAC TTCCACGGCC TGAGGTGGTT 420
TCTCTTGAGA CTCGTTACTG GGCATCAGTA GAAGAATATA TTCCCAAATG GGAACAGTTT 480
CTTTTAGGAA GAGCACCATA TCCTTTTGCT GTTGAAAATC AAAATGAAGC AGAAAA 536 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Met Thr Ser Leu Phe Ala Gin Glu He Arg Leu Ser Lys Arg His Glu 1 5 10 15
Glu He Val Ser Gin Arg Leu Met Leu Leu Gin Gin Met Glu Asn Lys 20 25 30
Leu Gly Asp Gin His Thr Glu Lys Ala Ser Gin Leu Gin Thr Val Glu 35 40 45
Thr Ala Phe Lys Arg Asn Leu Ser Leu Leu Lys Asp He Glu Ala Ala 50 55 60
Glu Lys Ser Leu Gin Thr Arg He His Pro Leu Pro Arg Pro Glu Val 65 70 75 80
Val Ser Leu Glu Thr Arg Tyr Trp Ala Ser Val Glu Glu Tyr He Pro 85 90 95
Lys Trp Glu Gin Phe Leu Leu Gly Arg Ala Pro Tyr Pro Phe Ala Val 100 105 110
Glu Asn Gin Asn Glu Ala Glu 115
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: TTTATTTAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAAAAAA 79 PAGE INTENTIONALLY LEFT BLANK
PAGINA DEJADA EN BLANCO INTENCIONALMENTE
PAGE LAISSEE INTENTIONNELLEMENT EN BLANC

Claims

What is claimed is:
1. A composition comprising an isolated protein encoded by a 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: 1 from nucleotide 28 to nucleotide 276;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AE402_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AE402_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
2. The composition of claim 1 , further comprising a pharmaceutically acceptable carrier.
3. 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 2.
4. 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; and
(c) the amino acid sequence encoded by the cDNA insert of clone AE402_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
5. The composition of claim 4, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
6. The composition of claim 4, further comprising a pharmaceutically acceptable carrier.
7. 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 6.
8. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 61 to nucleotide 513;
(c) a polynucleotide comprising the nucleotide sequence of SEQ LD NO:4 from nucleotide 322 to nucleotide 513;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AE610_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AE610_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:5; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:5 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
9. 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:5;
(b) fragments of the amino acid sequence of SEQ ID NO:5; and
(c) the amino acid sequence encoded by the cDNA insert of clone AE610_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
10. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ LD NO:7 from nucleotide 20 to nucleotide 523;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AH106_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AH106_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
11. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 8;
(b) fragments of the amino acid sequence of SEQ ID NO: 8; and
(c) the amino acid sequence encoded by the cDNA insert of clone AH106_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
12. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 130 to nucleotide 309;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AH 196_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AHl 96_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
13. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10;
(b) fragments of the amino acid sequence of SEQ ID NO: 10; and
(c) the amino acid sequence encoded by the cDNA insert of clone AH196_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
14. A composition comprising an isolated protein encoded by a 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 69 to nucleotide 467;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AI6_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AI6_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AI6_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AI6_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 13;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:l 3 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
15. 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 69 to amino acid 133;
(c) fragments of the amino acid sequence of SEQ ID NO: 13 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone AI6_1 i deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
16. A composition comprising an isolated protein encoded by a 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 55 to nucleotide 337;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ13_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ13_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ 13_1 i deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:17;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 17 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
17. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 17;
(b) the amino acid sequence of SEQ ID NO: 17 from amino acid 12 to amino acid 94;
(c) fragments of the amino acid sequence of SEQ ID NO: 17; and
(d) the amino acid sequence encoded by the cDNA insert of clone AJ13_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
18. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 33 to nucleotide 422;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 114 to nucleotide 422;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ27_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
19. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20;
(b) fragments of the amino acid sequence of SEQ ID NO:20; and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ27_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
20. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:22;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22 from nucleotide 47 to nucleotide 517;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:22 from nucleotide 116 to nucleotide 517;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ142_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:23;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:23 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
21. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:23;
(b) fragments of the amino acid sequence of SEQ ID NO:23; and
(c) the amino acid sequence encoded by the cDNA insert of clone AJ142_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
22. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 24 from nucleotide 312 to nucleotide 417;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK604_1 i deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK604_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:25;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:25 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
23. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:25;
(b) fragments of the amino acid sequence of SEQ ID NO: 25; and
(c) the amino acid sequence encoded by the cDNA insert of clone AK604_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
24. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:27;
(b) a polynucleotide composing the nucleotide sequence of SEQ ID NO:27 from nucleotide 76 to nucleotide 372;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK620_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK620_1 i deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:28;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:28 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
25. 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:28;
(b) fragments of the amino acid sequence of SEQ ID NO:28; and
(c) the amino acid sequence encoded by the cDNA insert of clone AK620_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
26. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29 from nucleotide 367 to nucleotide 552;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK650_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK650_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:30;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:30 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
27. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:30;
(b) fragments of the amino acid sequence of SEQ ID NO:30; and (c) the amino acid sequence encoded by the cDNA insert of clone AK650_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
28. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:32;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:32 from nucleotide 116 to nucleotide 310;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:32 from nucleotide 173 to nucleotide 310;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AM226_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AM226_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:33;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:33 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
29. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:33;
(b) fragments of the amino acid sequence of SEQ ID NO:33; and (c) the amino acid sequence encoded by the cDNA insert of clone AM226_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
30. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35 from nucleotide 281 to nucleotide 418;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35 from nucleotide 353 to nucleotide 418;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AR417_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AR417 li deposited under accession number ATCC
98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:36;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:36 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
31. 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: 36;
(b) fragments of the amino acid sequence of SEQ ID NO:36; and (c) the amino acid sequence encoded by the cDNA insert of clone AR417_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
32. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 496 to nucleotide 583;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 565 to nucleotide 583;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AU43_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AU43_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:39;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:39 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
33. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:39;
(b) fragments of the amino acid sequence of SEQ ID NO:39; and (c) the amino acid sequence encoded by the cDNA insert of clone AU43_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
34. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 55 to nucleotide 405;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 148 to nucleotide 405;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AW60_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AW60_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:42;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:42 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
35. 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:42;
(b) fragments of the amino acid sequence of SEQ ID NO: 42; and (c) the amino acid sequence encoded by the cDNA insert of clone AW60_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
36. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:44;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:44 from nucleotide 337 to nucleotide 525;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:44 from nucleotide 406 to nucleotide 525;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BA176_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BA176_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:45;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:45 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
37. 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:45;
(b) fragments of the amino acid sequence of SEQ ID NO:45; and (c) the amino acid sequence encoded by the cDNA insert of clone BA176_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
38. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:47;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:47 from nucleotide 536 to nucleotide 628;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BD140_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BD140_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:48;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:48 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
39. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:48;
(b) fragments of the amino acid sequence of SEQ ID NO:48; and
(c) the amino acid sequence encoded by the cDNA insert of clone BD140_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
40. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:50;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:50 from nucleotide 303 to nucleotide 617;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 50 from nucleotide 345 to nucleotide 617;
(d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BD407_li deposited under accession number ATCC 98190;
(e) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BD407_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:51 ;
(i) a polynucleotide encoding a protein composing a fragment of the amino acid sequence of SEQ ID NO:51 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
41. 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: 51 ;
(b) the amino acid sequence of SEQ ID NO:51 from amino acid 1 to amino acid 32;
(c) fragments of the amino acid sequence of SEQ ID NO:51 ; and (d) the amino acid sequence encoded by the cDNA insert of clone BD407_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
42. A composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:52;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:52 from nucleotide 178 to nucleotide 534;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BF290_li deposited under accession number ATCC 98190;
(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BF290_li deposited under accession number ATCC 98190;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:53;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:53 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(f) above; and
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
43. 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:53;
(b) fragments of the amino acid sequence of SEQ ID NO: 53; and
(c) the amino acid sequence encoded by the cDNA insert of clone BF290_li deposited under accession number ATCC 98190; the protein being substantially free from other mammalian proteins.
PCT/US1997/018032 1996-10-04 1997-10-03 Secreted proteins and polynucleotides encoding them WO1998014470A2 (en)

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US08/725,885 1996-10-04

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WO2001031009A2 (en) * 1999-10-22 2001-05-03 Tularik Inc. Chandra: a th1-specific gene
US6670464B1 (en) 1998-11-17 2003-12-30 Curagen Corporation Nucleic acids containing single nucleotide polymorphisms and methods of use thereof

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US6670464B1 (en) 1998-11-17 2003-12-30 Curagen Corporation Nucleic acids containing single nucleotide polymorphisms and methods of use thereof
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