WO1998056805A1 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998056805A1
WO1998056805A1 PCT/US1998/012076 US9812076W WO9856805A1 WO 1998056805 A1 WO1998056805 A1 WO 1998056805A1 US 9812076 W US9812076 W US 9812076W WO 9856805 A1 WO9856805 A1 WO 9856805A1
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WO
WIPO (PCT)
Prior art keywords
seq
polynucleotide
amino acid
protein
nucleotide
Prior art date
Application number
PCT/US1998/012076
Other languages
French (fr)
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
Steven H. Howes
Kim Fechtel
Original Assignee
Genetics Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to AU78352/98A priority Critical patent/AU7835298A/en
Priority to CA002293825A priority patent/CA2293825A1/en
Priority to JP50318099A priority patent/JP2002514089A/en
Priority to EP98926539A priority patent/EP1003768A4/en
Publication of WO1998056805A1 publication Critical patent/WO1998056805A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention provides novel 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) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 2178 to nucleotide 2513; the nucleotide sequence of SEQ ID NO:l from nucleotide 2364 to nucleotide 2513; the nucleotide sequence of SEQ ID NO:l from nucleotide 1980 to nucleotide 2311; the nucleotide sequence of the full-length protein coding sequence of clone ell 81 3 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67.
  • 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) consecutive 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 51 to amino acid 60 of SEQ ID NO:2.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67.
  • the present invention provides 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) consecutive amino acids of SEQ ID NO:2, or 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 51 to amino acid 60 of SEQ ID NO:2.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 207 to nucleotide 893; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 527; the nucleotide sequence of the full-length protein coding sequence of clone crl044_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone crl044_l deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107.
  • 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) consecutive 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 109 to amino acid 118 of SEQ ID NO:4.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107.
  • the present invention provides 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) consecutive amino acids of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4having biological activity, the fragment comprising the amino acid sequence from amino acid 109 to amino acid 118 of SEQ ID NO:4.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 400; the nucleotide sequence of SEQ ID NO:5 from nucleotide 118 to nucleotide 392; the nucleotide sequence of the full-length protein coding sequence of clone cz251_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone cz251_l deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105.
  • 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) consecutive amino acids 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 49 to amino acid 58 of SEQ ID NO:6.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105.
  • 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) consecutive amino acids of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6having biological activity, the fragment comprising the amino acid sequence from amino acid 49 to amino acid 58 of SEQ ID NO:6.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456.
  • 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) consecutive amino acids 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 comprising the amino acid sequence from amino acid 76 to amino acid 85 of SEQ ID NO:8.
  • 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 acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8having biological activity, the fragment comprising the amino acid sequence from amino acid 76 to amino acid 85 of SEQ ID NO:8.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 778 to nucleotide 1083; the nucleotide sequence of SEQ ID NO:9 from nucleotide 931 to nucleotide 1083; the nucleotide sequence of SEQ ID NO:9 from nucleotide 802 to nucleotide 1056; the nucleotide sequence of the full-length protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 93.
  • the present invention provides a polynucleotide encoding a protein 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) consecutive amino acids of SEQ ID NO:10, or a polynucleotide encoding 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 46 to amino acid 55 of SEQ ID NO:10.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 93.
  • the present invention provides a protein 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) consecutive amino acids of SEQ ID NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10having biological activity, the fragment comprising the amino acid sequence from amino acid 46 to amino acid 55 of SEQ ID NO:10.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:ll from nucleotide 390 to nucleotide 1355; the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1384 to nucleotide 1736; the nucleotide sequence of the full-length protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456.
  • 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) consecutive amino acids of SEQ ID NO: 12, or a polynucleotide 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 156 to amino acid 165 of SEQ ID NO:12.
  • 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 acid sequence of SEQ ID NO: 12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids 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 156 to amino acid 165 of SEQ ID NO:12.
  • 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:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 14;
  • a polynucleotide which is an allelic variant of a polynucleotide of
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 879 to nucleotide 1391; the nucleotide sequence of SEQ ID NO:13 from nucleotide 519 to nucleotide 1074; the nucleotide sequence of the full-length protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone gnll4 _1 deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 65.
  • 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) consecutive amino acids of SEQ ID NO:14, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 80 to amino acid 89 of SEQ ID NO:14.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 65.
  • the present invention provides 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) consecutive amino acids of SEQ ID NO: 14, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14having biological activity, the fragment comprising the amino acid sequence from amino acid 80 to amino acid 89 of SEQ ID NO:14.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 225 to nucleotide 1508; the nucleotide sequence of SEQ ID NO:15 from nucleotide 252 to nucleotide 1508; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 302; the nucleotide sequence of the full-length protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 26.
  • 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) consecutive amino acids 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 acid sequence from amino acid 209 to amino acid 218 of SEQ ID NO:16.
  • 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: 16 or the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 26.
  • the present invention provides 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) consecutive amino acids of SEQ ID NO:16, or a protein comprising a fragment of the amino acid sequence of SEQ ID
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
  • polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456.
  • 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) consecutive amino acids 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 acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO:18.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • fragments of the amino acid sequence of SEQ ID NO: 18 comprising eight consecutive amino acids of SEQ ID NO:18; and (c) the amino acid sequence encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
  • protein comprises the amino acid sequence of SEQ ID NO:18.
  • the present invention provides 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) consecutive amino acids 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 22 to amino acid 31 of SEQ ID NO:18.
  • 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:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:20;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:19 from nucleotide 96 to nucleotide 1145; the nucleotide sequence of SEQ ID NO:19 from nucleotide 109 to nucleotide 539; the nucleotide sequence of the full-length protein coding sequence of clone hm236_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone hm236_l deposited under accession number ATCC 98456.
  • the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148.
  • 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) consecutive 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 acid 170 to amino acid 179 of SEQ ID NO:20.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148.
  • the present invention provides 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) consecutive amino acids of SEQ ID NO:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20having biological activity, the fragment comprising the amino acid sequence from amino acid 170 to amino acid 179 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 1 A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
  • nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application.
  • the nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence.
  • "Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
  • cll81_3 A polynucleotide of the present invention has been identified as clone "cll81_3".
  • cll81_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cll81_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cll81_3 protein").
  • nucleotide sequence of cll81_3 as presently determined is reported in SEQ ID NO:l . What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cll81_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
  • Amino acids 50 to 62 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 63, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cll81 3 should be approximately 2800 bp.
  • cll81_3 The nucleotide sequence disclosed herein for cll81_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cll81_3 demonstrated at least some similarity with sequences identified as C16190 (Human aorta cDNA 5'-end GEN-241B04) and L36900 (Saccharomyces cerevisiae mitochondrion transfer RNA-Serl (tRNA-Ser) gene and varl gene, complete eds). Based upon sequence similarity, cll81_3 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cll81_3 protein sequence centered around amino acid 77 of SEQ ID NO:2. Clone "crl044 1"
  • crl044_l was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • crl044_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "crl044_l protein").
  • Amino acids 7 to 19 of SEQ ID NO:31 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20 of SEQ ID NO:31, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone crl044_l should be approximately 3200 bp.
  • crl044_l The nucleotide sequence disclosed herein for crl044_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/ BLASTX and FASTA search protocols.
  • crl044_l demonstrated at least some similarity with sequences identified as N99156 (zb81g04.sl Soares senescent fibroblasts NbHSF Homo sapiens cDNA clone 310038 3'), Q46852 (clone of recombinant human kappa casein gene fragment), and T20727 (Human gene signature HUMGS01945).
  • the predicted amino acid sequence disclosed herein for crl044_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted crl044_l protein demonstrated at least some similarity to sequences identified as Ml 6279 (antigen [Homo sapiens]) and U82164 (human CD99 type II). Based upon sequence similarity, crl044_l proteins and each similar protein or peptide may share at least some activity.
  • cz251 1 A polynucleotide of the present invention has been identified as clone "cz251_l".
  • cz251_l was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cz251_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cz251_l protein").
  • nucleotide sequence of cz251_l as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cz251_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cz251_l should be approximately 1200 bp.
  • cz251_l The nucleotide sequence disclosed herein for cz251_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cz251_l demonstrated at least some similarity with sequences identified as R55084 (yg87a06.rl Homo sapiens cDNA clone 40244 5') and U00930 (Human clone C4E 1.63 (CAC)n/(GTG)n repeat-containing mRNA). The predicted amino acid sequence disclosed herein for cz251_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted cz251_l protein demonstrated at least some similarity to the sequence identified as Z68751 (F01G4.1 [Caenorhabditis elegans]). Based upon sequence similarity, cz251_l proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of cz251_l may contain CAAA-like repeats.
  • ddl2_7 A polynucleotide of the present invention has been identified as clone "ddl2_7".
  • ddl2_7 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • ddl2_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ddl2_7 protein").
  • the nucleotide sequence of ddl2_7 as presently determined is reported in SEQ ID
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ddl2_7 should be approximately 1550 bp.
  • ddl2_7 The nucleotide sequence disclosed herein for ddl2_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ddl2_7 demonstrated at least some similarity with sequences identified as AA257999 (zs34a02.sl Soares NbHTGBC Homo sapiens cDNA clone 687050 3'). Based upon sequence similarity, ddl2_7 proteins and each similar protein or peptide may share at least some activity.
  • fnl91_3 A polynucleotide of the present invention has been identified as clone "fnl91_3".
  • fnl91_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • fnl91_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fnl91_3 protein").
  • nucleotide sequence of fnl91_3 as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the fnl91_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Amino acids 39 to 51 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 52, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone fnl91_3 should be approximately 3000 bp.
  • the nucleotide sequence disclosed herein for fnl91_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols.
  • fnl91_3 demonstrated at least some similarity with sequences identified as AA046787 (zk72f07.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 488389 5' similar to gb:L07077 ENOYL-COA HYDRATASE (HUMAN) ;contains Alu repetitive element), G13132 (human chromosome 7 STS sWSS3349; single read), T06013 (EST03902 Homo sapiens cDNA clone HFBDL25), T08594 (EST06486 Homo sapiens cDNA clone HIBBG72 5' end), and W28342 (45g9 Human retina cDNA randomly primed sublibrary Homo sapiens cDNA). Based upon sequence similarity, fnl91_3 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence in the 5' untranslated region of fnl91_3 may
  • gml96_4 A polynucleotide of the present invention has been identified as clone "gml96_4".
  • gml96_4 was isolated from a human adult uterus 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.
  • gml96_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gml96_4 protein").
  • nucleotide sequence of gml96_4 as presently determined is reported in SEQ ID NO:ll. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gml96_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12. Another potential gml96_4 reading frame and predicted amino acid sequence is encoded by basepairs 1364 to 2809 of SEQ ID NO:ll and is reported in SEQ ID NO:32. A frameshift in the nucleotide sequence of SEQ ID NO:ll could join the reading frames of SEQ ID NO:12 and SEQ ID NO:32.
  • the TopPredll computer program predicts two potential transmembrane domains within the amino acid sequence of SEQ ID NO:32.
  • Preferred fragments of the amino acid sequence of SEQ ID NO:32 comprise amino acids 1 to 163 or amino acids 236 to 245.of SEQ ID NO:32.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone gml96_4 should be approximately 3000 bp.
  • gml96_4 The nucleotide sequence disclosed herein for gml96_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gml96_4 demonstrated at least some similarity with sequences identified as AA233552 (zr43al0.sl Soares NhHMPu SI Homo sapiens cDNA clone 666138 3'), AB005666 (Homo sapiens mRNA for GTPase-activating protein, complete eds), F12887 (H.
  • gml96_4 sapiens partial cDNA sequence; clone c-3fh09), T25078 (Human gene signature HUMGS07218), and T75264 (yc88g09.rl Homo sapiens cDNA clone 23008 5').
  • the predicted amino acid sequence disclosed herein for gml96_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted gml96_4 protein demonstrated at least some similarity to the sequence identified as M64788 (GTPase activating protein [Homo sapiens]). Based upon sequence similarity, gml96_4 proteins and each similar protein or peptide may share at least some activity.
  • a polynucleotide of the present invention has been identified as clone "gnll4_l".
  • gnll4_l was isolated from a human adult blood (peripheral blood mononuclear cells treated with granulocyte-colony stimulating factor in vivo) 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.
  • gnll4_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gnll4_l protein").
  • nucleotide sequence of gnll4_l as presently determined is reported in SEQ ID NO: 13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gnll4_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14.
  • Amino acids 26 to 38 of SEQ ID NO:14 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 39 of SEQ ID NO:14, or are a transmembrane domain-
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone gnll4_l should be approximately 1500 bp.
  • the nucleotide sequence disclosed herein for gnll4_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols.
  • gnll4_l demonstrated at least some similarity with sequences identified as AA370547 (EST82206 Prostate gland I Homo sapiens cDNA 5' end), C04732 (Human Heart cDNA, clone 3NHC3910), and C05361 (Human Heart cDNA, clone 3NHC2451).
  • gnll4_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 gnll4_l protein sequence centered around amino acid 90 of SEQ ID NO:14.
  • hj968_2 A polynucleotide of the present invention has been identified as clone "hj968_2".
  • hj968_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 amino acid sequence of the encoded protein.
  • hj968_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hj968_2 protein").
  • nucleotide sequence of hj968_2 as presently determined is reported in SEQ ID NO:15. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hj968_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16. Amino acids 1 to 9 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 10, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hj968_2 should be approximately 1800 bp.
  • hj968_2 The nucleotide sequence disclosed herein for hj968_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hj968_2 demonstrated at least some similarity with sequences identified as AA071746 (mfl6h07.rl Life Tech mouse brain Mus musculus cDNA clone
  • hj968_2 The predicted amino acid sequence disclosed herein for hj968_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted hj968_2 protein demonstrated at least some similarity to the sequence identified as U23528 (translated cosmid B0034.[Caenorhabditis elegans]). Based upon sequence similarity, hj968_2 proteins and each similar protein or peptide may share at least some activity.
  • hkl0_3 A polynucleotide of the present invention has been identified as clone "hkl0_3".
  • hkl0_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • hkl0_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hkl0_3 protein").
  • nucleotide sequence of hkl0_3 as presently determined is reported in SEQ ID NO:17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hkl0_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18.
  • Amino acids 28 to 40 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 41, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone hkl0_3 should be approximately 1300 bp.
  • hkl0_3 The nucleotide sequence disclosed herein for hkl0_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hkl0_3 demonstrated at least some similarity with sequences identified as AA305975 (EST 176966 Jurkat T-cells VI Homo sapiens cDNA 5' end similar to S. cerevisiae hypothetical protein FAA3-BET1), F18016 (H. sapiens EST sequence 016-T), N36880 (yy37e07.sl Homo sapiens cDNA clone 273444 3'), and R87757 (yo45a08.sl Homo sapiens cDNA clone 180854 3').
  • the predicted amino acid sequence disclosed herein for hklO 3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted hkl0_3 protein demonstrated at least some similarity to the sequence identified as Z38113 .(CAI 0.21 [Saccharomyces cerevisiae]). Based upon sequence similarity, hkl0_3 proteins and each similar protein or peptide may share at least some activity. Clone "hm236 1"
  • hm236_l A polynucleotide of the present invention has been identified as clone "hm236_l".
  • hm236_l was isolated from a human adult testes (teratocarcinoma NCCIT) 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.
  • hm236_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hm236_l protein").
  • nucleotide sequence of hm236_l as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hm236_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone hm236_l should be approximately 1800 bp.
  • the nucleotide sequence disclosed herein for hm236_l was searched against the
  • hm236_l demonstrated at least some similarity with sequences identified as AA026169 (zk01b03.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 469229 5'), AA046211 (zk77e04.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 4888623'), AA223088 (PMY0368 KGla Lambda Zap Express cDNA Library Homo sapiens cDNA 5'), AB002368 and AC003010 (Human mRNA for KIAA0370 gene, partial eds), AC002399 (Human chromosome 16pll.2 BAC clone CIT987SK-A-481B3; HTGS phase 1, 18 unordered pieces, sequencing in progress), R86676 (ym86f03.
  • the predicted amino acid sequence disclosed herein for hm236_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted hm236_l protein demonstrated at least some similarity to sequences identified as AB002368 (KIAA0370 [Homo sapiens]). Based upon sequence similarity, hm236_l proteins and each similar protein or peptide may share at least some activity.
  • Clones cll81_3, crl044_l, cz251_l, ddl2_7, fnl91_3, gml96_4, gnll4_l, hj968_2, hkl0_3, and hm236_l were deposited on June 12, 1997 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 98456, from which each clone comprising a particular polynucleotide is obtainable.
  • Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' 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 pED6dpcl 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:
  • 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:
  • the oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • 6X SSC 20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al, Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • a bivalent form of the protein such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a protein - IgM fusion would generate a decavalent form of the protein of the invention.
  • the present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone.
  • the mature form(s) of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • the sequence(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
  • Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
  • the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol Sci. 15(7): 250-254; Lavarosky et al, 1997,
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
  • Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue” is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide.
  • polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canisfamiliaris, Oryctolagus cunicidus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (
  • allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
  • allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also mcludes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein Examples of stringency conditions are shown m the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybrid length is that anticipated for the hybridized reg ⁇ on(s) of the hybridizing polynucleotides
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE 0.15M NaCl, lOmM NaH 2 P0 4 , 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 minimizing sequence gaps.
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo205 cells human Colo205 cells
  • CV-1 cells other transformed primate cell lines
  • normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast eukaryotes
  • prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kod
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis. 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.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques;
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, those described in Gyuris et al, 1993, Cell 75: 791-803 and in Rossi et al, 1997, Proc. Natl Acad. Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. Cytokine and Cell Proliferation/Differentiation Activity
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cytokine
  • cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, TIO, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al, J. Immunol.
  • Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • To achieve sufficient immunosuppression or tolerance in a subject it may also be necessary to block the function of a combination of B lymphocyte antigens.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • 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 Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al, Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with 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
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. 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 follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • FSH follicle stimulating hormone
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
  • Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
  • Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
  • the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule.
  • the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
  • E-cadherin one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by 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 can be used to treat cancer.
  • Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
  • Fragments of proteins of the present invention with cadherin activity can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and /or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic f actor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
  • 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.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and
  • TGF- ⁇ 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 protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF I may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • AAAACATTGC CAATTTTGAT ATTTCCAGAG TTAATGTTAA ATTATTTGAA AGAAAATTAT 1140
  • AAACTGCACC CAGTTCTTCT GAACATATTA CCTTACAAAA CTTTCATTTA TGCCTGGTGA 2340
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 2 :
  • TCCAGGTCCA CATCAAGGTG ATTAGGCTCT TCCATGCACT CCACCTCCAG CTGCAAACCC 900
  • AAATGTCATC CAAACAAGAA ATAATGAGTG ACCAGCGGTT TAGACGGGTT 60 GCAAAGGACC CGAGATTTTG GGAAATGCCA GAAAAGGATC GAAAAGTCAA AATTGACAAG 120
  • ATGTGTAAAC AAAGATTCCA GGGCGTGGTT TTGCACTCCT GTTGTACTCT TTTAGAGGTG 720 GAAAAGAGGT GGATACTGAG ATCTAAGAGG AAAGGATAGT CATTCACGTT CTGAGATATG 780
  • GGTAACGGCC TCCAGATAAA GGGGTTATCC CTGTGGAAGT GACTTTTCCC CATTTGATCC 1500 CTTTTCAACT CTAAATGGCC AGGCCCAGAG CAGAAGAAGG GTTGGGTCTG GAAGGAAGGC 1560
  • CAGGCTGCCC AGTGCTTACC ATGCAGAAAG CAGTCAGCTG TACTCTGGAA GTTTCTGTTC 1980
  • CAATCACTCT AAAGGGCTGG ACGGGCTACC GTGGCGGTCT GGATACCAAA AATGATACCA 660
  • GTAATTTCTC AACAAAATGT GGCTTTTAGC CTGTCAGTGA TCTATTGGAC CAAACCTTCT 2940
  • AAAGTGGGCA TATTTGTGCC CTTGGACCAG ATGTAGCCCT TGATGCATTT TGCAGGAACA " 780
  • CTGGTATCCG CCTGGAGACC TGTGATGAAC
  • ACTCTGGAA CATTCAGGCC ACCATAGAGC 1080
  • TGCAGACCAG CCATGTGGCC AAGGGCTGTG ACCGTGACAA CTACTCAGAG CGGGCGCTGC 1140
  • CTCCCCTCCA GGCACTAGCC AGAGGGGGAA ACTGGCTTCT TGTCCCGCCT CTGTCACTGC 1500
  • CCAGCGTCTT CATGGGGAAG ATGAATGGAC CTGAGTAGCT GAAGGAAGGC CCCTCCCTAC 720 CCAAAGACTG GAGGCTTCTC AGCCTCAATT TCCCTGTCTG TACAGCTGAG GGCTCTGCCT 780
  • GTCCCCCACT GCTATCAGTA TGGAACCCCA GCTGGGGTCC CCTATTGAGT GCCGACTCCC 840
  • MOLECULE TYPE other nucleic acid
  • /desc "oligonucleotide"

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,XXX
(converted to a provisional application from non-provisional application 08/873,488), filed June 12, 1997, which is incorporated by reference herein.
FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed.
SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 2178 to nucleotide 2513;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 2364 to nucleotide 2513;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1980 to nucleotide 2311;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2; (j) 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 consecutive amino acids of SEQ ID NO:2;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 2178 to nucleotide 2513; the nucleotide sequence of SEQ ID NO:l from nucleotide 2364 to nucleotide 2513; the nucleotide sequence of SEQ ID NO:l from nucleotide 1980 to nucleotide 2311; the nucleotide sequence of the full-length protein coding sequence of clone ell 81 3 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67. In further preferred embodiments, 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) consecutive 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 51 to amino acid 60 of SEQ ID NO:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:l.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2; (b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising eight consecutive amino acids of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO:2, or 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 51 to amino acid 60 of SEQ ID NO:2.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 207 to nucleotide 893;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 527;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone crl044_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone crl044_l deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 207 to nucleotide 893; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 527; the nucleotide sequence of the full-length protein coding sequence of clone crl044_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone crl044_l deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107. In further preferred embodiments, 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) consecutive 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 109 to amino acid 118 of SEQ ID NO:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107;
(c) fragments of the amino acid sequence of SEQ ID NO:4 comprising eight consecutive amino acids of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4having biological activity, the fragment comprising the amino acid sequence from amino acid 109 to amino acid 118 of SEQ ID NO:4. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 400;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 118 to nucleotide 392;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cz251_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cz251_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 400; the nucleotide sequence of SEQ ID NO:5 from nucleotide 118 to nucleotide 392; the nucleotide sequence of the full-length protein coding sequence of clone cz251_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone cz251_l deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105. In further preferred embodiments, 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) consecutive amino acids 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 49 to amino acid 58 of SEQ ID NO:6.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:5. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising eight consecutive amino acids of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105. In further preferred embodiments, 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) consecutive amino acids of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6having biological activity, the fragment comprising the amino acid sequence from amino acid 49 to amino acid 58 of SEQ ID NO:6. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 13 to nucleotide 501;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 506;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:7 from nucleotide 13 to nucleotide 501; the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 506; the nucleotide sequence of the full-length protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456. In further preferred embodiments, 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) consecutive amino acids 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 comprising the amino acid sequence from amino acid 76 to amino acid 85 of SEQ ID NO:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) fragments of the amino acid sequence of SEQ ID NO:8 comprising eight consecutive amino acids of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone ddl2 7 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8having biological activity, the fragment comprising the amino acid sequence from amino acid 76 to amino acid 85 of SEQ ID NO:8. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 778 to nucleotide 1083;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 931 to nucleotide 1083;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 802 to nucleotide 1056;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 778 to nucleotide 1083; the nucleotide sequence of SEQ ID NO:9 from nucleotide 931 to nucleotide 1083; the nucleotide sequence of SEQ ID NO:9 from nucleotide 802 to nucleotide 1056; the nucleotide sequence of the full-length protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 93. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein 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) consecutive amino acids of SEQ ID NO:10, or a polynucleotide encoding 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 46 to amino acid 55 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:9.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 93;
(c) fragments of the amino acid sequence of SEQ ID NO:10 comprising eight consecutive amino acids of SEQ ID NO: 10; and
(d) the amino acid sequence encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 93. In further preferred embodiments, the present invention provides a protein 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) consecutive amino acids of SEQ ID NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10having biological activity, the fragment comprising the amino acid sequence from amino acid 46 to amino acid 55 of SEQ ID NO:10.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 390 to nucleotide 1355;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 1384 to nucleotide 1736;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:ll from nucleotide 390 to nucleotide 1355; the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1384 to nucleotide 1736; the nucleotide sequence of the full-length protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456. In further preferred embodiments, 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) consecutive amino acids of SEQ ID NO: 12, or a polynucleotide 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 156 to amino acid 165 of SEQ ID NO:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:ll. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12;
(b) fragments of the amino acid sequence of SEQ ID NO:12 comprising eight consecutive amino acids of SEQ ID NO:12; and
(c) the amino acid sequence encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:12. In further preferred embodiments, the present invention provides 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) consecutive amino acids 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 156 to amino acid 165 of SEQ ID NO:12.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 879 to nucleotide 1391;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 519 to nucleotide 1074; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456; (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 14; (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 879 to nucleotide 1391; the nucleotide sequence of SEQ ID NO:13 from nucleotide 519 to nucleotide 1074; the nucleotide sequence of the full-length protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone gnll4 _1 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 65. In further preferred embodiments, 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) consecutive amino acids of SEQ ID NO:14, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 80 to amino acid 89 of SEQ ID NO:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:13.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 14; (b) the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID NO:14 comprising eight consecutive amino acids of SEQ ID NO: 14; and
(d) the amino acid sequence encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 65. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO: 14, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14having biological activity, the fragment comprising the amino acid sequence from amino acid 80 to amino acid 89 of SEQ ID NO:14. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 225 to nucleotide 1508;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 252 to nucleotide 1508; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 1 to nucleotide 302;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456; (h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 16;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 225 to nucleotide 1508; the nucleotide sequence of SEQ ID NO:15 from nucleotide 252 to nucleotide 1508; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 302; the nucleotide sequence of the full-length protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 26. In further preferred embodiments, 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) consecutive amino acids 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 acid sequence from amino acid 209 to amino acid 218 of SEQ ID NO:16.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:15.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 26; (c) fragments of the amino acid sequence of SEQ ID NO: 16 comprising eight consecutive amino acids of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid 26. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO:16, or a protein comprising a fragment of the amino acid sequence of SEQ ID
NO:16having biological activity, the fragment comprising the amino acid sequence from amino acid 209 to amino acid 218 of SEQ ID NO:16.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 1113 to nucleotide 1274; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 1233 to nucleotide 1274;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 894 to nucleotide 1309;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hkl0_3 deposited under accession number
ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hkl0_3 deposited under accession number ATCC
98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 18;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:17 from nucleotide 1113 to nucleotide 1274; the nucleotide sequence of SEQ ID NO:17 from nucleotide 1233 to nucleotide 1274; the nucleotide sequence of SEQ ID NO: 17 from nucleotide 894 to nucleotide 1309; the nucleotide sequence of the full-length protein coding sequence of clone hkl0_3 deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone hkl0_3 deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456. In further preferred embodiments, 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) consecutive amino acids 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 acid sequence from amino acid 22 to amino acid 31 of SEQ ID NO:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:17.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 18;
(b) fragments of the amino acid sequence of SEQ ID NO: 18 comprising eight consecutive amino acids of SEQ ID NO:18; and (c) the amino acid sequence encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:18. In further preferred embodiments, the present invention provides 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) consecutive amino acids 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 22 to amino acid 31 of SEQ ID NO:18. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 96 to nucleotide 1145;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 109 to nucleotide 539; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hm236_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456; (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hm236_l deposited under accession number
ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:20; (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:19 from nucleotide 96 to nucleotide 1145; the nucleotide sequence of SEQ ID NO:19 from nucleotide 109 to nucleotide 539; the nucleotide sequence of the full-length protein coding sequence of clone hm236_l deposited under accession number ATCC 98456; or the nucleotide sequence of a mature protein coding sequence of clone hm236_l deposited under accession number ATCC 98456. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148. In further preferred embodiments, 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) consecutive 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 acid 170 to amino acid 179 of SEQ ID NO:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO: 19.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20; (b) the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising eight consecutive amino acids of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148. In further preferred embodiments, the present invention provides 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) consecutive amino acids of SEQ ID NO:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20having biological activity, the fragment comprising the amino acid sequence from amino acid 170 to amino acid 179 of SEQ ID NO:20. In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention.
Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
Clone "cll81 3"
A polynucleotide of the present invention has been identified as clone "cll81_3". cll81_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cll81_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cll81_3 protein").
The nucleotide sequence of cll81_3 as presently determined is reported in SEQ ID NO:l . What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cll81_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Amino acids 50 to 62 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 63, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cll81 3 should be approximately 2800 bp.
The nucleotide sequence disclosed herein for cll81_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cll81_3 demonstrated at least some similarity with sequences identified as C16190 (Human aorta cDNA 5'-end GEN-241B04) and L36900 (Saccharomyces cerevisiae mitochondrion transfer RNA-Serl (tRNA-Ser) gene and varl gene, complete eds). Based upon sequence similarity, cll81_3 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cll81_3 protein sequence centered around amino acid 77 of SEQ ID NO:2. Clone "crl044 1"
A polynucleotide of the present invention has been identified as clone "crl044_l". crl044_l was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. crl044_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "crl044_l protein").
The nucleotide sequence of crl044_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the crl044_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Amino acids 158 to 170 of SEQ ID NO:4 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 171 of SEQ ID NO:4, or are a transmembrane domain. Base pairs 175 to 237 of SEQ ID NO:3 are a possible intron; if this sequence were removed from SEQ
ID NO:3, another potential crl044_l reading frame and predicted amino acid sequence that could be encoded by basepairs 45 to 830 of SEQ ID NO:3 is reported in SEQ ID
NO:31. Amino acids 7 to 19 of SEQ ID NO:31 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20 of SEQ ID NO:31, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone crl044_l should be approximately 3200 bp.
The nucleotide sequence disclosed herein for crl044_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/ BLASTX and FASTA search protocols. crl044_l demonstrated at least some similarity with sequences identified as N99156 (zb81g04.sl Soares senescent fibroblasts NbHSF Homo sapiens cDNA clone 310038 3'), Q46852 (clone of recombinant human kappa casein gene fragment), and T20727 (Human gene signature HUMGS01945). The predicted amino acid sequence disclosed herein for crl044_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted crl044_l protein demonstrated at least some similarity to sequences identified as Ml 6279 (antigen [Homo sapiens]) and U82164 (human CD99 type II). Based upon sequence similarity, crl044_l proteins and each similar protein or peptide may share at least some activity.
Clone "cz251 1" A polynucleotide of the present invention has been identified as clone "cz251_l". cz251_l was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cz251_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cz251_l protein").
The nucleotide sequence of cz251_l as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cz251_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cz251_l should be approximately 1200 bp.
The nucleotide sequence disclosed herein for cz251_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cz251_l demonstrated at least some similarity with sequences identified as R55084 (yg87a06.rl Homo sapiens cDNA clone 40244 5') and U00930 (Human clone C4E 1.63 (CAC)n/(GTG)n repeat-containing mRNA). The predicted amino acid sequence disclosed herein for cz251_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted cz251_l protein demonstrated at least some similarity to the sequence identified as Z68751 (F01G4.1 [Caenorhabditis elegans]). Based upon sequence similarity, cz251_l proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of cz251_l may contain CAAA-like repeats.
Clone "dd!2 7"
A polynucleotide of the present invention has been identified as clone "ddl2_7". ddl2_7 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. ddl2_7 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ddl2_7 protein"). The nucleotide sequence of ddl2_7 as presently determined is reported in SEQ ID
NO:7. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ddl2_7 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ddl2_7 should be approximately 1550 bp.
The nucleotide sequence disclosed herein for ddl2_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ddl2_7 demonstrated at least some similarity with sequences identified as AA257999 (zs34a02.sl Soares NbHTGBC Homo sapiens cDNA clone 687050 3'). Based upon sequence similarity, ddl2_7 proteins and each similar protein or peptide may share at least some activity.
Clone "fn!91 3"
A polynucleotide of the present invention has been identified as clone "fnl91_3". fnl91_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. fnl91_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "fnl91_3 protein").
The nucleotide sequence of fnl91_3 as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the fnl91_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. Amino acids 39 to 51 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 52, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone fnl91_3 should be approximately 3000 bp. The nucleotide sequence disclosed herein for fnl91_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. fnl91_3 demonstrated at least some similarity with sequences identified as AA046787 (zk72f07.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 488389 5' similar to gb:L07077 ENOYL-COA HYDRATASE (HUMAN) ;contains Alu repetitive element), G13132 (human chromosome 7 STS sWSS3349; single read), T06013 (EST03902 Homo sapiens cDNA clone HFBDL25), T08594 (EST06486 Homo sapiens cDNA clone HIBBG72 5' end), and W28342 (45g9 Human retina cDNA randomly primed sublibrary Homo sapiens cDNA). Based upon sequence similarity, fnl91_3 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence in the 5' untranslated region of fnl91_3 may contain some repetitive elements.
Clone "gm!96 4" A polynucleotide of the present invention has been identified as clone "gml96_4". gml96_4 was isolated from a human adult uterus 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. gml96_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gml96_4 protein").
The nucleotide sequence of gml96_4 as presently determined is reported in SEQ ID NO:ll. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gml96_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12. Another potential gml96_4 reading frame and predicted amino acid sequence is encoded by basepairs 1364 to 2809 of SEQ ID NO:ll and is reported in SEQ ID NO:32. A frameshift in the nucleotide sequence of SEQ ID NO:ll could join the reading frames of SEQ ID NO:12 and SEQ ID NO:32. The TopPredll computer program predicts two potential transmembrane domains within the amino acid sequence of SEQ ID NO:32. Preferred fragments of the amino acid sequence of SEQ ID NO:32 comprise amino acids 1 to 163 or amino acids 236 to 245.of SEQ ID NO:32. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone gml96_4 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for gml96_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gml96_4 demonstrated at least some similarity with sequences identified as AA233552 (zr43al0.sl Soares NhHMPu SI Homo sapiens cDNA clone 666138 3'), AB005666 (Homo sapiens mRNA for GTPase-activating protein, complete eds), F12887 (H. sapiens partial cDNA sequence; clone c-3fh09), T25078 (Human gene signature HUMGS07218), and T75264 (yc88g09.rl Homo sapiens cDNA clone 23008 5'). The predicted amino acid sequence disclosed herein for gml96_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted gml96_4 protein demonstrated at least some similarity to the sequence identified as M64788 (GTPase activating protein [Homo sapiens]). Based upon sequence similarity, gml96_4 proteins and each similar protein or peptide may share at least some activity.
Clone "gn!14 1"
A polynucleotide of the present invention has been identified as clone "gnll4_l". gnll4_l was isolated from a human adult blood (peripheral blood mononuclear cells treated with granulocyte-colony stimulating factor in vivo) 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. gnll4_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gnll4_l protein").
The nucleotide sequence of gnll4_l as presently determined is reported in SEQ ID NO: 13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gnll4_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14. Amino acids 26 to 38 of SEQ ID NO:14 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 39 of SEQ ID NO:14, or are a transmembrane domain-
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone gnll4_l should be approximately 1500 bp. The nucleotide sequence disclosed herein for gnll4_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gnll4_l demonstrated at least some similarity with sequences identified as AA370547 (EST82206 Prostate gland I Homo sapiens cDNA 5' end), C04732 (Human Heart cDNA, clone 3NHC3910), and C05361 (Human Heart cDNA, clone 3NHC2451). Based upon sequence similarity, gnll4_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 gnll4_l protein sequence centered around amino acid 90 of SEQ ID NO:14.
Clone "hi968 2"
A polynucleotide of the present invention has been identified as clone "hj968_2". hj968_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 amino acid sequence of the encoded protein. hj968_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hj968_2 protein").
The nucleotide sequence of hj968_2 as presently determined is reported in SEQ ID NO:15. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hj968_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16. Amino acids 1 to 9 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 10, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hj968_2 should be approximately 1800 bp.
The nucleotide sequence disclosed herein for hj968_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hj968_2 demonstrated at least some similarity with sequences identified as AA071746 (mfl6h07.rl Life Tech mouse brain Mus musculus cDNA clone
405277 5') and AA325286 (EST28500 Cerebellum π Homo sapiens cDNA 5' end). The predicted amino acid sequence disclosed herein for hj968_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted hj968_2 protein demonstrated at least some similarity to the sequence identified as U23528 (translated cosmid B0034.[Caenorhabditis elegans]). Based upon sequence similarity, hj968_2 proteins and each similar protein or peptide may share at least some activity.
Clone "hklO 3"
A polynucleotide of the present invention has been identified as clone "hkl0_3". hkl0_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. hkl0_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hkl0_3 protein").
The nucleotide sequence of hkl0_3 as presently determined is reported in SEQ ID NO:17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hkl0_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18. Amino acids 28 to 40 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 41, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hkl0_3 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for hkl0_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hkl0_3 demonstrated at least some similarity with sequences identified as AA305975 (EST 176966 Jurkat T-cells VI Homo sapiens cDNA 5' end similar to S. cerevisiae hypothetical protein FAA3-BET1), F18016 (H. sapiens EST sequence 016-T), N36880 (yy37e07.sl Homo sapiens cDNA clone 273444 3'), and R87757 (yo45a08.sl Homo sapiens cDNA clone 180854 3'). The predicted amino acid sequence disclosed herein for hklO 3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted hkl0_3 protein demonstrated at least some similarity to the sequence identified as Z38113 .(CAI 0.21 [Saccharomyces cerevisiae]). Based upon sequence similarity, hkl0_3 proteins and each similar protein or peptide may share at least some activity. Clone "hm236 1"
A polynucleotide of the present invention has been identified as clone "hm236_l". hm236_l was isolated from a human adult testes (teratocarcinoma NCCIT) 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. hm236_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "hm236_l protein").
The nucleotide sequence of hm236_l as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the hm236_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone hm236_l should be approximately 1800 bp. The nucleotide sequence disclosed herein for hm236_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. hm236_l demonstrated at least some similarity with sequences identified as AA026169 (zk01b03.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 469229 5'), AA046211 (zk77e04.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 4888623'), AA223088 (PMY0368 KGla Lambda Zap Express cDNA Library Homo sapiens cDNA 5'), AB002368 and AC003010 (Human mRNA for KIAA0370 gene, partial eds), AC002399 (Human chromosome 16pll.2 BAC clone CIT987SK-A-481B3; HTGS phase 1, 18 unordered pieces, sequencing in progress), R86676 (ym86f03.rl Homo sapiens cDNA clone), and T21501 (Human gene signature HUMGS02874). The predicted amino acid sequence disclosed herein for hm236_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted hm236_l protein demonstrated at least some similarity to sequences identified as AB002368 (KIAA0370 [Homo sapiens]). Based upon sequence similarity, hm236_l proteins and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones cll81_3, crl044_l, cz251_l, ddl2_7, fnl91_3, gml96_4, gnll4_l, hj968_2, hkl0_3, and hm236_l were deposited on June 12, 1997 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 98456, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. § 1.808(b), and the term of the deposit will comply with 37 C.F.R. § 1.806.
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 pED6dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al, 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and 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.
Clone Probe Sequence cll81_3 SEQ ID NO:21 crl044_l SEQ ID NO:22 cz251_l SEQ ID NO:23 ddl2_7 SEQ ID NO:24 fnl91 3 SEQ ID NO:25 gml96_4 SEQ ID NO:26 gnll4_l SEQ ID NO:27 hj968_2 SEQ ID NO:28 hkl0_3 SEQ ID NO:29 hm236_l SEQ ID NO:30
In the sequences listed above which include an N at position 2, that position is occupied in preferred probes /primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1- dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole. The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al, Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention. The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form(s) of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol Sci. 15(7): 250-254; Lavarosky et al, 1997,
Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol Biol. 58: 1- 39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649464 Bl, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. Preferably, species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canisfamiliaris, Oryctolagus cunicidus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al, 1993, Nature Genetics 3:103-112; Johansson et al, 1995, Genomics 25: 682-690; Lyons et al, 1997, Nature Genetics 15: 47-56; O'Brien et al, 1997, Trends in Genetics 13(10): 393-399; Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein).
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein. The present invention also mcludes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein Examples of stringency conditions are shown m the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
Figure imgf000040_0001
* The hybrid length is that anticipated for the hybridized regιon(s) of the hybridizing polynucleotides When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
+: SSPE (lxSSPE is 0.15M NaCl, lOmM NaH2P04, and 1.25mM EDTA, pH 74) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
*TB - TR: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10 °C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5 + 16.6(log]0[Na+]) + 041 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC = 0.165 ).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al, eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
USES AND BIOLOGICAL ACTIVITY The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, those described in Gyuris et al, 1993, Cell 75: 791-803 and in Rossi et al, 1997, Proc. Natl Acad. Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, TIO, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al, J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al, J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and /or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and /or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens. The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease. Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/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 Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al, J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al, J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992. Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al, Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al, Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity
A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and /or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon /ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ). Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
Activin /Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin- β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis
Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
Receptor /Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al, J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin /Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by 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. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody. Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and /or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention. The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference. As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic f actor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and /or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and
TGF-β), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and /or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Treacy, Maurice Spaulding, Vikki Agostino, Michael J. Howes, Steven H. Fechtel, Kim
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CaπtbridgePark Drive
(C) CITY: Cambridge (D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2696 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
GTTGTACCAT TCTTGCCAAC TTCTGGGCTG GCAGTATGGA GTCATCTCCC TATCTTTCAT 60 TGCCTGTGTG AAATCTACTT TCTGAATTCT GCCATTTCCC TCTTCACACT GTCTCCTGGG 120
TTATCTTTGC TTCCTCACAT CCCTATCTCT CTTCCTATAA ACTGGCTCCC GTCACTTCCA 180
TGATCCCTTC AGTGGCTTCT GAGCTGGTCT CCCTGACCCC AAAGCCTCAG CCTTCCAGTC 240
TCCCTACAAA ATCTCAGCAA GTTCATTTTA GGTTAAAATT TGGACATATT TTAAATACGG 300
CTCACCACTT CATGTGAAAA TGATGGCACC CTACCAAGCA GTTTGCAGAG TTACGGTAAC 360 TGTTTCATGC TAATGATGTT ATTCATCCAG TTACAATTTT CTCAAAACTC CTTTGGGCAC 420
TCTTTATTTT TAATCAAATT TTAAAGCCAA TATTTCATTT TGAGAATATG AATTAAATTG 480
GGAAATTCAT CCTTGTGGTA CAGTTTACAG ATTTTTAATG TTTACCCATT TATCCTGTTT 540
TTTGATATAT TAATTTCCCA TATAGCTCCA GAGTTATGTG ATATTATTTC TTTGCCAGTA 600
TATTAGAAAA TGATTAATTT CTCATGACCA ACTTCTGAAA AGAAAGACCC AATGCAAAAT 660 GCAATCTATT ACAATTATTT TTTTGAATAA AAAAGAATAT ATTATAGTTC TTTAACATTT 720
GATATTTTAA ATTTGACATA TTCTTGATAT TTGTAAGAAA TTTCCACTGA ATGAATTTTA 780
CACAATTCAG ATACTACCAA TTAACTAATT CTAGCCTAAA CAAATAACAT TATTTTTAAA 840
TAACAAAATC TTTAAAAATA ATTTTCTATT TTGAACTTTT AGCCATAATG TAAGAAAATA 900
AAATTTTCTA GCAGAATAAT CAAAGAGTGA AACAAAGTTC CAACATGTTT TTTCTTTGCA 960 ATTAAACATG GCACTTTTAC AGTTATTTAT TATTCATATC AGTGCACTTA CCGACTTCAT 1020
ATTTTCAAAT CAAAATACAG TGTTTTTCTC CAGTGAAATC CTTATTCTCA TGACTGATAG 1080
AAAACATTGC CAATTTTGAT ATTTCCAGAG TTAATGTTAA ATTATTTGAA AGAAAATTAT 1140
TTAAAATAAT AAAAATAGAC ATTTCAAGAC TATTTCTTAT CACATAATTC AAAAAGTACT 1200
TGGATCAAAT CCTACAGAGT TTCTCCACTA AAATTCTACT TGTGCAGAGG GCATTGAAAC 1260 GCATGAAAAT CAACAGCAGC TTAGTTAGGT TAATTAATTC GGTTAATTAA GCACCTACTA 1320
CATGCTCAGC TCTATGCTAG GTGTCATGAG GAATTAAAAG GACATGTAAT GCACATTTTC 1380
TGATTTC AG GAGCTTTAAA TATTATTGTG TAGAAAAAGT TAACATCTAT GAAAATAGAA 1440 GTGGGGCAAT TTTGTGCTTA ATTCCATGGT CCAGATACAT CAAAAAATCA ATGTGGGCTG 1500
TCAAAGAAGG TTTCTTGATA GTCATGAGTC AGCCTGATTC TTGAAAGGAT ATGTGGAATA 1560 TAAAATTTTA TTTATATTCC TTTTGAGAAA ATACTGAGAA AACCATCTTC CCTGGAAAAG 1620
AGAACGTATT GTAAAGAAAG TACATGAAAT TGAAGGTTGA ATATCCAACA TCCCCCACAC 1680
TGCCCCAGTG TCTCTGCTCC CTTACTGAGC CTTACTATTA TTCTTCATAG CCCTATCACT 1740
ACCTAGTCTA GTATTCACTG AACTGTGTCA TCCACTAGAA TATGAGCATA ATGAGAGCAG 1800
AGACTACACC TGTCGGTTCA GTATTCTATC CTCAGCACAT AGAATGGTAC CTGGCACATA 1860 GCAGATGCTA AAATAAAATT TAAATGAATA AATTAATTCA ATCAACAACT TCAAGGTGTT 1920
ATTATTACCT ACAACTATTG TTTACAAGAG GTATGCACCG TGGAAGATCC TGGAGACACA 1980
ACAATGAATA AAGCCAAGCC AGTTCCTGCC CCCGTGGAGC TTGTAGTCAA GACATTGAAC 2040
AAGTGATCAG AAAGATGTTG ACTGCTGCAG CAGAGGGTTG CAAGCTGCTC ATGAGTATAT 2100
AACAAGTAGC CCTAACCAAA GCATTCTCTC CCTTGGTTTA ATGTCCACCC ATTGAGGTGA 2160 CTGCTAAATA CTAATCCATG ACTCTATCCC TTGGCATTCA AACTCACACA TCCACTTACC 2220
TGCCTCTCCA ACCTCATCTC CCTCCACTCA CAAGAGCCCA TCATATTATT CATCAAAATG 2280
AAACTGCACC CAGTTCTTCT GAACATATTA CCTTACAAAA CTTTCATTTA TGCCTGGTGA 2340
CTCTCATCAG GCATTCAAAA GCTTTCCCTC AGTGCTTCAG GGCTCTTCCT TTTCTTCCCT 2400
TTATACATAC ACCTTTATGT ATCTTCATAC GTACCCTGCA TAACCTCATA TATCTTAGCA 2460 TTTACCATAT TCTGTTGAAA AACTGTTTCC ATTTCTCTTT ACTTACTAGA ATGTAAACAG 2520
ATGCACAATG TTGAGAAAAT GAAAAGTGAC AACTTTGTTT ACAAGTTTAG AAATTATCAG 2580
ATTCTCACYT AAGCTCTAGT CTCTGTAAAG TCCACAACTA CTYAATAAAA GTGAAGAAAA 2640
ATGTTAACAG AGAGGGAGGA ATCAAAAACA AAGAACTATT TAAAAAAAAA AAAAAA 2696
(2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Thr Leu Ser Leu Gly lie Gin Thr His Thr Ser Thr Tyr Leu Pro 1 5 10 15
Leu Gin Pro His Leu Pro Pro Leu Thr Arg Ala His His lie lie His 20 25 30
Gin Asn Glu Thr Ala Pro Ser Ser Ser Glu His lie Thr Leu Gin Asn 35 40 45
Phe His Leu Cys Leu Val Thr Leu lie Arg His Ser Lys Ala Phe Pro 50 55 60 Gin Cys Phe Arg Ala Leu Pro Phe Leu Pro Phe lie His Thr Pro Leu 65 70 75 80
Cys lie Phe lie Arg Thr Leu His Asn Leu lie Tyr Leu Ser lie Tyr 85 90 95
His lie Leu Leu Lys Asn Cys Phe His Phe Ser Leu Leu Thr Arg Met 100 105 110
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3614 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : CGCGCTAACT GTGCTCCTCC GGGGCCCTCC GCCTGCTCCC AGCCATGGTG GCCTGGCGCT 60
CGGCGTTCCT TGTCTGCCTC GCTTTCTCCT TGGCCACCCT GGTCCAGCGA GGATCTGGGG 120
ACTTTGATGA TTTTAACCTG GAGGATGCAG TGAAAGAAAC TTCCTCAGTA AAGCGATCAC 180
TGTAAGGATG ACATGGGAGG AGCCATGTGA AGCACTCAGC ACAGTCCTTG GAACAAGAGC 240
CATGGGACCA CACCACCACC ACCACAACCA ATAGGCCAGG AACCACCAGA GCTCCGGCAA 300 AACCTCCAGG TAGTGGATTG GACTTGGCTG ATGCTTTGGA TGATCAAGAT GATGGCCGCA 360
GGAAACCGGG TATAGGAGGA AGAGAGAGAT GGAACCATGT AACCACCACG ACCAAGAGGC 420
CAGTAACCAC CAGAGCTCCA GCAAATACTT TAGGAAATGA TTTTGACTTG GCTGATGCCC 480 TGGATGATCG AAATGATCGA GATGATGGCC GCAGGAAACC AATTGCTGGA GGAGGAGGTT 540
TTTCAGACAA GGATCTTGAA GACATAGTAG GGGGTGGAGA ATACAAACCT GACAAGGGTA 600 AAGGTGATGG CCGGTACGGC AGCAATGACG ACCCTGGATC TGGCATGGTG GCAGAGCCTG 660
GCACCATTGC CGGGGTGGCC AGCGCCCTGG CCATGGCCCT CATCGGTGCC GTCTCCAGCT 720
ACATCTCCTA CCAGCAGAAG AAGTTCTGCT TCAGCATTCA GCAGGGTCTC AACGCAGACT 780
ACGTGAAGGG AGAGAACCTG GAAGCCGTGG TATGTGAGGA ACCCCAAGTG AAATACTCCA 840
CGTTGCACAC GCAGTCTGCA GAGCCGCCGC CGCCGCCCGA ACCAGCCCGG ATCTGAGGGC 900 CCTGTCCAGC TGCAGGCATG CACAATGGTG CCACCGCTTG TCACCCGGCT CCCCCCACCC 960
CTTCATTTGG ACCCGCAGCT GCTGTGCTGC TCTGTGCCGT CGGCTCCTTG TTGGTCTGAG 1020
TTTCCCGGAT GAGCTCTGGG TGTTTGTGAG TTTGGTTTCT CTGCCCTGCC CCAAGCGTGC 1080
TGAGACTTGG TGCCGAAATT CAAGAGCCAG CTCTGATAGA AAGCCAGCAC CAGCCTCGGG 1140
AGCTGCTGAG CCACCAACTC CCAAAGCCAG CCTGCCTCCA GCTTTACTGA GCACAGGATG 1200 CGGGGGCCAA GATGATGCTG AGGCCTGATG ACATTTATGC TTAGGGGACA AGAGTTTGAA 1260
CTCAAGGGAC TGTGACCCCT GCACACTGGA GTGGCTCATT GTGGCAGGTT TCTGCCAATA 1320
GACAGCCCCT GACAGTGGCC TCAAGGAGCT GCAGGTGGGG GGCTCAGCCT GCACCCACTT 1380
GGAGCCCCTG CAAGGAGCGA ACCGGTCAGC ACCAAGTAAC ACCACACACA CGCAGCACCC 1440
AGGATGATGG TTTCACTTCA GTCTTCCCCA TCCCAGGTTT TATGTTGCTG GGCTTCCGGA 1500 GAGCCGGTCC AAGCGGAGGC TTTCAGTGAT TTAAGTACAA ACATGCATCT CGTGATAGTC 1560
CTGCCTTGAG AGCTTAGGAA TCTTCCGGAT AAGTATGAAG CAATTCGTAG GCCTGTTTCC 1620
CATCTGATTC CATAGGGGGC TGGGTGTGGC TTCGGGTTGA CATGAGAAAG GTCTTTAGCA 1680
ATCATTTCTG CACCGGAGAT GAGTTTTATC CTGTGTTGGG GAGAGGTGCT CACCCTCCAC 1740
CCTGTGTCCC TGTTTTGGTA GCAAGAGTGA CCGATGTCAA GAACGAGCAT CAAAGCCAGA 1800 ATCCTGCTTG TTTGCTTAAA AATGTAATTG GGGGCGGCGG GGGAGGAGAG GGGAAAGAGA 1860
CATTCGCTTG GTTTAGTGAA ACGCAGGTGA CTTTGTAGCT CTGTGGTCAG CCTACTTGTC 1920
TGCTCTGAGG GAGAGTGCGT GGGGAGCCAT GCTCACCGTG GCAAACACAG GAACCCCATG 1980
ACTCGCCCCT CACCTGGCGT GGAGCTGCCT GGTTTGGGCT GGAGCAGAGC TGGTTTCCTG „ 2040
GAATGTTCCT TTGGCCCACA TATGGTTCTG TCCCGGTGAG CTCTGTTGTC AGAGGCTCAC 2100 GGGACAGAAC CACATGCTAG GGTCTAGGGC CCCTGTCTAC TGATAGTCAG TTTGCTGTGT 2160 CAGAAAGCAC TTCTGAAAGC AGATATGAGT CACCAGACAG GCAGGATCTT ACAAAACTCA 2220
CGGGCCTCTT TGGTCTGCAT GATGGCCCCA TGCGTTTCAT AGGCTGTCCA CTGAGCGGGA 2280 TTGTCTGCTG AGTGGGATGA GCCAACTCCA GTTTCTTAAG GAAACCACTG GAATCTGCAG 2340
CCCCCACATG CATCTGTCTA ACGCATGCCT CGTGTTCGTT TTGCAAACAT GCCTGTGGTG 2400
GAGGGTGGTC AGTTGTAGCC CTGTGCGTCT CAAGGCTGCC TTGTGAGGCC ATTCCCAGTG 2460
CGTGCCCTTG AGCTCCTTAC CACCCCTTTT CCTGCTCGGC CCTTTAATCC CTGACAGACC 2520
TGGACTGTGT GGCTGAAGGG GGACCTGCAG CACTGCAGAA ATGCCTCTGC GTGGTGCCAT 2580 GAAGGAAAGA AACCTTGGCC TGGTCTCGAG AAGCTTCCCA TGCTTCAGGA AGTTAGTAAG 2640
GGTGGGGTGG CTTGCAGGAT TGGCCTGTTT CCAGGGCCTC CCACACTCAT TGGCCAGATT 2700
GTGAACTTTG TCAGGCTTGT CCCTCCCTGA TACCAAGTAT GTCGAGAACC GATGGCCCCA 2760
CCCTCTGGCT GGTGCTGGGC CGGAGGTGGC TATGGAGGAT TTTGGCATGC GTGGCCTGTC 2820
GCCACCTGGA CAGCGTGACC TCAGGGGTTG TCCACTTTAC CTTTATGGTG AGGCCTGTCG 2880 GATGGCTAAG TCCTTGAAAC CCTAGAGCTG TGACGTAGAA TATGTGCTGT CTGTGAGACC 2940
GTGTTCCCAG GAGCACTGAC TGCAGTTGAG AGAGACCCAT TTTGCTCTCC CTTACCGCCC 3000
CCCGCCCCGG GTGCTTTCTG CACAAAGCCT AGAGCCTGGC ACTCAAGCCC ACCGGTGGCA 3060
GCTCCTAGTG ACTGGACATG CCTGGAAGAC CCCTCAGCCT TCTGTTTGCA GAACGTTCAT 3120
TTCAGGAGCT TCTCCTTCCC ACAGACATCT TACACTTGCT CGACACTGCC ACCTGCAGAA 3180 GCCTGGCGGG CTCTGGTCAC CATGTGTCTA TCTGAAGGTT GCACTGGCCA GCATGGGCCT 3240
GTCCCAAGCG AGAGGGGAGA CACAGTGGAC TGAAAGGACT GGTTGAAAGT GGCCAATCTC 3300
TGTCAGCTTA ATTTGGCAGA GAAAATTTGT AACAACTCTG AGCACATGCT GGGTGAAGTC 3360
ACAGCTCAAG GAAAGATAAA GCTGGGCGGA AGGAGGTGTG CGTGGCTTCT GGGGTGGGAC 3420
CCAGAGGGGA GGCTCTGGGA CAGGGGCTGG GGTTCAGTGC CAGGGCCCTG AGGAAGAAAT 3480 GGGGACTGAT CTCAAAATTC CAGAATTCCC TGTACATCTG TTCACGTGCT TGTGTCCAGG 3540
TGTGACTTGT AAACTGTCTA GTGTTTGCAT TAAATAAAAT GGCACCGAGC AAAAAAAAAA 3600
AAAAAAAAAA AAAA 3614
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Val Lys His Ser Ala Gin Ser Leu Glu Gin Glu Pro Trp Asp His Thr 1 5 10 15
Thr Thr Thr Thr Thr Asn Arg Pro Gly Thr Thr Arg Ala Pro Ala Lys 20 25 30
Pro Pro Gly Ser Gly Leu Asp Leu Ala Asp Ala Leu Asp Asp Gin Asp 35 40 45
Asp Gly Arg Arg Lys Pro Gly lie Gly Gly Arg Glu Arg Trp Asn His 50 55 60
Val Thr Thr Thr Thr Lys Arg Pro Val Thr Thr Arg Ala Pro Ala Asn 65 70 75 80
Thr Leu Gly Asn Asp Phe Asp Leu Ala Asp Ala Leu Asp Asp Arg Asn 85 90 95
Asp Arg Asp Asp Gly Arg Arg Lys Pro lie Ala Gly Gly Gly Gly Phe 100 105 110
Ser Asp Lys Asp Leu Glu Asp lie Val Gly Gly Gly Glu Tyr Lys Pro 115 120 125 Asp Lys Gly Lys Gly Asp Gly Arg Tyr Gly Ser Asn Asp Asp Pro Gly 130 135 140
Ser Gly Met Val Ala Glu Pro Gly Thr He Ala Gly Val Ala Ser Ala 145 150 155 160
Leu Ala Met Ala Leu He Gly Ala Val Ser Ser Tyr He Ser Tyr Gin 165 170 175
Gin Lys Lys Phe Cys Phe Ser He Gin Gin Gly Leu Asn Ala Asp Tyr 180 185 190
Val Lys Gly Glu Asn Leu Glu Ala Val Val Cys Glu Glu Pro Gin Val 195 200 205 Lys Tyr Ser Thr Leu His Thr Gin Ser Ala Glu Pro Pro Pro Pro Pro 210 215 220
Glu Pro Ala Arg He 225 (2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1077 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
AATCGGGAGT CCCAACAGCG TTAGGTTTTT TTTTTTGTTT GTTTGTTTGT TTTGTTTTTT 60
TCCAACCCTC TTTCGGATGG ACGGGGGAAA GAGAGAAAGA AAAACGAGGG AAAATCAACA 120 AAATGTGCGA TGCAAAGAGT CGATTTTCGC GGGGTTTGTC AACTTCGCCA CTGCCGCACG 180
CGAATCGACG TCGTCACGTG ACGGTCTGCC TCCGCCCTTA TTAACTCTCA GCCCAGCGGC 240
GGTTTCCAGG ACCTCAGACT TTTTGCCGAG GCGGCAGTCC CTAGACGAAG CGAAGGAGGC 300
GGCGCCTGCC CCGCCCACAA GAGCTGCCGC GCGCGGGTGT TATAGCTCCA CCCCATCTGC 360
AAAGGAAGGG GGAGCGGAAA GAGCGGGATC TAGCGTGGGA TAAAAGTGGG ACTACTACAG 420 TGTAACTGGG CATGCGCCCC TCCTAGAAAT GATGGGAATG CAAAAGCCCT TGACTGCTCC 480
AGGACTCGAG GGATCCTCGG TGCCAGGATG CTGGGTCAAG CGCTCCGCCG GGACAGAGGA 540
CTCATACCAG GGAAATGGAG CCCAGCCTCG TGATAAACTA CGACCCAAGC TGGGGGAGGA 600
ACCTAGTTTT CGAAAGGAAA ATAATATGCG CAAGCTTTAA CTGAGCAGTG GGATGGTCTT 660
AAATACCAAA GGAATGACTT TAAATCTTGC TGGATGGGAC TGCCACTCAC CGCTAGAAAT 720 CGGGGATCAA CAGCAAACTC TGGATGACCC TGTAACCACA TCTCCAGTTC AGCCCGGCGA 780
GGGGCATCCT CACCCACCAG CAAAGTACCA TCCACCTTAT TGATGACAGG GATCCGGGTC 840
TCCAGGTCCA CATCAAGGTG ATTAGGCTCT TCCATGCACT CCACCTCCAG CTGCAAACCC 900
AGAATCCACC CCCATGAGCA CATACTCTTC TTTGGGGGAG GGAGGGAGGG GGAGCAGGGC 960
CAATGGTAGT CATGAAATGA CTCTAGTATT TTCCATTCCC CCAGTCCCAC TGCCTTCATC 1020 AATTATTGGG AATAAAAAGA CAATCTAATC GTCAAAAAAA AAAAAAAAAA AAAAAAA 1077
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 108 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Asp Gly Gly Lys Arg Glu Arg Lys Thr Arg Glu Asn Gin Gin Asn 1 5 10 15 Val Arg Cys Lys Glu Ser He Phe Ala Gly Phe Val Asn Phe Ala Thr
20 25 30
Ala Ala Arg Glu Ser Thr Ser Ser Arg Asp Gly Leu Pro Pro Pro Leu 35 40 45
Leu Thr Leu Ser Pro Ala Ala Val Ser Arg Thr Ser Asp Phe Leu Pro 50 55 60
Arg Arg Gin Ser Leu Asp Glu Ala Lys Glu Ala Ala Pro Ala Pro Pro 65 70 75 80
Thr Arg Ala Ala Ala Arg Gly Cys Tyr Ser Ser Thr Pro Ser Ala Lys 85 90 95 Glu Gly Gly Ala Glu Arg Ala Gly Ser Ser Val Gly
100 105
(2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 588 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TAAGAATTAA AAATGTCATC CAAACAAGAA ATAATGAGTG ACCAGCGGTT TAGACGGGTT 60 GCAAAGGACC CGAGATTTTG GGAAATGCCA GAAAAGGATC GAAAAGTCAA AATTGACAAG 120
AGATTTCGAG CCATGTTTCA TGACAAGAAG TTCAAGTTGA ACTATGCCGT GGATAAAAGA 180
GGGCGCCCCA TTAGCCATAG CACTACAGAG GATTTGAAGC GTTTTTACGA CCTTTCAGAT 240 TCTGATTCCA ATCTCTCTGG TGAAGATAGC AAAGCATTGA GTCAAAAGAA AATAAAGAAG 300
AAAAAAACCC AGACTAAAAA AGAAATCGAT TCAAAAAATC TAGTTGAGAA AAAGAAAGAA 360 ACCAAGAAGG CTAATCACAA GGGTTCTGAA AATAAAACTG ATTTAGATAA TTCTATAGGA 420
ATTAAAAAAA TGAAAACCTC ATGTAAATTT AAGATAGATT CAAACATAAG TCCGAAGAAG 480
GATAGCAAAG AATTTACACA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 540
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 588
(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Ser Ser Lys Gin Glu He Met Ser Asp Gin Arg Phe Arg Arg Val 1 5 10 15
Ala Lys Asp Pro Arg Phe Trp Glu Met Pro Glu Lys Asp Arg Lys Val 20 25 30
Lys He Asp Lys Arg Phe Arg Ala Met Phe His Asp Lys Lys Phe Lys 35 40 45
Leu Asn Tyr Ala Val Asp Lys Arg Gly Arg Pro He Ser His Ser Thr 50 55 60 Thr Glu Asp Leu Lys Arg Phe Tyr Asp Leu Ser Asp Ser Asp Ser Asn 65 70 75 80
Leu Ser Gly Glu Asp Ser Lys Ala Leu Ser Gin Lys Lys He Lys Lys 85 90 95
Lys Lys Thr Gin Thr Lys Lys Glu He Asp Ser Lys Asn Leu Val Glu 100 105 110
Lys Lys Lys Glu Thr Lys Lys Ala Asn His Lys Gly Ser Glu Asn Lys 115 120 125
Thr Asp Leu Asp Asn Ser He Gly He Lys Lys Met Lys Thr Ser Cys 130 135 140
Lys Phe Lys He Asp Ser Asn He Ser Pro Lys Lys Asp Ser Lys Glu 145 150 155 160
Phe Thr Gin
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2773 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
GTATATTTTG GTTTACTTAC TCCTCTATTT CAGAAATTGA AAAAGATCCC CAAGGATCTG 60
TTACTACTGC ATTTCCTTCT TGCTCTGTCT ACAGCCTAGG CCAACTAGTC AGGGTCTGGA 120 CATGCATCTC CTAAAGGAAG AACTGTGTAG CACCATTGAT CACAATGTAA CATTTCCATG 180
CTGCATTAAG GGTGTCTCTC TCTAATCATG ATTGTACCTG TCTCTTCCTG GGTAAAGGGA 240
GATTTTTTTT TTTTAATGTG TAAAGAATTG ATGCSAGCCA GGAACATGTC TGTAGTCCCA 300
GCTACTTGGG CACACGCCTG TAGTCCMSCG CCACTCGAGC ACACACCTGT AGTACCAGCT 360
ACTCTGGAGG CTGAGGCAGG AGGATCACTT GAGCCCAGGA GATTAAGACT GTAGTATACT 420 ATGATCGTGC CTGTGGCTAG CCACTGTGCT CCAGCCTGGG CAACACCATC GTAAAAATAA 480
ATAAATAAAT AAATAAATTG GGGAGGACAG CCTCACTGGT ATCAGACTTA CAGGACCAGA 540
TAGACAAGAT GGGTATAAGG GGAGCTGAAG TCTGTGTTCA TATGAGGAAG AGAAGACCAA 600
GCCCTGGGAC TTTGGCTGAA TTCCTCCGTG GGGCTGGACG GCAGTGATCT CCTGTTCCCT 660
ATGTGTAAAC AAAGATTCCA GGGCGTGGTT TTGCACTCCT GTTGTACTCT TTTAGAGGTG 720 GAAAAGAGGT GGATACTGAG ATCTAAGAGG AAAGGATAGT CATTCACGTT CTGAGATATG 780
CGCTCTCTCT ATTGTTCTCG WACACAAAGG GATAGTCTCT TTTCTGGAGC TGATGTCCCT 840
GCTTGGAGGT TAGCCCCAAA ACATGGCTCT TGTATTGTTC TAAGAGAAAA GGCTTTCATT 900
TTGGTTCTTC TGATTGGTGT TACCTACTGC CTAATATGTG TTCATTTTTT GACAGAGAGG _ 960
CAGACTATTG AAAAAGTCTG TGTGAACAGA GAGCAGTTCA TTAAGCCCAT TGCTTTCAGT 1020 AATGTGGCCT TGACCCCTTC TGCTTCCCCC TTCTCCCATG GAGCATGGCA GGGCTTGGTT 1080 ATTTAGAGTC CATACATGCA AGCCATTGAG AGACTTGTTT GCTCAAATGC AAGTTTGCTC 1140
AAAAACAGGT CCTGAAGGCT TGCTTAGGAT TACAGGGATG CTGGGTAAGA ACACCGTTCC 1200 TCTCTCTCGC TGGAGAAATC CCTGTTTCTC TGACTCCCTT TGTGATCCTC ACAGTAATGT 1260
ATTCTGTGCC ACTGTAGGAC ACAAGGCTCT GGGCCAGTAG AACAGGCAGA GAGGTGACAC 1320
TGGGCAGCAA GCTGAGAGCT CTTTCTAAAT GGAGTGAAGG AATTCAGTGG CCTAGTTTCG 1380
CCATTCTCTA ATGAGAAACC AAGGCCAGGC TGAAAAGTGC AATTAGATGT GGTGGATTGT 1440
GGTAACGGCC TCCAGATAAA GGGGTTATCC CTGTGGAAGT GACTTTTCCC CATTTGATCC 1500 CTTTTCAACT CTAAATGGCC AGGCCCAGAG CAGAAGAAGG GTTGGGTCTG GAAGGAAGGC 1560
TCCAAAGGAT GAAAGCTTCT CCCTGATCAT AAGGAAGTGC ATCTTTATAG AATTGTTGTG 1620
CATAATGTCA GTAAATCCCT CTCACTTGAC AAGGGACTGG ATTCATCTTG CCTTGAGACG 1680
GGCCAGTAGT TATCAGTGAG TCAAAGCAAA GTGAAAGTTT CAGGAGATGG GACCAATGGT 1740
GCAATGCTCG CCATAACAAA ATTCCTTAAA AATAAAAAAG CTAATGTTAT AGCAACAAAA 1800 AAAGACTGAA GCAAAACCAC ACTGAAATGC ATCCCACTCC AGGAGAGGAA TTCTTAGCGT 1860
AACACTCTAA ATAAATGGAA GGAATCATCA CCTTCCTTAT TTTACCCCTG CCTTGTTCAC 1920
CAGGCTGCCC AGTGCTTACC ATGCAGAAAG CAGTCAGCTG TACTCTGGAA GTTTCTGTTC 1980
TTCTTTCCTG GGGCTTAGGA TATTCTGGGA GCTGTCTGAG CCTTGTGCCT AAGGCTTATC 2040
AGGTGATATA ATCTTCCTGT TCTGGGCTGC TTGCTGGAGG AATAGGAAGT GACATTTATA 2100 AGACACAGGC GGTGTGAGCA TCCATGTGTG GTCTTGGTCT AAACCAGCTC TTGAACAGGT 2160
TAAAGCAAAC AGCAATAACA AAACAAAAAC TACTGATGCT GAGCGTTTTG ATCCTAGTAA 2220
TATTTCAAAT ATTGTCCTTC TGCATATGTT CTATCCATAT TTGATTCCAA TATACATTAT 2280
TAAGCTTTCT TGGGTACTAT TTTGCTGGGG CTCTTGCGTG AAGGTGGTAC CTGTCTCATG 2340
ATCCTTAAAA GAGAGAGGCT TTTTTCATCC AAAGCTGTAG TGTTGGGAAC TGGGGTGGGA 2400 GAGGCACTTT TTGGAATTCT GAAAGAATCA TATCTGTGTA TATACATACT GAGTGGGGAA 2460
GGATGGGGGT TGGCAGGGGT TGAGGGAGGT GGGAACAAAC AGTGAGTATG GGAACAGGCA 2520
GTCACCTCGA GTGTGGGAGG TCACCTGGGT CCGTCGTCTT CCTTCTGTAT GGTGTTGGGT 2580
TTATGTACAC ACTATAACAC TTCCTGTGTG AGTTCATGTA CCTGTCTGTG AGTGCTTTGG _ 2640
TGTATTGAGC CTCAGTACAC TCCAAGGGCA TTAAAGTCAA GAACTAGAAC CTGGAAAAAA 2700 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2760 AAAAAAAAAA AAA 2773
(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Arg Ser Leu Tyr Cys Ser Arg Thr Gin Arg Asp Ser Leu Phe Ser 1 5 10 15
Gly Ala Asp Val Pro Ala Trp Arg Leu Ala Pro Lys His Gly Ser Cys 20 25 30
He Val Leu Arg Glu Lys Ala Phe He Leu Val Leu Leu He Gly Val 35 40 45
Thr Tyr Cys Leu He Cys Val His Phe Leu Thr Glu Arg Gin Thr He 50 55 60
Glu Lys Val Cys Val Asn Arg Glu Gin Phe He Lys Pro He Ala Phe 65 70 75 80
Ser Asn Val Ala Leu Thr Pro Ser Ala Ser Pro Phe Ser His Gly Ala 85 90 95
Trp Gin Gly Leu Val He 100
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3119 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGAAACATTA TGGATCTGTG GAGCTGCTTA TTTCCAGTGA TGCTGATGGA GCCATCCAAA 60 GGGCTGGAAG ATTCAGAGTG GAAAATGGCT CTTCAGATGA GAATGCAACT GCCCTGCCTG 120
GTACTTGGCG AAGAACAGAC GTGCACTTAG AGAACCCAGA ATACCACACC AGATGGTATT 180 TCAAATATTT TTTAGGACAA GTCCATCAGA ACTACATTGG AAACGATGCC GAGAAGAGCC 240
CTTTCTTCTT GTCCGTGACC CTTTCTGACC AAAACAATCA ACGTGTCCCT CAATACCGTG 300
CAATTCTTTG GAGAAAAACA GGTACCCAGA AAATATGCCT TCCCTACAGT CCCACAAAAA 360
CTCTTTCTGT GAAGTCCATC TTAAGTGCCA TGAATCTGGA CAAATTTGAG AAAGGCCCCA 420
GGGAAATTTT TCATCCTGAA ATACAAAAGG ACTTGCTGGT TCTTGAAGAA CAAGAGGGCT 480 CTGTGAATTT CAAGTTTGGG GTTCTTTTTG CCAAAGATGG GCAGCTCACT GATGATGAGA 540
TGTTCAGCAA TGAAATTGGA AGCGAGCCTT TTCAAAAATT TTTAAATCTT CTGGGTGACA 600
CAATCACTCT AAAGGGCTGG ACGGGCTACC GTGGCGGTCT GGATACCAAA AATGATACCA 660
CAGGGATACA TTCAGTTTAT ACTGTGTACC AAGGGCATGA GATCATGTTT CATGTTTCCA 720
CCATGTTGCC ATATTCCAAA GAGAACAAAC AGCAGGTGGA AAGGAAACGC CACATTGGAA 780 ACGATATCGT CACCATTGTG TTCCAAGAAG GAGAGGAATC TTCTCCTGCC TTTAAGCCTT 840
CCATGATCCG CTCCCACTTT ACACATATTT TTGCCTTAGT GAGATACAAT CAACAAAATG 900
ACAATTACAG GCTGAAAATA TTTTCAGAAG AGAGCGTACC ACTCTTTGGC CCTCCCTTGC 960
CAACTCCACC AGTGTTTACA GACCACCAGG AATTCAGGGA CTTTTTGCTA GTGAAATTAA 1020
TTAATGGTGA AAAAGCCACT TTGGAAACCC CAACATTTGC CCAGAAACGT CGGCGTACCC 1080 TGGATATGTT GATTAGATCT TTACACCAGG ATTTGATGCC AGATTTGCAT AAGAACATGC 1140
TTAATAGACG ATCTTTTAGT GATGTCTTAC CAGAGTCACC CAAGTCAGCG CGGAAGAAAG 1200
AGGAGGCCCG CCAGGCGGAG TTTGTTAGAA TAGGGCAGGC ACTAAAACTG AAATCCATTG 1260
TGAGAGGGGA TGCTCCATCA AGCTTGGCAG CTTCAGGGAT CTGTAAAAAA GAGATGACCT 1320
TCCATCAGTG CCCGTGTTTG ACAGAACTCT GCCAGTGAAG CAAATGCATG TGCTTGAGAC 1380 CCTGGACCTT CTGGTTCTCA GAGCAGACAA AGGAAAAGAT GCTCGCCTCT TTGTCTTCAG 1440
GCTAAGTGCT CTGCAAAAGG GCCTTGAGGG GAAGCAGGCT GGGAAGAGCA GGTCTGACTG 1500
CAGAGAAAAC AAGTTGGAGA AAACAAAAGG CTGCCACCTG TATGCTATTA ACACTCACCA 1560
CAGCAGAGAG CTGAGGATTG TGGTTGCAAT TCGGAATAAA CTGCTTCTGA TCACAAGAAA . 1620
ACACAACAAG CCAAGCGGGG TCACCAGCAC CTCATTGTTA TCTCCCCTGT CTGAGTCACC 1680 TGTTGAAGAA TTCCAGTACA TCAGGGAGAT CTGTCTGTCT GACTCTCCCA TGGTGATGAC 1740 CTTAGTGGAT GGGCCAGCTG AAGAGAGTGA CAATCTCATC TGTGTGGCTT ATCGACACCA 1800
ATTTGATGTG GTGAATGAGA GCACAGGAGA AGCCTTCAGG CTGCACCACG TGGAGGCCAA 1860 CAGGGTTAAT TTTGTTGCAG CTATTGATGT GTACGAAGAT GGAGAAGCTG GTTTGCTGTT 1920
GTGTTACAAC TACAGTTGCA TCTATAAAAA GGTTTGCCCC TTTAATGGTG GCTCTTTTTT 1980
GGTTCAACCT TCTGCGTCAG ATTTCCAGTT CTGTTGGAAC CAGGCTCCCT ATGCAATTGT 2040
CTGTGCTTTC CCGTATCTCC TGGCCTTCAC CACCGACTCC ATGGAGATCC GCCTGGTGGT 2100
GAACGGGAAC CTGGTCCACA CTGCAGTCGT GCCGCAGCTG CAGCTGGTGG CCTCCAGGTC 2160 GGATATATAC TTCACAGCAA CTGCAGCTGT GAATGAGGTC TCATCTGGAG GCAGCTCCAA 2220
GGGGGCCAGT GCCCGAAATT CTCCTCAGAC ACCCCCGGGC CGAGATACTC CAGTATTTCC 2280
TTCTTCCCTG GGGGAAGGTG AAATTCAATC AAAAAATCTG TACAAGATTC CACTTAGAAA 2340
CCTCGTGGGC AGAAGCATCG AACGACCTCT GAAGTCACCC TTAGTCTCCA AGGTCATCAC 2400
CCCACCCACT CCCATCAGTG TGGGCCTTGC TGCCATTCCA GTCACGCACT CCTTGTCCCT 2460 GTCTCGCATG GAGATCAAAG AAATAGCAAG CAGGACCCGC AGGGAACTAC TGGGCCTCTC 2520
GGATGAAGGT GGACCCAAGT CAGAAGGAGC GCCAAAGGCC AAATCAAAAC CCCGGAAGCG 2580
GTTAGAAGAA AGCCAAGGAG GCCCCAAGCC AGGGGCAGTG AGGTCATCTA GCAGTGACAG 2640
GATCCCATCA GGCTCCTTGG AAAGTGCTTC TACTTCCGAA GCCAACCCTG AGGGGCACTC 2700
AGCCAGCTCT GACCAGGACC CTGTGGCAGA CAGAGAGGGC AGCCCGGTCT CCGGCAGCAG 2760 CCCCTTCCAG CTCACGGCTT TCTCCGATGA AGACATTATA GACTTGAAGT AACAGAGTTG 2820
AATCTCATTT GCCATCTTTA GTTTTCTTAT GGAGGTTTAT ACTCTTTAAA CAGTTCTGAT 2880
GTAATTTCTC AACAAAATGT GGCTTTTAGC CTGTCAGTGA TCTATTGGAC CAAACCTTCT 2940
GCACACTCGG CCAGTTCCCT CTCCAATGTC CGGTGCCATC TTTCCTGACC TTTGTTTCTT 3000
TCTGTTCAGG AACCATCAGT CCCCTTGTAA TAAAGGTGGT AGATTTCATT GAGGTTTTAG 3060 ATTGAAACTT TGAATAAATC AAAAATACTC ATTCTTAAAA AAAAAAAAAA AAAAAAAAA 3119
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 322 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Asn Leu Asp Lys Phe Glu Lys Gly Pro Arg Glu He Phe His Pro 1 5 10 15
Glu He Gin Lys Asp Leu Leu Val Leu Glu Glu Gin Glu Gly Ser Val 20 25 30
Asn Phe Lys Phe Gly Val Leu Phe Ala Lys Asp Gly Gin Leu Thr Asp 35 40 45 Asp Glu Met Phe Ser Asn Glu He Gly Ser Glu Pro Phe Gin Lys Phe 50 55 60
Leu Asn Leu Leu Gly Asp Thr He Thr Leu Lys Gly Trp Thr Gly Tyr 65 70 75 80
Arg Gly Gly Leu Asp Thr Lys Asn Asp Thr Thr Gly He His Ser Val 85 90 95
Tyr Thr Val Tyr Gin Gly His Glu He Met Phe His Val Ser Thr Met 100 105 110
Leu Pro Tyr Ser Lys Glu Asn Lys Gin Gin Val Glu Arg Lys Arg His 115 120 125 He Gly Asn Asp He Val Thr He Val Phe Gin Glu Gly Glu Glu Ser 130 135 140
Ser Pro Ala Phe Lys Pro Ser Met He Arg Ser His Phe Thr His He 145 150 155 160
Phe Ala Leu Val Arg Tyr Asn Gin Gin Asn Asp Asn Tyr Arg Leu Lys 165 170 175
He Phe Ser Glu Glu Ser Val Pro Leu Phe Gly Pro Pro Leu Pro Thr 180 185 190
Pro Pro Val Phe Thr Asp His Gin Glu Phe Arg Asp Phe Leu Leu Val 195 200 205
Lys Leu He Asn Gly Glu Lys Ala Thr Leu Glu Thr Pro Thr Phe Ala 210 215 220
Gin Lys Arg Arg Arg Thr Leu Asp Met Leu He Arg Ser Leu His Gin 225 230 235 240
Asp Leu Met Pro Asp Leu His Lys Asn Met Leu Asn Arg Arg Ser Phe 245 250 255"
Ser Asp Val Leu Pro Glu Ser Pro Lys Ser Ala Arg Lys Lys Glu Glu 260 265 270 Ala Arg Gin Ala Glu Phe Val Arg He Gly Gin Ala Leu Lys Leu Lys 275 280 285
Ser He Val Arg Gly Asp Ala Pro Ser Ser Leu Ala Ala Ser Gly He 290 295 300
Cys Lys Lys Glu Met Thr Phe His Gin Cys Pro Cys Leu Thr Glu Leu 305 310 315 320 Cys Gin
(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1592 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GACTTTTATA AAAAAGTCAA GCAGTACAAA AGGGTGTAAA GTGAAGTTAC TGTCCTTCCC 60 CTCCATAAAC CCCCTGACCT TGGGAAACTG TTGTTAACAG TTACTTGGGT AACTTTTCAG 120
ATATTTTGTA TGCATGTACA AATGTGAGTA TCTAATGTAA AAAAATCAAA CCAAGATAAA 180
GTGTAAACTG CTATGATGGA ATCCTGCCTT GTTCTGCTAT TAGTCTTCTG TTTAATAATC 240
AGCTTTGGTA TTAGGACAGT GGTAGGAAGA AGCCAGTATG TCCTGCAACA TAATTTGTGG 300
TTCTGGACTG GTCAGGATTT CCTGAATGCA GCCTTTATCT GGAAGCTCTG CCCTTCTCCA 360 TCTGGGATAC GCTTTTTCAT CCATCAAAAC TGTCATCTCC CTCTGTGAAG CCTTCCCTGA 420
CTATTCTCTG TCCCTCTTTC CTCTCTTCCC ACAAACACAA CTGTGTACGC GTGTCACCAA 480
AGAGTTAATC GTGCTTTTCT CTGTGCTACT TTTATACSTA GTATATGGTC CATTGTTTTG 540
CACTTAATAC ACTCTCTTGT AATGATTTGT TTACATGTCA GTCTCCCAGC CAGACTGAGA 600
GCTCACCAAG GGCAGAAGCC GTGTTTTGTT TACTGCTGTA TTCCTGGTAC CTGGTACAAT 660 GCTTGGCATA CAGTTGGATG AACGGGAAAG TAATCTGAGC TGCCGGTGCT GTGGCAGTGC 720
AAAGTGGGCA TATTTGTGCC CTTGGACCAG ATGTAGCCCT TGATGCATTT TGCAGGAACA " 780
CGGCTTAGTT ATTGTTTACT TTGAAGCCCT TTTGCCTCTA CTCTCTCCCA TATATCTTCT 840 CCTGACAGGG TGAAGTCACC TATAGCATTT CCTAGTGTAT GGAAGTATTA ATTTCTTTCT 900
TTACTGGAAG AGCTACTAGC TTTTCTTCAT ACAGTTTCCT CTGCTCCAGT TTCATAAGTT 960 TCTTTTTGGC TTGTATCTGT TTAGGATCAG GTGATATGGC TTCATTTCTC ATGACTGAAG 1020
CCCGGCAACA TAACACTGAA ATTCGAATGG CAGTCAGCAA AGTGGCTGAT AAAATGGATC 1080
ATCTCATGAC TAAGGTTGAA GAGTTACAGA AACATAGTGC TGGCAATTCC ATGCTTATTC 1140
CTAGCATGTC AGTTACAATG GAAACAAGCA TGATTATGAG CAACATCCAG CGAATCATTC 1200
AGGCCAAGGT GACAGAGGAG TTAGCAGCGG CCACTGCACA GKTCTCTCAT CTGCAGCTGA 1260 AAATGACTTG CTCACCAAAA AAAGGAAACA GAGCTGCAGA TGCAGCTGAC AGAAAGCCTG 1320
AAGGAGACAG ATCTTCTCAG GGGCCAGCTC ACCAAAGTGC AGGCAAAGCT CTCAGAGCTC 1380
CAAGAAACYT CTGAGCAAGC ACAGTCCAAA TTCAAAAGTG AAAAGCAGAA CCGGAAACAA 1440
CTGGAACTCA AGGTGACATC CCTGGAGGAG GAACTGACTG ACCTTCGAGT TGAGAAGGAG 1500
TCCTTGGAAA AGGTAAGCTC TACAACCCAG TTTGCCAGAA TTAGCTGTTT AATAAACATT 1560 TTTATTTTCC TTTTACAAAA AAAAAAAAAA AA 1592
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Glu Val Leu He Ser Phe Phe Thr Gly Arg Ala Thr Ser Phe Ser 1 5 10 15 Ser Tyr Ser Phe Leu Cys Ser Ser Phe He Ser Phe Phe Leu Ala Cys
20 25 30
He Cys Leu Gly Ser Gly Asp Met Ala Ser Phe Leu Met Thr Glu Ala 35 40 45
Arg Gin His Asn Thr Glu He Arg Met Ala Val Ser Lys Val Ala Asp 50 55 60
Lys Met Asp His Leu Met Thr Lys Val Glu Glu Leu Gin Lys His Ser 65 70 75 80 Ala Gly Asn Ser Met Leu He Pro Ser Met Ser Val Thr Met Glu Thr 85 90 95
Ser Met He Met Ser Asn He Gin Arg He He Gin Ala Lys Val Thr 100 105 110
Glu Glu Leu Ala Ala Ala Thr Ala Gin Xaa Ser His Leu Gin Leu Lys 115 120 125 Met Thr Cys Ser Pro Lys Lys Gly Asn Arg Ala Ala Asp Ala Ala Asp 130 135 140
Arg Lys Pro Glu Gly Asp Arg Ser Ser Gin Gly Pro Ala His Gin Ser 145 150 155 160
Ala Gly Lys Ala Leu Arg Ala Pro Arg Asn Xaa 165 170
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1694 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GGGAAACGGG AAGCCGCTGC AAGTCCACCG CCTCAGCTAC CCAGATTGGG ATCTGCCCAG 60
GCCCGCTTTA TGGACTAGTG TGGGCGGCAG GCTCCTTTCC GTCCCTGCCC TGCTGTACCC 120
CGCTCCTTGG AGACCCCCTG TATCCCTCCC GCAAGGTGGA ATCCGCAGGC TGGAGGCTCC 180 CAGGGGAGGC AAACGCCTGG CCCTGCCCTG CCCCACGCCG CACCATGACC CTCCTGCTGC 240
TGCCCCTTCT GCTGGCCTCT CTGCTCGCGT CCTGCTCCTG TAACAAAGCC AACAAGCACA 300
AGCCATGGAT TGAGGCAGAG TACCAGGGCA TCGTCATGGA GAATGACAAC ACGGTCCTAC 360
TGAATCCACC ACTCTTTGCC TTGGACAAGG ATGCCCCGCT GCGCTATGCA GGTGAGATCT 420
GCGGCTTCCG GCTCCATGGG TCTGGGGTGC CCTTTGAGGC TGTGATCCTT GACAAGGCGA 480 CAGGAGAGGG GCTGATCCGG GCCAAGGAGC CTGTGGACTG CGAGGCCCAG AAGGAACACA 540
CCTTCACCAT CCAGGCCTAT GACTGTGGCG AGGGCCCCGA CGGGGCCAAC ACCAAGAAGT " 600
CCCACAAGGC CACTGTGCAT GTGCGGGTCA ACGATGTGAA CGAGTTTGCC CCAGTGTTTG 660 TGGAACGGCT GTATCGTGCG GCTGTGACAG AGGGGAAGCT GTACGATCGC ATCCTGCGGG 720
TGGAAGCCAT TGACGGTGAC TGCTCCCCCC AGTACAGCCA GATCTGCTAC TATGAGATTC 780 TCACACCCAA CACCCCTTTC CTCATTGACA ATGACGGGAA CATTGAGAAC ACAGAGAAGC 840
TGCAGTACAG TGGTGAGAGG CTCTATAAGT TTACAGTGAC AGCTTATGAC TGTGGGAAGA 900
AGCGGGCAGC AGATGATGCT GAGGTGGAGA TTCAGGTGAA GCCCACCTGT AAACCCAGCT 960
GGCAAGGCTG GAACAAAAGG ATCGAATATG CACCAGGTGC TGGGAGCTTG GCTTTGTTCC 1020
CTGGTATCCG CCTGGAGACC TGTGATGAAC CACTCTGGAA CATTCAGGCC ACCATAGAGC 1080 TGCAGACCAG CCATGTGGCC AAGGGCTGTG ACCGTGACAA CTACTCAGAG CGGGCGCTGC 1140
GGAAACTCTG TGGTGCTGCC ACTGGGGAGG TGGATCTGTT GCCCATGCCT GGCCCCAATG 1200
CCAACTGGAC AGCAGGACTC TCGGTGCACT ACAGCCAGGA CAGCAGCCTG ATCTACTGGT 1260
TCAATGGCAC CCAGGCTGTG CAGGTGCCCC TGGGTGGCCC CAGTGGGCTG GGCTCTGGGC 1320
CCCAGGACAG CCTCAGTGAC CACTTCACCC TGTCCTTCTG GATGAAGCAT GGCGTAACTC 1380 CCAACAAGGG CAAGAAGGAA GAGGAAACCA TCGTATGTAA CACTGTCCAG AATGGTGAGC 1440
CTCCCCTCCA GGCACTAGCC AGAGGGGGAA ACTGGCTTCT TGTCCCGCCT CTGTCACTGC 1500
CCAGTGTGTG ACTGTGAACA GGTCACTTCC CCTCTCTTCA TTTGTGAGGT GCAAGTGCCA 1560
GGTGTGATAT GCCTTGATTC TGTGCTTTAT CCCCAACATG ACATGTTGGA TCGTAAAAAA 1620
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1680 AAAAAAAAAA AAAA 1694
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 428 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Thr Leu Leu Leu Leu Pro Leu Leu Leu Ala Ser Leu Leu Ala Ser
1 5 10 15 Cys Ser Cys Asn Lys Ala Asn Lys His Lys Pro Trp He Glu Ala Glu 20 25 30
Tyr Gin Gly He Val Met Glu Asn Asp Asn Thr Val Leu Leu Asn Pro 35 40 45
Pro Leu Phe Ala Leu Asp Lys Asp Ala Pro Leu Arg Tyr Ala Gly Glu 50 55 60
He Cys Gly Phe Arg Leu His Gly Ser Gly Val Pro Phe Glu Ala Val 65 70 75 80
He Leu Asp Lys Ala Thr Gly Glu Gly Leu He Arg Ala Lys Glu Pro 85 90 95 Val Asp Cys Glu Ala Gin Lys Glu His Thr Phe Thr He Gin Ala Tyr
100 105 110
Asp Cys Gly Glu Gly Pro Asp Gly Ala Asn Thr Lys Lys Ser His Lys 115 120 125
Ala Thr Val His Val Arg Val Asn Asp Val Asn Glu Phe Ala Pro Val 130 135 140
Phe Val Glu Arg Leu Tyr Arg Ala Ala Val Thr Glu Gly Lys Leu Tyr 145 150 155 160
Asp Arg He Leu Arg Val Glu Ala He Asp Gly Asp Cys Ser Pro Gin 165 170 175
Tyr Ser Gin He Cys Tyr Tyr Glu He Leu Thr Pro Asn Thr Pro Phe 180 185 190
Leu He Asp Asn Asp Gly Asn He Glu Asn Thr Glu Lys Leu Gin Tyr 195 200 205
Ser Gly Glu Arg Leu Tyr Lys Phe Thr Val Thr Ala Tyr Asp Cys Gly 210 215 220
Lys Lys Arg Ala Ala Asp Asp Ala Glu Val Glu He Gin Val Lys Pro 225 230 235 240
Thr Cys Lys Pro Ser Trp Gin Gly Trp Asn Lys Arg He Glu Tyr Ala 245 250 255 Pro Gly Ala Gly Ser Leu Ala Leu Phe Pro Gly He Arg Leu Glu Thr
260 265 270
Cys Asp Glu Pro Leu Trp Asn He Gin Ala Thr He Glu Leu Gin Thr 275 280 285
Ser His Val Ala Lys Gly Cys Asp Arg Asp Asn Tyr Ser Glu Arg Ala 290 295 300
Leu Arg Lys Leu Cys Gly Ala Ala Thr Gly Glu Val Asp Leu Leu Pro 305 310 315 320 Met Pro Gly Pro Asn Ala Asn Trp Thr Ala Gly Leu Ser Val His Tyr
325 330 335
Ser Gin Asp Ser Ser Leu He Tyr Trp Phe Asn Gly Thr Gin Ala Val 340 345 350
Gin Val Pro Leu Gly Gly Pro Ser Gly Leu Gly Ser Gly Pro Gin Asp 355 360 365 Ser Leu Ser Asp His Phe Thr Leu Ser Phe Trp Met Lys His Gly Val 370 375 380
Thr Pro Asn Lys Gly Lys Lys Glu Glu Glu Thr He Val Cys Asn Thr 385 390 395 400
Val Gin Asn Gly Glu Pro Pro Leu Gin Ala Leu Ala Arg Gly Gly Asn 405 410 415
Trp Leu Leu Val Pro Pro Leu Ser Leu Pro Ser Val 420 425
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1309 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
CTGGTCCTCC TTTGCAGAGG TGGTGCGGAG CTCCTGTTTG ACGGTATTAA GAAACATCGA 60
GTCACTTTGC CTGGACAGGA GGAACCCTGG GACATCCGGA ACCTGCTCAT CTGGATCAAG 120
AAGAATTTGC TAAAAGAGCG GCCAGAGTTG TTCATCCAGG GAGACAGCGT GCGGCCAGGA 180
ATTCTGGTGC TGATTAACGA TGCCGACTGG GAGCTACTGG GTGAGCTGGA CTACCAGCTT 240 CAGGACCAGG ACAGCGTCCT CTTCATCTCC ACTCTGCACG GCGGCTGAGG GCCCTTCTCT 300
GGGCCTGGGC ACCCTTAGAG GGGAGAACGA AGCAATCAGA CATCCCCTTG GGCCCTGCTT 360
CCAGGTCTCC CTGTCCCCCT TGCCTGCCTT CTTCCCTGCT CTGTCCCCTA AGCTCCCTCC 420
AGGCAGGGAA AAGAGGCCAG GTGCTAAAAA TGAGCCTTTC TCAAGCACGT GAGCAGCGGA 480
AGGCAGACAG GCGCCAGAGC CCAGCACTCC CTTTTCCAGC AGCTGTGGTG GGGGAGGGTT 540 CCCCTCCAGT TTGTCAAGAG TTGAAGGAGG CTCTGTGGCC AGGTGACCTG GCTGCCTTCC 600 ACTCCTTGTA CCTCAGTCTA AACATGGAGT GGCCGCTGAC AAGGCGCTCC AGCCCCAGAG 660
CCAGCGTCTT CATGGGGAAG ATGAATGGAC CTGAGTAGCT GAAGGAAGGC CCCTCCCTAC 720 CCAAAGACTG GAGGCTTCTC AGCCTCAATT TCCCTGTCTG TACAGCTGAG GGCTCTGCCT 780
GTCCCCCACT GCTATCAGTA TGGAACCCCA GCTGGGGTCC CCTATTGAGT GCCGACTCCC 840
CCCACCGCCA GCAGCTGCTC CTCCAGCCAC ACCCTTCCTG CTCCCCCCAC CCYTAGCCCT 900
TGACCCTGGC TGGCCTGCCC CGCTCCACAG GCCACCAGAT GGGCTCCTGA GACCCTCCCC 960
AGGCTGCTTA CAGCTCATTC TGCTGGGGGT AGAGATGAGG GGAGGGAGTA AGTTAAACCT 1020 TGGACTAGCA AGTAGAAGCC TGGGGGGATG CGTGTGCCTC AGTTTCCTCC TCCACAACTG 1080
AATATAGTGG CTGAAAACTG GGGAGATACT TGATGGCGCG AATGTCCGTT TTCTCTCCCT 1140
TCCCACCTCC TGCAGGAAGC AGGACGGGGC AGGCAGCACC TGGTAGGCAC AGTGCTTTGC 1200
CCCTCCTCCC CTTCCCTTCT GGAAGTCTTG GGGCCTCAGT GCTTGCAACA GCCGGCCTTG 1260
GGCAAATAAA AGACTAGGTT GTTTACTAAA AAAAAAAAAA AAAAAAAAA 1309 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Met Ala Arg Met Ser Val Phe Ser Pro Phe Pro Pro Pro Ala Gly Ser 1 5 10 15
Arg Thr Gly Gin Ala Ala Pro Gly Arg His Ser Ala Leu Pro Leu Leu 20 25 30
Pro Phe Pro Ser Gly Ser Leu Gly Ala Ser Val Leu Ala Thr Ala Gly 35 40 45
Leu Gly Gin He Lys Asp 50
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1740 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GGCCCGTGCG CTCCATCAAC CACGCCAGCC TCATCTCTGC ACTCTCCCGG GACTATCGCA 60
ACCTGAAGCC CAGTGCTGTT GCCCCACAGA GAAAGATGCC ACTGGATGAC ACCAAACTGA 120
TTATCCACCA GACACTCAGC GTCTTAGAAG ATATTGTGGA GAATATCTCG GGGGAGTCCA 180
CCAAGTCTCG ACAGATTTGC TACCAGTCGC TGCAGGAATC TGTTCAGGTC TCCCTGGCCC 240 TCTTTCCAGC TTTTATCCAT CAGTCAGATG TGACTGATGA GATGCTGAGC TTCTTCCTCA 300
CTCTGTTTCG AGGCCTTAGA GTACAGATGG GTGTGCCTTT CACTGAGCAA ATCATACAGA 360
CTTTCCTCAA CATGTTTACC AGAGAGCAGT TAGCCGAGAG CATCCTCCAC GAGGGCAGCA 420
CAGGCTGCCG GGTGGTGGAG AAGTTTCTGA AGATCCTGCA GGTGGTGGTC CAGGAGCCAG 480
GCCAGGTGTT CAAGCCCTTC CTCCCCAGCA TCATCGCCCT GTGCATGGAG CAAGTGTATC 540 CCATCATTGC CGAGCGTCCC TCCCCTGATG TGAAGGCCGA GCTGTTTGAG CTCCTTTTCC 600
GGACGCTCCA TCACAACTGG AGGTACTTCT TCAAGTCCAC CGTGCTGGCC AGTGTCCAGA 660
GGGGGATCGC TGAGGAGCAG ATGGAGAATG AGCCCCAGTT CAGTGCCATC ATGCAGGCTT 720
TCGGACAGTC CTTTCTCCAG CCCGACATCC ACCTTTTTAA ACAAAATCTC TTCTACTTGG 780
AGACTCTCAA CACCAAGCAG AAGCTGTACC ACAAGAAGAT CTTCCGGACT GCCATGCTGT 840 TCCAGTTTGT GAACGTGCTG CTCCAGGTCC TGGTCCACAA GTCCCATGAT CTTCTGCAGG 900
AGGAGATTGG CATCGCCATC TACAACATGG CCTCAGTCGA CTTTGATGGC TTCTTTGCCG 960
CCTTCCTCCC AGAGTTCCTG ACCAGCTGTG ATGGTGTGGA TGCCAACCAG AAAAGTGTGC 1020
TGGGGCGGAA TTTCAAGATG GATCGGGACC TGCCCTCATT CACCCAGAAT GTGCACAGGC 1080
TGGTCAACGA CCTGCGCTAC TACAGACTCT GCAACGACAG CCTGCCCCCT GGCACTGTGA 1140 AGCTCTAGGC CTGCTACTGC CTGGGGACAC GGACTTCTGC TGCTGCCACC TGCGCCAGCC 1200
CTACCTTCCA CCACAGATGT CTCCCAGATG GGCCTTGGTC ACACTCCTTG GCTTCTCCCA 1260
CCGCAAGCAA CGCTGCCTGC CTCTGCCGCT CCTCCACATC TTGCCGCTGC CCAGCAGAGC 1320 TGGCTTCTGG GTCCACCTGA GCACTGGACG GTGCTCCCAG GGCGTTGGAG CAGGCGGAGG 1380
GGTGTGTGGC CAGGTACTAG GAGGCACCAG GAAATCCCGC GGGGTGGCCC ATGCAGACCA 1440 GGCGCACGTG GCTCATGGGG CAGAATTGCC AAGGACAGCT CACGACAGTG CMACCTTCTC 1500
ACCATTCCAG CCAAGGAGAG ATGTGACGTT GGAAMTGYTY TGGCAMTTYT GTCAAGCCTC 1560
CCCCGCCCCA ATTGCCTTGA RATYTYTGCT CTTTGTCAGA GATTTGCAAA GACTCAMGTT 1620
TTTGTTGTTT TCTCATCATT CCATTGTGAT ACTAAGAAAC TAAGAAGCTT AATGAAAAGA 1680
AATAAAATGC CTATGTTGTT GTTCTAGGRR AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1740
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Pro Leu Asp Asp Thr Lys Leu He He His Gin Thr Leu Ser Val 1 5 10 15
Leu Glu Asp He Val Glu Asn He Ser Gly Glu Ser Thr Lys Ser Arg 20 25 30
Gin He Cys Tyr Gin Ser Leu Gin Glu Ser Val Gin Val Ser Leu Ale 35 40 45
Leu Phe Pro Ala Phe He His Gin Ser Asp Val Thr Asp Glu Met Leu 50 55 60
Ser Phe Phe Leu Thr Leu Phe Arg Gly Leu Arg Val Gin Met Gly Val 65 70 75 80
Pro Phe Thr Glu Gin He He Gin Thr Phe Leu Asn Met Phe Thr Arg 85 90 95
Glu Gin Leu Ala Glu Ser He Leu His Glu Gly Ser Thr Gly Cys Arg 100 105 110
Val Val Glu Lys Phe Leu Lys He Leu Gin Val Val Val Gin Glu Pro 115 120 125 Gly Gin Val Phe Lys Pro Phe Leu Pro Ser He He Ala Leu Cys Met 130 135 140
Glu Gin Val Tyr Pro He He Ala Glu Arg Pro Ser Pro Asp Val Lys 145 150 155 160
Ala Glu Leu Phe Glu Leu Leu Phe Arg Thr Leu His His Asn Trp Arg 165 170 175
Tyr Phe Phe Lys Ser Thr Val Leu Ala Ser Val Gin Arg Gly He Ala 180 185 190
Glu Glu Gin Met Glu Asn Glu Pro Gin Phe Ser Ala He Met Gin Ala 195 200 205 Phe Gly Gin Ser Phe Leu Gin Pro Asp He His Leu Phe Lys Gin Asn 210 215 220
Leu Phe Tyr Leu Glu Thr Leu Asn Thr Lys Gin Lys Leu Tyr His Lys
225 230 235 240
Lys He Phe Arg Thr Ala Met Leu Phe Gin Phe Val Asn Val Leu Leu
245 250 255
Gin Val Leu Val His Lys Ser His Asp Leu Leu Gin Glu Glu He Gly 260 265 270
He Ala He Tyr Asn Met Ala Ser Val Asp Phe Asp Gly Phe Phe Ala 275 280 285
Ala Phe Leu Pro Glu Phe Leu Thr Ser Cys Asp Gly Val Asp Ala Asn 290 295 300
Gin Lys Ser Val Leu Gly Arg Asn Phe Lys Met Asp Arg Asp Leu Pro 305 310 315 320
Ser Phe Thr Gin Asn Val His Arg Leu Val Asn Asp Leu Arg Tyr Tyr 325 330 335
Arg Leu Cys Asn Asp Ser Leu Pro Pro Gly Thr Val Lys Leu 340 345 350
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ANTGTCTTGA CTACAAGCTC CACGGGGGC 29
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TNGCCAAGGA GAAAGCGAGG CAGACAAGG 29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ANATCGACTC TTTGCATCGC ACATTTTGT 29
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CNTTCTTCGG ACTTATGTTT GAATCTATC 29 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: CNTTCCTCTT AGATCTCAGT ATCCACCTC 29
(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: CNCAGACAGG GGAGATAACA ATGAGGTGC 29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TNCTATAGGT GACTTCACCC TGTCAGGAG 29
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TNTTACAGGA GCAGGACGCG AGCAGAGAG 29
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ANTCAGTTGT GGAGGAGGAA ACTGAGGCA 29
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CNTCGAAACA GAGTGAGGAA GAAGCTCAG 29 (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met Val Ala Trp Arg Ser Ala Phe Leu Val Cys Leu Ala Phe Ser Leu 1 5 10 15 Ala Thr Leu Val Gin Arg Gly Ser Gly Asp Phe Asp Asp Phe Asn Leu
20 25 30
Glu Asp Ala Val Lys Glu Thr Ser Ser Val Lys Gin Pro Trp Asp His 35 40 45
Thr Thr Thr Thr Thr Thr Asn Arg Pro Gly Thr Thr Arg Ala Pro Ala 50 55 60
Lys Pro Pro Gly Ser Gly Leu Asp Leu Ala Asp Ala Leu Asp Asp Gin 65 70 75 80
Asp Asp Gly Arg Arg Lys Pro Gly He Gly Gly Arg Glu Arg Trp Asn 85 90 95
His Val Thr Thr Thr Thr Lys Arg Pro Val Thr Thr Arg Ala Pro Ala 100 105 110
Asn Thr Leu Gly Asn Asp Phe Asp Leu Ala Asp Ala Leu Asp Asp Arg 115 120 125
Asn Asp Arg Asp Asp Gly Arg Arg Lys Pro He Ala Gly Gly Gly Gly 130 135 140
Phe Ser Asp Lys Asp Leu Glu Asp He Val Gly Gly Gly Glu Tyr Lys 145 150 155 160
Pro Asp Lys Gly Lys Gly Asp Gly Arg Tyr Gly Ser Asn Asp Asp Pro 165 170 175 Gly Ser Gly Met Val Ala Glu Pro Gly Thr He Ala Gly Val Ala Ser
180 185 190
Ala Leu Ala Met Ala Leu He Gly Ala Val Ser Ser Tyr He Ser Tyr 195 200 205 Gin Gin Lys Lys Phe Cys Phe Ser He Gin Gin Gly Leu Asn Ala Asp 210 215 220
Tyr Val Lys Gly Glu Asn Leu Glu Ala Val Val Cys Glu Glu Pro Gin 225 230 235 240
Val Lys Tyr Ser Thr Leu His Thr Gin Ser Ala Glu Pro Pro Pro Pro 245 250 255 Pro Glu Pro Ala Arg He
260
(2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met His Val Leu Glu Thr Leu Asp Leu Leu Val Leu Arg Ala Asp Lys 1 5 10 15
Gly Lys Asp Ala Arg Leu Phe Val Phe Arg Leu Ser Ala Leu Gin Lys 20 25 30
Gly Leu Glu Gly Lys Gin Ala Gly Lys Ser Arg Ser Asp Cys Arg Glu 35 40 45
Asn Lys Leu Glu Lys Thr Lys Gly Cys His Leu Tyr Ala He Asn Thr 50 55 60 His His Ser Arg Glu Leu Arg He Val Val Ala He Arg Asn Lys Leu 65 70 75 80
Leu Leu He Thr Arg Lys His Asn Lys Pro Ser Gly Val Thr Ser Thr 85 90 95
Ser Leu Leu Ser Pro Leu Ser Glu Ser Pro Val Glu Glu Phe Gin Tyr 100 105 110
He Arg Glu He Cys Leu Ser Asp Ser Pro Met Val Met Thr Leu Val 115 120 125
Asp Gly Pro Ala Glu Glu Ser Asp Asn Leu He Cys Val Ala Tyr Arg 130 135 140
His Gin Phe Asp Val Val Asn Glu Ser Thr Gly Glu Ala Phe Arg Leu 145 150 155 160
His His Val Glu Ala Asn Arg Val Asn Phe Val Ala Ala He Asp Val 165 170 175
Tyr Glu Asp Gly Glu Ala Gly Leu Leu Leu Cys Tyr Asn Tyr Ser Cys 180 185 190
He Tyr Lys Lys Val Cys Pro Phe Asn Gly Gly Ser Phe Leu Val Gin 195 200 205
Pro Ser Ala Ser Asp Phe Gin Phe Cys Trp Asn Gin Ala Pro Tyr Ala 210 215 220 He Val Cys Ala Phe Pro Tyr Leu Leu Ala Phe Thr Thr Asp Ser Met 225 230 235 240
Glu He Arg Leu Val Val Asn Gly Asn Leu Val His Thr Ala Val Val
245 250 255
Pro Gin Leu Gin Leu Val Ala Ser Arg Ser Asp He Tyr Phe Thr Ala
260 265 270
Thr Ala Ala Val Asn Glu Val Ser Ser Gly Gly Ser Ser Lys Gly Ala 275 280 285
Ser Ala Arg Asn Ser Pro Gin Thr Pro Pro Gly Arg Asp Thr Pro Val 290 295 300
Phe Pro Ser Ser Leu Gly Glu Gly Glu He Gin Ser Lys Asn Leu Tyr 305 310 315 320
Lys He Pro Leu Arg Asn Leu Val Gly Arg Ser He Glu Arg Pro Leu 325 330 335
Lys Ser Pro Leu Val Ser Lys Val He Thr Pro Pro Thr Pro He Ser 340 345 350
Val Gly Leu Ala Ala He Pro Val Thr His Ser Leu Ser Leu Ser Arg 355 360 365
Met Glu He Lys Glu He Ala Ser Arg Thr Arg Arg Glu Leu Leu Gly 370 375 380 Leu Ser Asp Glu Gly Gly Pro Lys Ser Glu Gly Ala Pro Lys Ala Lys 385 390 395 400
Ser Lys Pro Arg Lys Arg Leu Glu Glu Ser Gin Gly Gly Pro Lys Pro 405 410 415
Gly Ala Val Arg Ser Ser Ser Ser Asp Arg He Pro Ser Gly Ser Leu 420 425 430
Glu Ser Ala Ser Thr Ser Glu Ala Asn Pro Glu Gly His Ser Ala Ser 435 440 445 Ser Asp Gin Asp Pro Val Ala Asp Arg Glu Gly Ser Pro Val Ser Gly 450 455 460
Ser Ser Pro Phe Gin Leu Thr Ala Phe Ser Asp Glu Asp He He Asp 465 470 475 480
Leu Lys

Claims

What is claimed is:
1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 2178 to nucleotide 2513;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 2364 to nucleotide 2513;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1980 to nucleotide 2311;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cll81_3 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(j) 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 consecutive amino acids of SEQ ID NO:2;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
2. The polynucleotide of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with the polynucleotide of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein encoded by the polynucleotide of claim 2, which process comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and
(b) purifying said protein from the culture.
6. A protein produced according to the process of claim 5.
7. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising eight consecutive amino acids of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone cll81_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
8. The protein of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
9. The protein of claim 7, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 67.
10. A composition comprising the protein of claim 7 and a pharmaceutically acceptable carrier.
11. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:l.
12. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 207 to nucleotide 893;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1 to nucleotide 527;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone crl044_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone crl044_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
13. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 107; (c) fragments of the amino acid sequence of SEQ ID NO:4 comprising eight consecutive amino acids of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone crl044_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
14. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
15. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 400;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 118 to nucleotide 392;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cz251_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cz251_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
16. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 15 to amino acid 105;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising eight consecutive amino acids of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone cz251_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
17. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:5.
18. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 13 to nucleotide 501;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 1 to nucleotide 506;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ddl2_7 deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
19. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) fragments of the amino acid sequence of SEQ ID NO:8 comprising eight consecutive amino acids of SEQ ID NO:8; and
(c) the amino acid sequence encoded by the cDNA insert of clone ddl2_7 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
20. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
21. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 778 to nucleotide 1083;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 931 to nucleotide 1083;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 802 to nucleotide 1056; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone fnl91_3 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
22. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to amino acid 93;
(c) fragments of the amino acid sequence of SEQ ID NO:10 comprising eight consecutive amino acids of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone fnl91_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
23. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
24. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 390 to nucleotide 1355;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 1384 to nucleotide 1736;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gml96_4 deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
25. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12;
(b) fragments of the amino acid sequence of SEQ ID NO: 12 comprising eight consecutive amino acids of SEQ ID NO: 12; and (c) ' the amino acid sequence encoded by the cDNA insert of clone gml96_4 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
26. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:ll.
27. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 879 to nucleotide 1391;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 519 to nucleotide 1074;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gnll4_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 14;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
28. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 14;
(b) the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 65;
(c) fragments of the amino acid sequence of SEQ ID NO: 14 comprising eight consecutive amino acids of SEQ ID NO:14; and
(d) the amino acid sequence encoded by the cDNA insert of clone gnll4_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
29. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:13.
30. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 225 to nucleotide 1508;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 252 to nucleotide 1508;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 302;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hj968_2 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:16;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
31. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 16;
(b) the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 26;
(c) fragments of the amino acid sequence of SEQ ID NO:16 comprising eight consecutive amino acids of SEQ ID NO:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone hj968_2 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
32. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:15.
33. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 1113 to nucleotide 1274;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 1233 to nucleotide 1274;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 894 to nucleotide 1309; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hkl0_3 deposited under accession number ATCC 98456;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hkl0_3 deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO: 18;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j).
34. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 18;
(b) fragments of the amino acid sequence of SEQ ID NO:18 comprising eight consecutive amino acids of SEQ ID NO: 18; and
(c) the amino acid sequence encoded by the cDNA insert of clone hkl0_3 deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
35. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:17.
36. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19 from nucleotide 96 to nucleotide 1145;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 109 to nucleotide 539;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone hm236_l deposited under accession number ATCC 98456;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone hm236_l deposited under accession number ATCC 98456;
(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
37. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20;
(b) the amino acid sequence of SEQ ID NO:20 from amino acid 6 to amino acid 148; (c) fragments of the amino acid sequence of SEQ ID NO:20 comprising eight consecutive amino acids of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone hm236_l deposited under accession number ATCC 98456; the protein being substantially free from other mammalian proteins.
38. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:19.
PCT/US1998/012076 1997-06-12 1998-06-12 Secreted proteins and polynucleotides encoding them WO1998056805A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU78352/98A AU7835298A (en) 1997-06-12 1998-06-12 Secreted proteins and polynucleotides encoding them
CA002293825A CA2293825A1 (en) 1997-06-12 1998-06-12 Secreted proteins and polynucleotides encoding them
JP50318099A JP2002514089A (en) 1997-06-12 1998-06-12 Secreted proteins and polynucleotides encoding them
EP98926539A EP1003768A4 (en) 1997-06-12 1998-06-12 Secreted proteins and polynucleotides encoding them

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US8623497P 1997-06-12 1997-06-12
US60/086,234 1998-06-11

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Citations (1)

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

Patent Citations (1)

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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MPSRCH GENBANK 1 January 1900 (1900-01-01), FUJIWARA T, ET AL: "HUMAN AORTA CDNA 5'-END GEN-241B04, MRNA SEQUENCE", XP002910368, Database accession no. C16190 *
See also references of EP1003768A4 *

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