Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:2;
• a polynucleotide which is an allelic variant of a polynucleotide of
• polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID NO:l.
• polynucleotide comprises the nucleotide sequence of SEQ ID NO:
• polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cc359_4 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:2, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 321 to amino acid 330 of SEQ ID NO:2.
• inventions provide isolated polynucleotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of:
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:l, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:l to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:l , but excluding the poly(A) tail at the 3' end of SEQ ID NO:l.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:l from nucleotide 216 to nucleotide 2174, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:l from nucleotide 216 to nucleotide 2174, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:l from nucleotide 216 to nucleotide 2174.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:l from nucleotide 339 to nucleotide 2174, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:l from nucleotide 339 to nucleotide 2174, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:l from nucleotide 339 to nucleotide 2174.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:2, or a protein comprising a fragment of the amino acid sequence of SEQ
• the fragment comprising the amino add sequence from amino acid 321 to amino acid 330 of SEQ ID NO:2.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:4;
• (k) a polynudeotide which encodes a species homologue of the protein of (h) or (i) above ; (1) a polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3.
• polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ct547_2 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:4, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 30 to amino acid 39 of SEQ ID NO:4.
• Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
• inventions provide isolated polynucleotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of: (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
• the polynucleotide isolated according to the above process comprises a nudeotide sequence corresponding to the cDNA sequence of SEQ ID NO:3, and extending contiguously from a nudeotide sequence corresponding to the 5' end of SEQ ID NO:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:3 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:3.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:3 from nucleotide 202 to nucleotide 414, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:3 from nucleotide 202 to nucleotide 414, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 202 to nucleotide 414.
• the polynucleotide isolated according to the above process comprises a nudeotide sequence corresponding to the cDNA sequence of SEQ ID NO:3 from nucleotide 370 to nucleotide 414, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:3 from nucleotide 370 to nucleotide 414, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 370 to nucleotide 414.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 30 to amino acid 39 of SEQ ID NO:4.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
• polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of done en553_l deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:6, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 30 to amino acid 39 of SEQ ID NO:6.
• inventions provide the gene corresponding to the cDNA sequence of SEQ ID NO:5. Further embodiments of the invention provide isolated polynudeotides produced according to a process selected from the group consisting of:
• the polynucleotide isolated according to the above process comprises a nudeotide sequence corresponding to the cDNA sequence of SEQ ID NO:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:5 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:5.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:5 from nucleotide 206 to nucleotide 415, and extending
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:5 from nucleotide 293 to nucleotide 415, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:5 from nudeotide 293 to nucleotide 415, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:5 from nucleotide 293 to nucleotide 415.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising the amino add sequence from amino acid 30 to amino acid 39 of SEQ ID NO:6.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h) and that has a length that is at least
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nudeotide 120 to nucleotide 1202; the nucleotide sequence of the full-length protein coding sequence of clone nn296_2 deposited under accession number ATCC 98715; or the nudeotide sequence of a mature protein coding sequence of clone nn296_2 deposited under accession number ATCC 98715.
• the polynudeotide encodes the full-length or a mature protein encoded by the cDNA insert of done nn296_2 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:8, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:8, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having
• the fragment comprising the amino add sequence from amino add 175 to amino acid 184 of SEQ ID NO:8.
• inventions provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
• Further embodiments of the invention provide isolated polynudeotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of:
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:7 to
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:7 from nucleotide 120 to nucleotide 1202, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:7 from nucleotide 120 to nucleotide 1202, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:7 from nucleotide 120 to nucleotide 1202.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
• the protein comprises the amino acid sequence of SEQ ID NO:8.
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising the amino acid sequence from amino acid 175 to amino acid 184 of SEQ ID NO:8.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; (1) a polynudeotide which encodes a spedes homologue of the protein of (g)-(j) above ;
• (n) a polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j) and that has a length that is at least
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 597 to nucleotide 704; the nucleotide sequence of the full-length protein coding sequence of clone nq27_13 deposited under accession number ATCC 98715; or the nucleotide sequence of a mature protein coding sequence of clone nq27_13 deposited under accession number ATCC 98715.
• the polynudeotide encodes the full-length or a mature protein encoded by the cDNA insert of clone nq27_13 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein
• fragment 15 comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:10, or a polynudeotide encoding a protein comprising a fragment of the amino add sequence of SEQ ID NO: 10 having biological activity, the fragment comprising the amino acid sequence from amino acid 13 to amino acid 22 of SEQ ID NO.T0.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:9, and extending contiguously from a nudeotide sequence corresponding to the 5' end of SEQ ID NO:9 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:9 , but excluding the poly (A) tail at the 3' end of SEQ ID NO:9.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:9 from nucleotide 597 to nucleotide 704, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:9 from nucleotide 597 to nucleotide 704, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:9 from nucleotide 597 to nucleotide 704.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of :
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 13 to amino acid 22 of SEQ ID NO:10.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• a polynudeotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:12;
• polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides spedfied in (a)-(h) and that has a length that is at least 25% of the length of SEQ ID NO:ll.
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 11 from nucleotide 44 to nucleotide 475; the nucleotide sequence of the full-length protein coding sequence of done pk65_4 deposited under accession number ATCC 98715; or the nucleotide sequence of a mature protein coding sequence of clone pk65_4 deposited under accession number ATCC 98715.
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:12, or a polynudeotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 67 to amino acid 76 of SEQ ID NO:12.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:ll, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:ll to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:ll , but excluding the poly (A) tail at the 3' end of SEQ ID NO:ll.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO: 11 from nucleotide 44 to nucleotide 475, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO: 11 from nucleotide 44 to nucleotide 475, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO: 11 from nucleotide 44 to nucleotide 475.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:12, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 67 to amino acid 76 of SEQ ID NO:12.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynudeotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID NO:13.
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 285 to nucleotide 590; the nucleotide sequence of SEQ ID NO:13 from nucleotide 408 to nucleotide 590; the nucleotide sequence of the full-length protein coding sequence of clone pk855_l deposited under accession number ATCC 98715; or the
• the polynudeotide encodes the full-length or a mature protein encoded by the cDNA insert of clone pk855_l deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO: 14, or a polynudeotide encoding a protein comprising a fragment of the amino add sequence of SEQ ID NO:14 having biological activity, the fragment comprising the amino acid sequence from amino add 46 to amino acid 55 of SEQ ID NO:14.
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:13 to a nudeotide sequence corresponding to the 3' end of SEQ ID NO:13 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:13.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:13 from nudeotide 285 to nucleotide 590, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO: 13 from nucleotide 285 to nucleotide 590, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide 285 to nucleotide 590.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:13 from nucleotide 408 to nucleotide 590, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO: 13 from nudeotide 408 to nudeotide 590, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide 408 to nucleotide 590.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
• the protein comprises the amino acid sequence of SEQ ID NO: 14.
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO: 14 having biological activity, the fragment preferably
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 193 to nudeotide 2223; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1045 to nucleotide 2223; the nucleotide sequence of the full-length protein coding sequence of clone PL776_6 deposited under accession number ATCC
• the polynudeotide encodes the full-length or a mature protein encoded by the cDNA insert of done PL776_6 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:16, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising the amino add sequence from amino add 333 to amino acid 342 of SEQ ID NO:16.
• inventions provide isolated polynucleotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of:
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:15 .
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:15 from nucleotide 193 to nudeotide 2223, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:15 from nucleotide 193 to nudeotide 2223, to a nudeotide sequence corresponding to the 3' end of said sequence of
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO: 15 from nucleotide 1045 to nucleotide 2223, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:15 from nucleotide 1045 to nucleotide 2223, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO: 15 from nucleotide 1045 to nucleotide 2223.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• PL776_6 deposited under accession number ATCC 98715; the protein being substantially free from other mammalian proteins.
• the protein comprises the amino acid sequence of SEQ ID NO:16.
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:16, or a protein comprising a fragment of the amino add sequence of SEQ
• the fragment comprising the amino add sequence from amino acid 333 to amino acid 342 of SEQ ID NO:16.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• (k) a polynudeotide which encodes a spedes homologue of the protein of (h) or (i) above ; (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17.
• polynudeotide encodes the full-length or a mature protein encoded by the cDNA insert of clone pm4_13 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:18, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising the amino add sequence from amino add 149 to amino acid 158 of SEQ ID NO:18.
• Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
• inventions provide isolated polynudeotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of: (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
• the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:17 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:17.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:17 from nucleotide 198 to nudeotide 1121, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:17 from nudeotide 198 to nucleotide 1121, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 198 to nucleotide 1121.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:17 from nucleotide 381 to nucleotide 1121, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO: 17 from
• nucleotide 381 to nucleotide 1121 to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 381 to nucleotide 1121.
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO: 18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:18, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 149 to amino acid 158 of SEQ ID NO:18.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• a polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h) and that has a length that is at least
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:19.
• polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of done pt326_4 deposited under accession number ATCC 98715.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino add 317 to amino acid 326 of SEQ ID NO:20.
• inventions provide isolated polynucleotides produced according to a process selected from the group consisting of: (a) a process comprising the steps of:
• the polynucleotide isolated according to the above process comprises a nudeotide sequence corresponding to the cDNA sequence of SEQ ID NO:19, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:19 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:19.
• the polynudeotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO: 19 from nucleotide 19 to nucleotide 1953, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:19 from nucleotide 19 to nucleotide 1953, to a nucleotide
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino add sequence selected from the group consisting of:
• the present invention provides a protein comprising a fragment of the amino add sequence of SEQ ID NO:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino adds of SEQ ID NO:20, or a protein comprising a fragment of the amino add sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 317 to amino acid 326 of SEQ ID NO:20.
• the polynucleotide is operably linked to an expression control sequence.
• the invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein. Processes are also provided for producing a protein, which comprise:
• the protein produced according to such methods is also provided by the present invention.
• Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier.
• Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
• Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
• Figures 1A and IB are schematic representations of the pED6 and pNOTs vertors, respectively, used for deposit of clones disclosed herein.
• nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application.
• the nudeotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino add sequence (both full-length and mature forms) can then be determined from such nucleotide sequence.
• the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and dete ⁇ rtining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
• a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, induding transport as a result of signal sequences in its amino acid sequence.
• "Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed.
• “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
• cc359 4" A polynudeotide of the present invention has been identified as clone "cc359_4".
• cc359_4 was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• cc359_4 is a full-length done,
• nucleotide sequence of cc359_4 as presently determined is reported in SEQ ID NO:l, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino add sequence of the cc359_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
• Arnino adds 29 to 41 of SEQ ID NO:2 are a predicted leader/signal sequence, with the predicted mature amino add sequence beginning at amino add 42. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predided leader/signal sequence not be separated from the remainder of the cc359_4 protein.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing done cc359_4 should be approximately 2200 bp.
• cc359_4 The nucleotide sequence disclosed herein for cc359_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cc359_4 demonstrated at least some similarity with sequences identified as H23117 (ym51gl2.sl Homo sapiens cDNA clone 51970 3'). Based upon sequence similarity, cc359_4 proteins and each similar protein or peptide may share at least some activity. The cc359_4 protein sequence contains four chaoptin domains (at amino acids 94-115, 118-139, 142-163, and 261-282 of SEQ ID NO:2).
• Chaoptin is a cell- surface glycoprotein required for Drosophila photoreceptor cell morphogenesis. Chaoptin is largely composed of 41 potentially amphipathic repeats. Chaoptin repeats that have been reported in both yeast and human proteins are remarkably similar, suggesting their general importance as a structural and/or functional motif. Additionally, the cc359_4 protein was found to contain a leucine zipper motif. The TopPredll computer program predicts five potential transmembrane domains within the cc359_4 protein sequence, centered around amino acids 20, 410, 490, 530, and 590 of SEQ ID NO:2, respectively.
• ct547 2 A polynucleotide of the present invention has been identified as clone "ct547_2".
• ct547_2 was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the arnino acid sequence of the encoded protein.
• ct547_2 is a full-length clone
• nucleotide sequence of ct547_2 as presently determined is reported in SEQ ID NO:3, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ct547_2 protein corresponding to the foregoing nudeotide sequence is reported in SEQ ID NO:4. Amino adds 44 to 56 of SEQ ID NO:4 are a predided leader/signal sequence, with the predicted mature amino add sequence beginning at amino acid 57. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the ct547_2 protein.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing done ct547_2 should be approximately 1600 bp.
• ct547_2 The nucleotide sequence disclosed herein for ct547_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ct547_2 demonstrated at least some similarity with sequences identified as AA428546 (zw47d03.rl Soares total fetus Nb2HF8 9w Homo sapiens cDNA done 773189 5'), R20032 (yg31g04.rl Homo sapiens cDNA clone 34069 5'), R27350 (yh53cl2.rl Homo sapiens cDNA done 1334625'), and R66971 (yi29el2.rl Homo sapiens cDNA clone 1406865'). Based upon sequence similarity, ct547_2 proteins and each similar protein or peptide may share at least some activity.
• a polynucleotide of the present invention has been identified as clone "en553_l".
• en553_l was isolated from a human fetal brain cDNA library and was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino add sequence of the encoded protein.
• en553_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "en553_l protein").
• nucleotide sequence of en553_l as presently determined is reported in SEQ ID NO:5, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the en553_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. Amino adds 17 to 29 of SEQ ID NO:6 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 30. Due to the hydrophobic nature
• the EcoRI/NotI restriction fragment obtainable from the deposit containing clone en553_l should be approximately 2675 bp.
• en553_l The nucleotide sequence disdosed herein for en553_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. en553_l demonstrated at least some similarity with sequences identified as No hits were found in the databases. The nucleotide sequence of en553_l indicates that it may contain one or more copies of the following repetitive elements: Alu, MIR.
• nn296_2 A polynucleotide of the present invention has been identified as clone "nn296_2".
• nn296_2 was isolated from a human fetal kidney (293 cell line) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino add sequence of the encoded protein.
• nn296_2 is a full- length clone, including the entire coding sequence of a secreted protein (also referred to herein as "nn296_2 protein").
• nucleotide sequence of nn296_2 as presently determined is reported in SEQ ID NO:7, and indudes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the nn296_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing done nn296_2 should be approximately 4500 bp.
• nucleotide sequence disclosed herein for nn296_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
• nn296_2 demonstrated at least some similarity with sequences identified as AA054538 (zk83e02.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 4894343'), AA175407 (ms80c06.rl Soares mouse 3NbMS Mus musculus cDNA clone 617866 5' similar to WP R08C7.2 CE07425), AA510586 (vg33c05.rl Soares mouse
• nn296_2 protein demonstrated at least some similarity to sequences identified as U61953 (cosmid R08C7 [Caenorhabditis elegans]). Based upon sequence similarity, nn296_2 proteins and each similar protein or peptide may share at least some activity.
• the TopPred ⁇ computer program predicts two potential transmembrane domains within the nn296_2 protein sequence, one centered around amino acid 50 and another around amino acid 340 of SEQ ID NO:8.
• the nucleotide sequence of nn296_2 indicates that it may contain at least one repetitive element.
• nq27_13 A polynucleotide of the present invention has been identified as clone "nq27_13".
• nq27_13 was isolated from a human adult blood (erythroleukemia TF-1) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• nq27_13 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "nq27_13 protein").
• nq27_13 The nucleotide sequence of nq27_13 as presently determined is reported in SEQ ID NO:9, and indudes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the nq27_13 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Another potential nq27_13 reading frame and predicted amino acid sequence is encoded by basepairs 371 to 616 of SEQ ID NO:9 and is reported in SEQ ID NO:32.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing clone nq27_13 should be approximately 900 bp.
• the nucleotide sequence disclosed herein for nq27_13 was searched against the
• nq27_13 demonstrated at least some similarity with sequences identified as AA015599 (ze20hl0.sl Soares fetal heart NbHH19W Homo sapiens cDNA clone 359587 3'), AA465112 (aa32c05.rl NCI_CGAP_GCB1 Homo sapiens cDNA clone
• nq27_13 proteins and each similar protein or peptide may share at least some activity.
• the TopPredll computer program predicts a potential transmembrane domain at the N-terminus of the nq27_13 protein sequence, centered around amino add 13 of SEQ ID NO:10. nq27_13 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 11 kDa was detected in membrane fractions using SDS polyacrylamide gel electrophoresis.
• a polynucleotide of the present invention has been identified as clone "pk65_4".
• pk65_4 was isolated from a human fetal kidney (293 cell line) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino add sequence of the encoded protein.
• pk65_4 is a full- length done, including the entire coding sequence of a secreted protein (also referred to herein as "pk65_4 protein").
• the nucleotide sequence of pk65_4 as presently determined is reported in SEQ ID NO:ll, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pk65_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing clone pk65_4 should be approximately 1500 bp.
• pk65_4 The nucleotide sequence disclosed herein for pk65_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols.
• pk65_4 demonstrated at least some similarity with sequences identified as AA309041 (EST179822 Colon carcinoma (Caco-2) cell line I Homo sapiens cDNA 5' end), AA316187 (EST187903 HCC cell line (matastasis to liver in mouse) II Homo sapiens cDNA 5' end), AA488843 (aa55al0.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:824826 3'), AF022811 (Mus musculus cornichon mRNA, complete eds), D16980 (Human HepG2 partial cDNA, clone hmd2a04m5), N44081 (yy32a02.r
• pk65_4 39 sapiens cDNA clone 2729065' similar to PIR A56724 A56724 cni protein - fruit fly).
• the predicted amino acid sequence disclosed herein for pk65_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
• the predicted pk65_4 protein demonstrated at least some similarity to sequences identified as AB006191 (cornichon-like protein [Mus musculus]), AF022811 (cornichon [Mus musculus]), and U28069 (cni gene produd [Drosophila melanogaster]). Based upon sequence similarity, pk65_4 proteins and each similar protein or peptide may share at least some activity.
• the TopPredll computer program predicts three potential transmembrane domains within the pk65_4 protein sequence, centered around amino acids 16, 67, and 133 of SEQ ID NO:12, respectively.
• pk65_4 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 15 kDa was detected in membrane fractions using SDS polyacrylamide gel electrophoresis.
• a polynudeotide of the present invention has been identified as clone "pk855_l".
• pk855_l was isolated from a human fetal kidney (293 cell line) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino add sequence of the encoded protein.
• pk855_l is a full- length clone, including the entire coding sequence of a secreted protein (also referred to herein as " ⁇ k855_l protein").
• nucleotide sequence of pk855_l as presently determined is reported in SEQ ID NO:13, and indudes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pk855_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14. Amino adds 29 to 41 of SEQ ID NO:14 are a predicted leader/signal sequence, with the predicted mature amino add sequence beginning at amino acid 42. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pk855_l protein.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing clone pk855_l should be approximately 1400 bp.
• pk855_l demonstrated at least some similarity with sequences identified as AA236320 (zr53h03.rl Soares NhHMPu SI Homo sapiens cDNA clone 667157 5'), AA491296 (aa53d03.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:8246453'), and W76249 (zd66e06.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 345634 5').
• pk855_l proteins and each similar protein or peptide may share at least some activity.
• the TopPredll computer program predicts an additional potential transmembrane domain within the pk855_l protein sequence centered around amino acid 78 of SEQ ID NO: 14.
• the nucleotide sequence of pk855_l indicates that it may contain some repeat elements (including some short stretches of poly(T)).
• PL776_6 was isolated from a human fetal kidney (293 cell line) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• PL776_6 is a full- length clone, including the entire coding sequence of a secreted protein (also referred to herein as "PL776_6 protein").
• nucleotide sequence of PL776_6 as presently determined is reported in SEQ ID NO:15, and indudes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the PL776_6 protein corresponding to the foregoing nudeotide sequence is reported in SEQ ID NO:16.
• Amino acids 272 to 284 of SEQ ID NO:16 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 285. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the PL776_6 protein.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing clone PL776_6 should be approximately 3600 bp.
• nucleotide sequence disclosed herein for PL776_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
• PL776_6 demonstrated at least some similarity with sequences identified as AA009770 (zi04c06.sl Soares fetal liver spleen 1NFLS SI Homo sapiens cDNA clone 4298023'), AA056343 (zl66f04.sl Stratagene colon (#937204) Homo sapiens cDNA clone 509599 3'), AA076433 (zml9g07.sl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526140 3'), AA076517 (zml9g07.rl Stratagene pancreas (#937208) Homo sapiens cDNA done 5261405'), M93661 (Rat notch 2 mRNA), and R36803 (CBS-216 Homo sapiens cDNA clone CBS-2165' end). Based upon sequence similarity, PL776_6 proteins and each similar protein or
• pm4_13 A polynudeotide of the present invention has been identified as clone "pm4_13".
• pm4_13 was isolated from a human fetal kidney (293 cell line) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• pm4_13 is a full- length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pm4_13 protein").
• the nucleotide sequence of pm4_13 as presently determined is reported in SEQ ID NO:17, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pm4_13 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18. Amino adds 49 to 61 of SEQ ID NO:18 are a predicted leader/signal sequence, with the predicted mature amino add sequence beginning at arnino add 62. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pm4_13 protein.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing done pm4_13 should be approximately 3100 bp.
• pm4_13 demonstrated at least some similarity with sequences identified as H98853 (yxl5dl0.sl Homo sapiens cDNA clone 261811 3'), R43710 (ygl9fl2.sl Homo sapiens cDNA clone 32692 3'), and T22962 (Human gene signature HUMGS04687).
• the predicted amino acid sequence disclosed herein for pm4_13 was
• the predicted pm4_13 protein demonstrated at least some similarity to sequences identified as U20865 (ylr246wp [Saccharomyces cerevisiae]), Z29121 (ZK757.1 [Caenorhabditis elegans]), Z46259 (N0325 gene product [Saccharomyces cerevisiae]), Z68507 (ml8.8 [Caenorhabditis elegans]), and Z71602 (orf ynl326c [Saccharomyces cerevisiae]).
• pm4_13 proteins and each similar protein or peptide may share at least some activity.
• the TopPredll computer program predicts three additional potential transmembrane domains within the pm4_13 protein sequence, centered around amino acids 86, 184, and 225 of SEQ ID NO:18, respectively.
• a polynudeotide of the present invention has been identified as clone "pt326_4".
• pt326_4 was isolated from a human adult blood (lymphoblastic leukemia MOLT-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• pt326_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pt326_4 protein").
• nudeotide sequence of pt326_4 as presently determined is reported in SEQ ID NO:19, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pt326_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
• the EcoRI/NotI restriction fragment obtainable from the deposit containing done pt326_4 should be approximately 3100 bp.
• the nucleotide sequence disclosed herein for pt326_4 was searched against the
• pt326_4 demonstrated at least some similarity with sequences identified as AA361324 (EST70660 T-cell lymphoma Homo sapiens cDNA 5' end), H94651 (yvl9cl2.sl Homo sapiens cDNA clone 243190 3'), T22309 (Human gene signature HUMGS03882), and U17907 (Human chromosome 17q21 mRNA clone B169).
• the predicted amino acid sequence disclosed herein for pt326_4 was searched against the GenPept and GeneSeq arnino acid sequence databases using the BLASTX search protocol.
• the predicted pt326_4 protein demonstrated at least some similarity to sequences identified as D89646 (GTBP-ALT [Homo sapiens]) and U28946 (G/T mismatch binding
• GTBP 43 protein (GTBP) [Homo sapiens]). Based upon sequence similarity, pt326_4 proteins and each similar protein or peptide may share at least some activity.
• Clones cc359_4, ct547_2, en553_l, nn296_2, nq27_13, pk65_4, pk855_l, PL776_6, pm4_13, and pt326_4 were deposited on March 31, 1998 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an original deposit under the Budapest Treaty and were given the accession number ATCC 98715, from which each done 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' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Figures 1A and IB, respectively.
• the pED6dpc2 vector (“pED6" was derived from pED ⁇ dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al, 1991, Nucleic Acids Res.
• the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site.
• the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate.
• the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector.
• the cDNA may also be expressed from the vectors in which they were deposited.
• Bacterial cells containing a particular clone can be obtained from the composite deposit as follows: An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of an oligonucleotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
• the design of the oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
• the oligonucleotide should preferably be labeled with ⁇ - 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
• 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 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
• Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
• fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
• a fusion could be to the Fc portion of an IgG molecule.
• Other immunoglobulin isotypes may also be used to generate such fusions.
• a protein - IgM fusion would generate a decavalent form of the protein of the invention.
• the present invention also provides both full-length and mature forms of the disclosed proteins.
• the full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone.
• the mature form(s) of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
• the sequence(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form.
• the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
• “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore indude but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
• Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
• An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
• the chromosomal location corresponding to the polynucleotide sequences disclosed herein may also be determined, for example by hybridizing appropriately labeled polynucleotides of the present invention to chromosomes in situ. It may also be possible to determine the corresponding chromosomal location for a disclosed polynucleotide by identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs), that have already been mapped to particular chromosomal locations. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotide of the present invention have been listed by database accession number.
• ESTs expressed sequence tags
• Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
• the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol 58: 1- 39; all of which are incorporated by reference herein).
• Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
• organisms are provided in which the gene(s) corresponding to the polynudeotide sequences disdosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s).
• Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive /negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S.
• These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s).
• 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.
• 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
• the TopPredll computer program can be used to predict the location of transmembrane domains in an amino add sequence, domains which are described by the location of the center of the transmsmbrane domain, with at least ten transmembrane amino acids on each side of the reported central residue(s).
• Proteins and protein fragments of the present invention include proteins with amino add sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
• sequence identity may be determined using WU-BLAST
• search programs BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX
• WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes.
• the gapped alignment routines are integral to the database search itself, and thus yield much better sensitivity and selectivity while producing the more easily interpreted output. Gapping can optionally be turned off in all of these programs, if desired.
• the default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized. Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
• a "species homologue” is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide.
• polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while mir mizing sequence gaps.
• Spedes homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
• spedes homologues are those isolated from mammalian species.
• species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus,
• allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynudeotides.
• allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while ⁇ unimizing sequence gaps.
• Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
• the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
• the present invention also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein.
• stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
• the hybnd length is that anti ⁇ pated for the hybndized reg ⁇ on(s) of the hybndizing polynucleotides.
• the hybnd length is assumed to be that of the hybndizing polynucleotide.
• the hybnd length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementanty. f .
• SSPE 0.15M NaCl, lOmM NaH ⁇ C and 1 25mM EDTA, pH 74
• SSC 0 15M NaCl and 15mM sodium citrate
• each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while rriinimizing sequence gaps.
• the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
• an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
• Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
• operably linked means that the isolated polynudeotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
• Mammalian host cells include, for example, monkey COS cells, Chinese Hamster
• Ovary (CHO) cells human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
• yeast eukaryotes
• prokaryotes e.g., yeast or any yeast strain capable of expressing heterologous proteins.
• Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial
• heterologous proteins 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 polynudeotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
• suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
• Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). incorporated herein by reference.
• an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
• the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
• the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
• the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
• the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
• Kits for expression and purification of such fusion proteins are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and Invitrogen Corporation (Carlsbad, CA), 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 the Eastman Kodak Company (New Haven, CT).
• RP- HPLC reverse-phase high performance liquid chromatography
• hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
• Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
• the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
• the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nudeotide 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.
• 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.
• polynudeotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
• Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
• the polynucleotides provided by the present invention can be used by the research community for various purposes.
• the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques;
• the polynudeotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
• the polynucleotide can also be used in interaction trap assays (such as, for example, those described in Gyuris et al, 1993, Cell 75: 791-803 and in Rossi et al, 1997, Proc. Natl. Acad.
• 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 deterrnine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
• the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
• the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
• Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses indude without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
• the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
• the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
• a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or irihibiting) or cell differentiation (either inducing or inhibiting) activity or may
• cytokine activity 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- Intersdence (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.
• Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine
• Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
• a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
• a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
• SCID severe combined immunodeficiency
• These immune defidendes 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 HTV, 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.
• 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 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
• B7 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
• 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, Sdence 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, £9:11102-11105 (1992).
• murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
• Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
• Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
• Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
• Administration of reagents which block costimulation of T cells by disrupting 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.
• 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/lprApr 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
• 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
• 61 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.
• 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 infeded 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 reintiOducing 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.
• 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.
• 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.
• 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.
• 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 indude 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-Intersdence (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. Sd. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
• T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: 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.
• 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,
• 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.
• 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., CeU 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. 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
• 64 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
• Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
• Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al eds. Vol pp. 23-39,
• 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 craniofadal 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
• 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.
• compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or Ugament defects.
• the compositions may also indude 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, induding 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 invention may also exhibit angiogenic activity.
• a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
• a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
• the activity of a protein of the invention may, among other means, be measured by the following methods:
• Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/ 16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
• Assays for wound healing activity include, without limitation, those described in:
• 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 follide stimulating hormone (FSH).
• FSH follicle stimulating hormone
• FSH follide stimulating hormone
• a protein of the present invention alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
• 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.
• a protein of the invention may, among other means, be measured by the following methods:
• Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
• a protein of the present invention may have chemotactic or diemokinetic 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 iemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
• Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
• a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
• the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
• the activity of a protein of the invention may, among other means, be measured by the following methods:
• Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
• Suitable assays for movement and adhesion indude without limitation, those described in: Current Protocols in Immunology, Ed by
• a protein of the invention may also exhibit hemostatic or thrombolytic activity.
• 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 (induding 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.
• the activity of a protein of the invention may, among other means, be measured by the following methods:
• Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Biereretal., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
• Proteins of the present invention may also exhibit anti-inflammatory activity.
• the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
• Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation assodated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
• infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
• ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease
• Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
• the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved
• cadherin domains 71 extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule.
• the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino adds in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specifidty of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin.
• some cadherins engage in heterophilic adhesion with other cadherins.
• E-cadherin one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types.
• proteins of the present invention with cadherin activity and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynudeotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
• Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
• Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or enminating the tendency of the cells to metastasize.
• proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
• Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
• Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
• Fragments of proteins of the present invention with cadherin activity preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects.
• fragments of proteins of the present invention with cadherin activity preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
• Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
• a protein of the invention may exhibit other anti-tumor activities.
• a protein may inhibit tumor growth directly or indirectly (such as, for example, via antibody-dependent cell-mediated cytotoxidty (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 fadors, agents or cell types which inhibit tumor growth, or by suppressing, eUminating or inhibiting factors, agents or cell types which promote tumor growth.
• ADCC antibody-dependent cell-mediated cytotoxidty
• 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 (induding 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.
• hyperproliferative disorders such as, for example, psoriasis
• immunoglobulin-like activity such as, for example, the ability to bind antigens or complement
• 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- , 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 nunimize 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.
• multimers e.g., heterodimers or homodimers
• complexes with itself or other proteins.
• compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
• the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
• the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
• B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
• T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
• TCR T cell receptor
• MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
• the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
• antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
• the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
• Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysoledthin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disdosed, 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 eadi 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.
• 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 fador(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
• 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.lng to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
• the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physidan 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 whidi specifically react with the protein.
• antibody includes without limitation a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, a CDR-grafted antibody, a humanized antibody, or fragments thereof which bind to the indicated protein.
• Such term also includes any other species derived from an antibody or antibody sequence which is capable of binding the indicated protein.
• Antibodies to a particular protein can be produced by methods well known to those skilled in the art.
• monoclonal antibodies can be produced by generation of antibody-producing hybridomas in accordance with known methods (see for example, Goding, 1983, Monoclonal antibodies: principles and practice, Academic Press Inc., New York; and Yokoyama, 1992, "Production of Monoclonal Antibodies” in Current Protocols in Immunology, Unit 2.5, Greene Publishing Assoc. and John Wiley & Sons).
• Polyclonal sera and antibodies can be produced by inoculation of a mammalian subject with the relevant protein or fragments thereof in accordance with known methods.
• Fragments of antibodies, receptors, or other reactive peptides can be produced from the corresponding antibodies by cleavage of and collection of the desired fragments in accordance with known methods (see for example, Goding, supra; and Andrew et al., 1992, "Fragmentation of Immunoglobulins” in Current Protocols in Immunology, Unit 2.8, Greene Publishing Assoc. and John Wiley & Sons).
• Chimeric antibodies and single chain antibodies can also be produced in accordance with known recombinant methods (see for example, 5,169,939, 5,194,594, and 5,576,184).
• Humanized antibodies can also be made from corresponding murine antibodies in accordance with well known methods (see for example, U.S. Patent Nos.
• human antibodies may be produced in non-human animals such as mice that have been genetically altered to express human antibody molecules (see for example Fishwild et al, 1996, Nature Biotechnology 14: 845-851; Mendez etal, 1997, Nature Genetics 15: 146-156 (erratum Nature Genetics 16: 410); and U.S. Patents 5,877,397 and 5,625,126).
• Such antibodies maybe 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
• 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
• 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.
• compositions 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 protem-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 canonic 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 benefidal to the treatment of the bone and/or cartilage defed, wound, or tissue in question.
• agents include various growth factors such as epidermal growth factor
• EGF platelet derived growth factor
• TGF- ⁇ transforming growth factors
• TGF- ⁇ TGF- ⁇
• IGF insulin-like growth factor
• the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
• the dosage regimen of a protem-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 fadors, sud as IGF I (insulin like growth factor I), to the final composition, may also effect
• 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 nudeic add into a cell or organism (induding, 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.

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