Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• 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;
• such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 69 to nucleotide 908; the nucleotide sequence of SEQ ID NO:l from nucleotide 270 to nucleotide 908; the nucleotide sequence of the full-length protein coding sequence of clone bn97_l deposited under accession number ATCC 98535; or the nucleotide sequence of a mature protein coding sequence of clone bn97_l deposited under accession number ATCC 98535.
• 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 135 to amino acid 144 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:
• 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 bn268_ll deposited under accession number ATCC 98535.
• 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 37.
• 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 31 to amino acid 40 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:
• 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:5 from nucleotide 286 to nucleotide 1686; the nucleotide sequence of SEQ ID NO:5 from nucleotide 544 to nucleotide 1686; the nucleotide sequence of SEQ ID NO:5 from nucleotide 365 to nucleotide 1160; the nucleotide sequence of the full-length protein coding sequence of clone cb96_10 deposited under accession number ATCC 98535; or the nucleotide sequence of a mature protein coding sequence of clone cb96_10 deposited under accession number ATCC 98535.
• 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 228 to amino acid 237 of SEQ ID NO:6.
• protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 28 to amino acid 395.
• 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:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 228 to amino acid 237 of SEQ ID NO:6.
• 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:7 from nucleotide 99 to nucleotide 1049; the nucleotide sequence of SEQ ID NO:7 from nucleotide 222 to nucleotide 1049; the nucleotide sequence of SEQ ID NO:7 from nucleotide 632 to nucleotide 998; the nucleotide sequence of the full-length protein coding sequence of clone cb213_ll deposited under accession number ATCC 98535; or the nucleotide sequence of a mature protein coding sequence of clone cb213_ll deposited under accession number ATCC 98535.
• 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 153 to amino acid 162 of SEQ ID NO:8.
• 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: 1
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cj457_4 deposited under accession number ATCC 98535.
• 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 37.
• 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 37.
• 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:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 17 to amino acid 26 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: 1
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone cz653_l 1 deposited under accession number ATCC 98535.
• the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 147 to amino acid
• 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 174 to amino acid 183 of SEQ ID NO:12.
• Other embodiments provide the gene corresponding to the cDNA sequence 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: (a) the amino acid sequence of SEQ ID NO:12;
• amino acid sequence of SEQ ID NO:12 comprising eight consecutive amino acids of SEQ ID NO:12; and (d) the amino acid sequence encoded by the cDNA insert of clone cz653_ll deposited under accession number ATCC 98535; the protein being substantially free from other mammalian proteins.
• protein comprises the amino acid sequence of SEQ ID NO:12 or the amino acid sequence of SEQ ID NO:12 from amino acid 147 to amino acid 358.
• 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 174 to amino acid 183 of SEQ ID NO:12.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide 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: 1
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone dxl38_4 deposited under accession number ATCC 98535.
• the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 83 to amino acid
• 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 185 to amino acid 194 of SEQ ID NO:14.
• Other embodiments provide the gene corresponding to the cDNA sequence 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: (a) the amino acid sequence of SEQ ID NO:14;
• 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 dxl38_4 deposited under accession number ATCC 98535; the protein being substantially free from other mammalian proteins.
• protein comprises the amino acid sequence of SEQ ID NO:14 or the amino acid sequence of SEQ ID NO:14 from amino acid 83 to amino acid 229.
• 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: 14 having biological activity, the fragment comprising the amino acid sequence from amino acid 185 to amino acid 194 of SEQ ID NO:14.
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• 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;
• 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:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 24 to amino acid 33 of SEQ ID NO:16.
• 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:
• 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.
• a polynucleotide of the present invention has been identified as clone "bn97_l”.
• bn97_l was isolated from a human adult placenta cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• bn97_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bn97_l protein").
• bn97_l demonstrated at least some identity with sequences identified as AA046689 (zk72h06.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 488411 3'), D30934 (Human fetal-lung cDNA 5'-end sequence), R78820 (yi90b03.rl Homo sapiens cDNA clone 146477 5'), and R91687 (yql0h09.sl Homo sapiens cDNA clone 196577 3').
• the predicted amino acid sequence disclosed herein for bn97_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
• the predicted bn97_l protein demonstrated at least some identity with sequences identified as A10431 (Hepatitis-B virus surface antigen P31).
• the bn97_l protein also shows some identity (30% identity, 50% conserved amino acids) to both bovine and human lectin-like receptor for oxidized LDL (low-density lipoprotein). While this homology is weak, it gets stronger (44% identity and 62% conserved amino acids) in the lectin-like domain.
• the 3' untranslated region of the bovine receptor has seven mRNA unstabilising sequences (ATTTA) and bn97_l has four in its 3' untranslated region.
• This lectin-like receptor for oxidized LDL (designated LOX-1, Sawamura et al, 1997, Nature 386: 73-77) is an integral membrane protein which binds oxidized low-density lipoproteins, internalizes them into the endothelial cells and destroys them, thus playing a crucial role in the pathogenesis of atherosclerosis.
• bn97_l proteins and each identical protein or peptide may share at least some activity.
• a polynucleotide of the present invention has been identified as clone "bn268_ll".
• bn268_ll was isolated from a human adult placenta cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• bn268_ll is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bn268_ll protein").
• the nucleotide sequence of bn268_ll as presently determined is reported in SEQ.
• the EcoRI/Notl restriction fragment obtainable from the deposit containing clone bn268_ll should be approximately 1050 bp.
• the predicted bn268_ll protein demonstrated at least some identity with sequences identified as D83171 (GDP-GTP exchange protein for Rholp [Saccharomyces cerevisiae]). Based upon identity, bn268_ll proteins and each identical protein or peptide may share at least some activity.
• the TopPredll computer program predicts a potential transmembrane domain within the bn268_ll protein sequence centered around amino acid 33 of SEQ ID NO:4; this region may also function as a signal sequence.
• cb96_10 A polynucleotide of the present invention has been identified as clone "cb96_10".
• cb96_10 was isolated from a human fetal brain cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• cb96_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cb96_10 protein").
• nucleotide sequence of cb96_10 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 cb96_10 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
• Amino acids 74 to 86 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 87, or are a transmembrane domain.
• the EcoRI/Notl restriction fragment obtainable from the deposit containing clone cb96_10 should be approximately 2100 bp.
• cb96_10 Homo sapiens cDNA clone 245221 3'), and N57339 (yw81h07.rl Homo sapiens cDNA clone 258685 5').
• the predicted amino acid sequence disclosed herein for cb96_10 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
• the predicted cb96_10 protein demonstrated at least some identity with sequences identified as X80036 (ascorbate peroxidase [Arabidopsis thaliana]). Based upon identity, cb96_10 proteins and each identical protein or peptide may share at least some activity.
• cb96_10 is likely to be an integral membrane protein with multiple helices in the membrane; it also contains the sequence motif of the actinin-type actin-binding domains that are believed to anchor actin to the cell membrane.
• cb213_ll A polynucleotide of the present invention has been identified as clone "cb213_ll".
• cb213_ll was isolated from a human fetal brain cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• cb213_ll is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cb213_ll protein").
• cb213_ll protein demonstrated at least some identity with sequences identified as U39847 (AO13 ankyrin [Caenorhabditis elegans]). Based upon identity, cb213_ll proteins and each identical protein or peptide may share at least some activity.
• amino acids 11 to 23 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 24, or are a transmembrane domain.
• cj457_4 The nucleotide sequence disclosed herein for cj457_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cj457_4 demonstrated at least some identity with sequences identified as T92881 (ye22al0.sl Homo sapiens cDNA clone 1 18458 3') and T92488 (ye21g09.rl Homo sapiens cDNA clone 118432 5'). Based upon identity, cj457_4 proteins and each identical protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cj457_4 protein sequence, centered around amino acid 17 of SEQ ID NO:10; this region may also function as a signal sequence.
• cz653_ll demonstrated at least some identity with sequences identified as AA024740 (ze76c09.s 1 Soares fetal heart NbHH 19W Homo sapiens cDNA clone 364912 3'), AA203204 (zx57b04.rl Soares fetal liver spleen INFLS S I Homo sapiens cDNA clone 446575 5' similar to contains element MSR1 repetitive element), W72894 (zd59e06.sl Soares fetal heart NbHH19W Homo sapiens cDNA clone 344962 3'), and W76099 (zd59e06.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone 344962 5').
• the predicted cz653_ll demonstrated similarity to various WD-40 repeat containing proteins such as beta transducin-like protein (L28125) and coatomer, beta-prime subunit (AJ006523). The homology appears to be due to the presence of the Beta-transducin family Trp-Asp repeats signature (WD-40) beginning at residue 262 of SEQ ID NO:12. The WD-40 repeat has been thought to be a protein-protein interaction domain. Based upon identity, cz653_ll proteins and each identical protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cz653_ll protein sequence centered around amino acid 200 of SEQ ID NO:12.
• dxl38_4 A polynucleotide of the present invention has been identified as clone "dxl38_4".
• dxl38_4 was isolated from a human adult testes cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• dxl38_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dxl38_4 protein").
• dxl38_4 proteins and each identical protein or peptide may share at least some activity.
• a polynucleotide of the present invention has been identified as clone "ijl67_5".
• ijl67_5 was isolated from a human adult blood (peripheral blood mononuclear cells treated in vivo with G-CSF) cDNA library was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
• ijl67_5 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ijl67_5 protein").
• nucleotide sequence of ijl67_5 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 ijl67_5 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16. Amino acids 12 to 24 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 25, or are a transmembrane domain.
• the EcoRI/Notl restriction fragment obtainable from the deposit containing clone ijl67_5 should be approximately 1050 bp.
• Clones bn97_l, bn268_ll, cb96_10, cb213_.ll, cj457_4, cz653_.ll, dxl38_4, and ijl67_5 were deposited on September 4, 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 98535, 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/Notl digestion (5' site, EcoRl; 3' site, Notl) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Figures 1A and IB, respectively.
• the pED6dpc2 vector (“pED6" was derived from pED ⁇ dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al, 1991, Nucleic Acids Res.
• the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Moi. 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 EcoRl and Notl. However, Notl will then produce the 5' site and EcoRl 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 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.
• 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.
• 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.
• 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.
• linker 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.
• 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.
• the chromosomal location corresponding to the polynucleotide sequences disclosed herein may also be determined, for example by hybridizing appropriately labeled polynucleotides of the present invention to chromosomes in situ. It may also be possible to determine the corresponding chromosomal location for a disclosed polynucleotide by identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs), that have already been mapped to particular chromosomal locations. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotide of the present invention have been listed by database accession number.
• ESTs expressed sequence tags
• Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
• the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al, 1997, Biochem. Moi Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Moi Biol. 58: 1- 39; all of which are incorporated by reference herein).
• organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. 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.
• WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.wustl.edu/blast/executables.
• the complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at that site, in addition to several support programs.
• WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes.
• the default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
• Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
• a "species homologue” is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide.
• allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
• allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
• the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
• the present invention also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein.
• stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
• the hybrid length is that anticipated for the hybridized region(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 7.4
• IxSSC 0.15M NaCl and 15mM sodium citrate
• each such hybridizing polynucleotide has a length that is at least
• 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).
• Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
• yeast 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. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
• Kits for expression and purification of such fusion proteins are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and Invitrogen Corporation (Carlsbad, CA), respectively.
• the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
• One such epitope (“Flag") is commercially available from the Eastman Kodak Company (New Haven, CT).
• RP-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.
• modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
• Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
• one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
• such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
• Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
• 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 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.
• a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
• cytokine cytokine
• cell proliferation either inducing or inhibiting
• cell differentiation either inducing or inhibiting
• the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
• 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; BertagnoUi et al., J. Immunol.
• 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.
• 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.
• a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody
• B7- 1, B7-3 or blocking antibody e.g., B7- 1, B7-3 or blocking antibody
• 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, 59:11102-11105 (1992).
• murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
• Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
• Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
• Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
• Administration of reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
• blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
• 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.
• 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: MaUszewski, 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.
• Assays for embryonic stem ceU 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 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 (coUagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
• 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).
• FSH follicle stimulating hormone
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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
• the cadherin superfamily includes well over forty members, each with a distinct pattern of expression.
• AU 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.
• some cadherins engage in heterophilic adhesion with other cadherins.
• 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.
• 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
• 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
• 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 hyperpro
• 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.
• 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.
• 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 factor(s), thrombolytic or anti-thrombotic factors.
• Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
• protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
• the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
• the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
• a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
• the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
• the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
• protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
• parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
• a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium 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
• 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.
• 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.

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• 1998
  • 1998-09-08 WO PCT/US1998/018724 patent/WO1999013066A1/en not_active Application Discontinuation
  • 1998-09-08 EP EP98945966A patent/EP1012263A4/de not_active Withdrawn
  • 1998-09-08 CA CA002302705A patent/CA2302705A1/en not_active Abandoned
  • 1998-09-08 AU AU93095/98A patent/AU9309598A/en not_active Abandoned
  • 1998-09-08 JP JP2000510854A patent/JP2001515717A/ja not_active Withdrawn

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