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WO2003048326A2 - Procedes et matieres associes a de nouveaux polypeptides et polynucleotides - Google Patents

Procedes et matieres associes a de nouveaux polypeptides et polynucleotides Download PDF

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Publication number
WO2003048326A2
WO2003048326A2 PCT/US2002/038526 US0238526W WO03048326A2 WO 2003048326 A2 WO2003048326 A2 WO 2003048326A2 US 0238526 W US0238526 W US 0238526W WO 03048326 A2 WO03048326 A2 WO 03048326A2
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WIPO (PCT)
Prior art keywords
seq
polypeptide
amino acid
polynucleotide
adiponectin
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
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PCT/US2002/038526
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English (en)
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WO2003048326A3 (fr
Inventor
Malabika Ghosh
Y. Tom Tang
Jian Rui Wang
Zhiwei Wang
Qing A. Zhao
Chongjun Xu
Julio J. Mulero
Bryan J. Boyle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hyseq Inc
Nuvelo Inc
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Hyseq Inc
Nuvelo Inc
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Priority to AU2002360460A priority Critical patent/AU2002360460A1/en
Priority to US10/496,905 priority patent/US20050192215A1/en
Publication of WO2003048326A2 publication Critical patent/WO2003048326A2/fr
Priority to US10/758,846 priority patent/US20040248156A1/en
Anticipated expiration legal-status Critical
Publication of WO2003048326A3 publication Critical patent/WO2003048326A3/fr
Ceased legal-status Critical Current

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Classifications

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

Definitions

  • the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.
  • DNA/amino acid sequences for proteins that are known to have biological activity for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.
  • Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping, identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.
  • Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed.
  • compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides, and cells genetically engineered to express such polynucleotides.
  • compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485- 486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577- 578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631; or a fragment thereof that retains a
  • the polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345- 347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587,
  • polynucleotide which is an allelic variant of any polynucleotides recited above having at least 70% polynucleotide sequence identity to the polynucleotides; a polynucleotide which encodes a species homolog (e.g.
  • the collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array.
  • segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment.
  • the array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.
  • the collection can also be provided in a computer-readable format.
  • This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors.
  • Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.
  • the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
  • the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
  • Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
  • compositions of the present invention include polypeptides comprising, but not limited to, an isolated polypeptide selected from the group comprising the amino acid sequence of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380-
  • Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631; or (b) polynucle
  • polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recomb
  • compositions comprising a polypeptide of the invention.
  • Pharmaceutical compositions of the invention may comprise a polypeptide of the invention and an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • the invention also relates to methods for producing a polypeptide of the invention comprising culturing host cells comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein or peptide from the culture or from the host cells.
  • Preferred embodiments include those in which the protein produced by such a process is a mature form of the protein.
  • Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like.
  • polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
  • the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al, Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
  • polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.
  • a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.
  • Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.
  • the polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
  • Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier.
  • the methods of the invention also provide methods for the treatment of disorders as recited herein which comprise the administration of a therapeutically effective amount of a composition comprising a polynucleotide or polypeptide of the invention and a pharmaceutically acceptable carrier to a mammalian subject exhibiting symptoms or tendencies related to disorders as recited herein.
  • the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering a composition comprising compounds and other substances that modulate the overall activity of the target gene products and a pharmaceutically acceptable carrier.
  • Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.
  • methods are provided for preventing, treating or ameliorating a medical condition, including viral diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) the polypeptides of the invention.
  • a binding partner of e.g., antibody specifically reactive for
  • polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of cellular function.
  • a polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies.
  • protein or other active ingredients of the present invention may be included in formulations of a particular agent to minimize side effects of such an agent.
  • the invention further provides methods for manufacturing medicaments useful in the above-described methods.
  • the present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.
  • the invention provides a method for detecting a polypeptide of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting formation of the complex, so that if a complex is formed, the polypeptide is detected.
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.
  • the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
  • the invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.
  • Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g. , bind to) the polypeptides of the invention.
  • the invention provides a method for identifying a compound that binds to the polypeptide of the present invention comprising contacting the compound with the polypeptide under conditions and for a time sufficient to form a polypeptide/compound complex and detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of the invention is identified.
  • Also provided is a method for identifying a compound that binds to a polypeptide of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex wherein the complex drives expression of a reporter gene sequence in the cell and detecting the complex by detecting reporter gene sequence expression so that if the polypeptide/compound complex is detected a compound that binds to the polypeptide of the invention is identified.
  • Figure 1 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 5 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 2 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 5 and human adiponectin SEQ ID NO: 404 (Patent No.
  • Figure 3 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 15 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 4 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 15 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 5 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 28 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 6 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 28 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 7 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 160 and adiponectin SEQ ID NO: 402 (Hotta et al. Diabetes
  • Figure 8 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 160 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 9 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 186 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 10 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 186 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 11 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 215 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes
  • Figure 12 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 215 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 13 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 241 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 14 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 241 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 15 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 272 and adipose tissue-specific protein AdipoQ SEQ ID NO: 403 (Sato et al, J. Biol Chem. 276:28849-28856 (2001)).
  • Figure 16 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 272 and adipose tissue-specific protein AdipoQ SEQ ID NO: 403 (Sato et al, J. Biol. Chem. 276:28849-28856 (2001)).
  • Figure 17 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 272 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 18 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 302 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 19 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 302 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 20 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 323 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 21 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 323 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 22 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 348 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes
  • Figure 23 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 348 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 24 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 355 and adiponectin SEQ ID NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 25 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 355 and human adiponectin SEQ ID NO: 404 (Patent No. JP3018186-B1).
  • Figure 26 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 378 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)).
  • Figure 27 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ID NO: 378 and human adiponectin SEQ ID NO: 404 (Patent No.
  • Figure 28 shows the BLASTP amino acid sequence alignment of the first high scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 and SERPINB12 SEQ ID NO: 416 (Askew et al, J. Biol. Chem. 276:49320-49330 (2001), herein incorporated by reference in its entirety).
  • HSP high scoring pair
  • Figure 29 shows the BLASTP amino acid sequence alignment of the second high scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 and SERPINB12 SEQ ID NO: 416 (Askew et al, J. Biol Chem. 276:49320-49330 (2001), herein incorporated by reference in its entirety).
  • Figure 30 shows the BLASTP amino acid sequence alignment of the first high scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 and human SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, herein incorporated by reference in its entirety).
  • Figure 31 shows the BLASTP amino acid sequence alignment of the second high scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 and human SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, herein incorporated by reference in its entirety).
  • Figure 32 shows a schematic diagram illustrating the major structural features of the
  • Nogo receptor NgR
  • Nogo receptor homolog NgRHy
  • Figure 33 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 419 (i.e. SEQ ID NO: 420), NgRHy, and the human NgR (SEQ ID NO: 440).
  • Figure 34 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 443 (i.e. SEQ ID NO: 444), scavenger receptor-like polypeptide and mouse macrophage scavenger receptor type I (SEQ ID NO: 481).
  • Figure 35 shows a BLASTP amino acid sequence alignment between neural IgCAM-like polypeptide (SEQ ID NO: 487) and another member of the family, mouse PANG (SEQ ID NO: 502).
  • Figure 36 shows a BLASTP amino acid sequence alignment between neural IgCAM- like polypeptide (SEQ ID NO: 505) and bovine NCAM-140 (SEQ ID NO: 513).
  • Figure 37 shows a multiple amino acid sequence alignment between neural IgCAM- like polypeptide (SEQ ID NO: 505), neural IgCAM-like polypeptide (SEQ ID NO: 542) and bovine NCAM-140 (SEQ ID NO: 513).
  • Figure 38 shows a BLASTP amino acid sequence alignment between neural IgCAM- like polypeptide (SEQ ID NO: 516) and another member of the family, mouse DDM36 (SEQ ID NO: 52).
  • Figure 39 shows a BLASTP amino acid sequence alignment between neural IgCAM-like polypeptide (SEQ ID NO: 530) and another member of the family, rat BIG-2
  • Figure 40 shows a BLASTP amino acid sequence alignment between growth hormone-like polypeptide (SEQ ID NO: 548) and human chorionic somatomammotropin hormone-like 1, isoform 3 precursor (SEQ ID NO: 554).
  • Figure 41 shows a BLASTP amino acid sequence alignment between growth hormone-like polypeptide (SEQ ID NO: 548) and human chorionic somatomammotropin hormone-like 1, isoform 5 precursor (SEQ ID NO: 555).
  • Figure 42 shows a BLASTP amino acid sequence alignment between growth hormone-like polypeptide (SEQ ID NO: 557) and human chorionic somatomammotropin hormone 1, isoform 2 precursor (SEQ ID NO: 568).
  • Figure 43 shows a BLASTP amino acid sequence alignment between growth hormone-like polypeptide (SEQ ID NO: 557) and human growth hormone 2, isoform 2 precursor (SEQ ID NO: 569).
  • Figure 44 shows a multiple sequence alignment between NGAL-like polypeptides (SEQ ID NO: 572 and 579) and other members of the family: (SEQ ID NO: 585 and 586, respectively).
  • Figure 45 shows a BLASTP amino acid sequence alignment of mucolipin-like polypeptide (SEQ ID NO: 588) and human mucolipin 1 (SEQ ID NO: 592).
  • Figure 46 shows a multiple amino acid sequence alignment of mucolipin-like polypeptide (SEQ ID NO: 588) and other members of the family: mouse mucolipin 2 (SEQ ID NO: 591), human mucolipin 1 (SEQ ID NO: 592), human mucolipin 3 (SEQ ID NO: 593), C. elegans CUP-5 (SEQ ID NO: 595).
  • Figure 47 shows an alignment of the conserved serine lipase active site between mucolipin-like polypeptide (SEQ ID NO: 596) and mucolipin 1 (SEQ ID NO: 597), as well as other lipolytic enzymes: H. liph triacylglycerol lipase, hepatic precursor (SEQ ID NO: 598), H. liph lipoprotein lipase precursor (SEQ ID NO: 599), and H. lcat phosphatidylcholine-sterol acyltransferase precursor (SEQ ID NO: 600).
  • Figure 48 shows a BLASTP amino acid sequence alignment between a peroxidasin-like polypeptide (SEQ ID NO: 602) and another member of the family, human peroxidasin-like protein MG50 (SEQ ID NO: 616).
  • Figure 49 shows a multiple sequence alignment between peroxidasin-like polypeptides SEQ ID NO: 602, 618, 622, and 626.
  • Figure 50 shows a BLASTP amino acid sequence alignment between a second peroxidasin-like polypeptide (SEQ ID NO: 607) and another member of the family, human peroxidasin-like protein MG50 (SEQ ID NO: 616).
  • Figure 51 shows a BLASTP amino acid sequence alignment between a third peroxidasin-like polypeptide (SEQ ID NO: 612) and another member of the family, human peroxidasin-like protein MG50 (SEQ ID NO: 616).
  • Figure 52 shows a BLASTP amino acid sequence alignment between SAPAP- like polypeptide (SEQ ID NO: 630) and rat SAPAP3 (SEQ ED NO: 633). 4. DETAILED DESCRIPTION OF THE INVENTION
  • Table 1 is a correlation table of the novel polynucleotide sequences (1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,
  • Patent Applications bearing the serial numbers of: 10/005,499 filed on December 03, 2001, 60/341,362 filed on December 17, 2001, 60/379,875 filed on May 10, 2002, 60/379,834 filed May 10, 2002, 60/384,450 filed on May 31, 2002, 60/384,665 filed on May 31, 2002, 60/389,715 filed on June 17, 2002, 60/393,722 filed on July 02, 2002, 60/390,531 filed on June 21, 2002, and 60/391,326 filed on June 24, 2002.
  • HYS-46_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-46, U.S. Serial No. 10/005,499 filed 12/03/2001, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-47_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-47, U.S. Serial No. 60/341,362 filed 12/17/2001, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-52 XXX SEQ ID NO: XXX of Attorney Docket No. HYS-52, U.S. Serial No.
  • HYS-54 XXX SEQ ID NO: XXX of Attorney Docket No. HYS-54, U.S. Serial No. 60/379,834 filed 05/10/2002, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-55_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-55, U.S. Serial No. 60/384,450 filed 05/31/2002, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-57_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-57, U.S. Serial No. 60/384,665 filed 05/31/02, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-58_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-58, U.S. Serial No. 60/389,715 filed 06/17/2002, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-60_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-60, U.S. Serial No.
  • HYS-61_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-61, U.S. Serial No. 60/390,531 filed 06/21/2002, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • HYS-62_XXX SEQ ID NO: XXX of Attorney Docket No. HYS-62, U.S. Serial No. 60/391,326 filed 06/24/2002, the entire disclosure of which, including sequence listing, is incorporated herein by reference.
  • Adipose tissue primarily serves as an energy reservoir by storing fat and is involved in regulating available energy to the body.
  • adipocytes synthesize and secrete many important proteins, including leptin, adipsin, complement components such as C3a and properdin, tumor necrosis factor (TNF)- ⁇ , plasminogen-activator inhibitor type 1 (PAI-1), and resistin.
  • TNF tumor necrosis factor
  • PAI-1 plasminogen-activator inhibitor type 1
  • resistin resistin.
  • These adipocyte proteins are collectively called adipocytokines (Yamauchi et al, Nature Med. 7:941-946 (2001), herein incorporated by reference).
  • Adiponectin also known as adipocyte complement-related protein, Acrp30, gelatin- binding protein (GBP28), or APM1
  • GBP28 gelatin- binding protein
  • APM1 is such an adipocytokine that was identified by differential display cloning of preadipocytes and adipocytes in mouse cells. In humans, it was identified as an adipocyte-specific gene.
  • Adiponectin has a modular design: a cleaved amino-terminal sequence, a region without homology to known proteins, a collagen-like region, and a C-terminal complement factor ClQ-like globular domain (Fruebis et al, Proc. Natl. Acad. Sci. USA 98:2005-2010 (2001), herein incorporated by reference).
  • the globular domain forms homotrimers like TNF- ⁇ , and the collagen-like domains can further form higher order structures.
  • adiponectin was found to suppress TNF- ⁇ -induced monocyte adhesion to human aortic endothelial cells (Ouchi et al, Circulation 100:2473-2476 (1999), herein incorporated by reference). They also reported that adiponectin suppressed the increased expression of VCAM-1, ICAM-1, and E-selectin, suggesting that adiponectin may attenuate the inflammatory responses associated with atherosclerosis. More recently, authors also reported that plasma levels of adiponectin were significantly lower in patients with coronary artery disease than in age and body mass index-matched normal subjects (Ouchi et al, Circulation 102:1296-1301 (2000), herein incorporated by reference).
  • adiponectin suppressed TNF- ⁇ -induced nuclear factor Kappa B (NF- ⁇ B) activation accompanied by cAMP accumulation.
  • Adiponectin also inhibited myelomonocytic progenitor cell proliferation, at least in part due to apoptotic mechanisms in hematopoietic colony formation assays.
  • macrophages adiponectin suppressed the expression of class A macrophage scavenger receptors (MSR) and altered cholesterol metabolism.
  • MSR macrophage scavenger receptors
  • adiponectin reduced intracellular cholesteryl ester content of the macrophages (Ouchi et al, Circulation 103:1057-63 (2001), herein incorporated by reference).
  • the findings suggested that adiponectin protein suppressed the transformation of macrophages to foam cells.
  • Insulin resistance induced by high-fat diet and associated with obesity is a major risk factor for diabetes and cardiovascular diseases. It has been shown that adipocytokines play a crucial role in these processes. TNF- ⁇ overproduced in adipose tissue contributes to insulin resistance. Leptin, another adipocytokine, which contributes to the regulation of food intake and energy expenditure, also affects insulin sensitivity and may lead to hypertension. Similarly, serum adiponectin concentrations are decreased in homozygous obese (ob/ob) mice, obese humans, diabetic patients, and patients with coronary artery diseases (Hotta et al. Arterioscler. Thromb. Vase. Biol. 20:1595-1599 (2000), herein incorporated by reference).
  • gAcrp30 proteolytically generated globular domain of Acrp30
  • gAcrp30 reduced plasma fatty acid levels caused by administration of a high- fat test meal
  • adiponectin decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity (Yamauchi et al, Nature Med. 7:941-946 (2001), herein incorporated by reference).
  • Adiponectin decreased the levels of triglycerides in muscle and liver in obese mice. These effects were due to increased fatty acid combustion and energy dissipation in muscle.
  • the authors further showed that insulin resistance was completely reversed in lipoatrophic mice by administering combination of physiological doses of adiponectin and leptin, but only partially with either adiponectin or leptin alone.
  • adiponectin knock-out mice The role of adiponectin was further studied in the adiponectin knock-out (KO) mice by Matsuda et al. (J. Biol. Chem. 277:37487-37491 (2002)) and Kubota et al (J. Biol Chem. 277:25863-25866 (2002), both herein incorporated by reference).
  • the adiponectin-deficient mice in each study showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries.
  • adiponectin plays a direct role in neointimal thickening of arteries, a key feature of the restenosis phenomenon observed after balloon angioplasty.
  • adiponectin attenuated DNA synthesis induced a variety of growth factors such as PDGF, HB-EGF, bFGF and EGF and cell proliferation and migration induced by HB-EGF.
  • adiponectin attenuated HB-EGF expression stimulated by TNF ⁇ (Matsuda et al, J. Biol Chem.
  • TNF ⁇ decreased FATPl mRNA, IRS 1 -associated PI3-kinase activity and glucose uptake whereas adiponectin increased these parameters supporting the similar observations in mice (Maeda et al, Nature Med. 8:731-737, (2002), herein incorporated by reference).
  • Hotta et al have shown that plasma levels of adiponectin are decreased in Type 2 diabetes patients with coronary artery disease (CAD) complications and may cause the develoment of insulin resistance in these patients.
  • CAD coronary artery disease
  • adiponectin levels independently negatively correlated with serum triglyceridemia levels suggesting decreased adiponectin is associated with hypertriglyceridemia which is known to play a significant role in the deveopment of atherosclerosis.
  • sex differences were observed in adiponectin concentrations in the diabetic subjects without CAD with higher levels in clinically normal women as well as in diabetic women suggesting that sex hormones including estrogen, progesterone and androgen may affect plasma adiponectin levels (Hotta et al, Arterioscler . Thromb. Vase. Biol. 20:1595-1599 (2000), herein incorporated by reference).
  • adiponectin The plasma levels of adiponectin are also reduced in cardiovascular patients with end stage renal disease and the incidence of cardiovascular death is higher in renal failure patients with low plasma adiponectins compared with those with higher plasma adiponectin levels (Zoccali et al, JAm Soc Nephrol. 13:134-41 (2002), herein incorporated by reference). These data clearly show that adiponectin is involved in metabolic disorders including diabetes cardiovascular disease with and without renal complications.
  • therapeutics that increase plasma adiponectin should be useful in preventing metabolic disorders, diabetes, cardiovascular and other related disorders such as atherogenesis, hypertriglyceridemia, vascular stenosis after angioplasty.
  • the adiponectin-like polypeptides and polynucleotides of the invention may be used to treat obesity, diabetes, lipoatrophy, coronary artery diseases, atherosclerosis, and other obesity and diabetes-related cardiovascular pathologies.
  • Adiponectin-like polypeptides and polynucleotides of the invention may also be used in treatment of autoimmune diseases and inflammation, to modulate immune responses, and to treat transplant patients.
  • Adiponectin- like polypetides may also be used in the treatment of tumors such as solid tumors and leukemia.
  • SEQ ED NO: 15 (and encoding nucleotide sequence SEQ ED NO: 14), amino acid SEQ ED NO: 28 (and encoding nucleotide SEQ ED NO: 27), amino acid SEQ ED NO: 160 (and encoding nucleotide sequence 159), amino acid SEQ ED NO: 186 (and encoding nucleotide sequence SEQ ID NO: 185), amino acid SEQ ED NO: 215 (and encoding nucleotide sequence SEQ ID NO: 214), amino acid sequence SEQ ED NO: 241 (and encoding nucleotide sequence SEQ ED NO: 240), amino acid SEQ ED NO: 272 (and encoding nucleotide sequence SEQ ED NO: 271), amino acid SEQ ED NO: 302 (and encoding nucleotide sequence SEQ ED NO: 301), amino acid SEQ ED NO: 323 (and encoding nucleotide sequence SEQ ED NO: 322)
  • the first adiponectin-like polypeptide of SEQ ED NO: 5 is an approximately 800- amino acid protein with a predicted molecular mass of approximately 90-kDa unglycosylated.
  • the initial methionine starts at position 511 of SEQ ED NO: 4 and the putative stop codon begins at positions 2911 of SEQ ED NO: 4.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 5 is homologous to adiponectin.
  • adiponectin-like polypeptide of SEQ ID NO: 5 revealed its structural homology to Clq domain. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • Figure 2 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 5 and human adiponectin SEQ ID NO: 404 (Patent No.
  • JP3018186-B1 JP3018186-B1
  • A Alanine
  • C Cysteine
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ID NO: 5 was determined to have following eMATRIX domain hits.
  • the second adiponectin-like polypeptide of SEQ ID NO: 15 is an approximately 710- amino acid protein with a predicted molecular mass of approximately 80-kDa unglycosylated.
  • the initial methionine starts at position 511 of SEQ ED NO: 14 and the putative stop codon begins at positions 2641 of SEQ ED NO: 14.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol 36:290-300 (1993) and
  • Figure 3 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 15 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the two sequences share 47% similarity over 136 amino acid residues and 29% identity over the same 136 amino acid residues, wherein
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ID NO: 15 was determined to have following eMATRIX domain hits.
  • the third adiponectin-like polypeptide of SEQ ED NO: 28 is an approximately 744- amino acid protein with a predicted molecular mass of approximately 83-kDa unglycosylated.
  • the initial methionine starts at position 235 of SEQ ED NO: 27 and the putative stop codon begins at positions 2467 of SEQ ED NO: 27.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ID NO: 28 is homologous to adiponectin.
  • adiponectin-like polypeptide of SEQ ID NO: 28 revealed its structural homology to Clq, and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • Figure 5 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 28 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 28 was determined to have following eMATRIX domain hits.
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • a predicted approximately twenty seven-residue signal peptide is encoded from approximately residue 1 to residue 27 of SEQ LD NO: 28 (SEQ ED NO: 30).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • SEQ ED NO: 31 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 28.
  • the fourth adiponectin-like polypeptide of SEQ ID NO: 160 is an approximately 289-amino acid protein with a predicted molecular mass of approximately 32-kDa unglycosylated.
  • the initial methionine starts at position 80 of SEQ ED NO: 159 and the putative stop codon begins at positions 947 of SEQ ED NO: 159.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 160 is homologous to adiponectin.
  • adiponectin-like polypeptide of SEQ ID NO: 160 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • Figure 7 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 160 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 160 was determined to have following eMATRIX domain hits.
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • a predicted approximately sixteen-residue signal peptide is encoded from approximately residue 1 to residue 16 of SEQ ED NO: 160 (SEQ ED NO: 162).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • SEQ ED NO: 163 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 160.
  • the fifth adiponectin-like polypeptide of SEQ ED NO: 186 is an approximately 288- amino acid protein with a predicted molecular mass of approximately 32-kDa unglycosylated.
  • the initial methionine starts at position 18 of SEQ ED NO: 185 and the putative stop codon begins at positions 882 of SEQ ID NO: 185.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and
  • Figure 9 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 186 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the two sequences share 63% similarity over 204 amino acid residues and 50% identity over the same 204 amino acid residues, wherein
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ID NO: 186 was determined to have following eMATRIX domain hits.
  • SEQ ED NO in sequence listing, e-value, subtype, Accession number, name, position of the domain in the full-length protein, and the amino acid sequence and are shown in Table 6 below
  • the sixth adiponectin-like polypeptide of SEQ ED NO: 215 is an approximately 300- amino acid protein with a predicted molecular mass of approximately 34-kDa unglycosylated.
  • the initial methionine starts at position 18 of SEQ ED NO: 214 and the putative stop codon begins at positions 918 of SEQ ED NO: 214.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol Evol 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 215 is homologous to adiponectin.
  • adiponectin-like polypeptide of SEQ ID NO: 215 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 215 was determined to have following eMATRIX domain hits.
  • the seventh adiponectin-like polypeptide of SEQ ED NO: 241 is an approximately 314-amino acid protein with a predicted molecular mass of approximately 35-kDa unglycosylated.
  • the initial methionine starts at position 25 of SEQ ED NO: 240 and the putative stop codon begins at positions 1024 of SEQ ED NO: 240.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and
  • Figure 13 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 241 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the two sequences share 63% similarity over 202 amino acid residues and 50% identity over the same 202 amino acid residues, wherein
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 241 was determined to have following eMATRIXdomain hits.
  • the eighth adiponectin-like polypeptide of SEQ ID NO: 272 is an approximately 306-amino acid protein with a predicted molecular mass of approximately 34-kDa unglycosylated.
  • the initial methionine starts at position 25 of SEQ ED NO: 271 and the putative stop codon begins at positions 943 of SEQ ED NO: 271.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 272 is homologous to adiponectin.
  • adiponectin-like polypeptide of SEQ ID NO: 272 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • Figure 16 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 272 and adipose tissue-specific protein AdipoQ SEQ ID NO:
  • adiponectin-like polypeptide of SEQ ED NO: 272 was determined to have following eMATRIX domain hits.
  • a predicted approximately nineteen-residue signal peptide is encoded from approximately residue 1 to residue 19 of SEQ ED NO: 186, 215, 241, and 272 (SEQ LD NO: 188).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 189 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 186.
  • SEQ ID NO: 217 is the resulting peptide when the signal peptide is removed from SEQ ID NO: 215.
  • SEQ ED NO: 243 is the resulting peptide when the signal peptide is removed from SEQ ID NO: 241.
  • SEQ ED NO: 274 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 272.
  • the ninth adiponectin-like polypeptide of SEQ ED NO: 302 is an approximately 338- amino acid protein with a predicted molecular mass of approximately 38-kDa unglycosylated.
  • the initial methionine starts at position 199 of SEQ ED NO: 301 and the putative stop codon begins at positions 1213 of SEQ ED NO: 301.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and
  • Figure 18 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 302 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the two sequences share 52% similarity over 220 amino acid residues and 37% identity over the same 220 amino acid residues, wherein
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 302 was determined to have following eMATRIX domain hits.
  • the tenth adiponectin-like polypeptide of SEQ ED NO: 323 is an approximately 244- amino acid protein with a predicted molecular mass of approximately 27-kDa unglycosylated.
  • the initial methionine starts at position 161 of SEQ ED NO: 322 and the putative stop codon begins at positions 893 of SEQ ED NO: 322.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol Evol. 36:290-300 (1993) and
  • Figure 20 shows the BLASTP amino acid sequence alignment between adiponectin- like polypeptide SEQ ED NO: 323 and adiponectin SEQ ED NO: 402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the two sequences share 52% similarity over 220 amino acid residues and 37% identity over the same 220 amino acid residues, wherein
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 323 was determined to have following eMATRIX domain hits.
  • a predicted approximately nineteen-residue signal peptide is encoded from approximately residue 1 to residue 19 of SEQ ED NO: 323 (SEQ ED NO: 325). The extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst.
  • SEQ ED NO: 326 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 323.
  • the eleventh adiponectin-like polypeptide of SEQ ED NO: 348 is an approximately 513-amino acid protein with a predicted molecular mass of approximately 57-kDa unglycosylated.
  • the initial methionine starts at position 1 of SEQ ED NO: 347 and the putative stop codon begins at positions 1540 of SEQ ED NO: 347.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol Biol.
  • SEQ ED NO: 348 is homologous to adiponectin.
  • Pfam software program Nonnhammer et al, Nucleic Acids Res., 26:320-322 (1998) herein inco ⁇ orated by reference
  • adiponectin-like polypeptide of SEQ ID NO: 348 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu .
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 348 was determined to have following eMATRIX domain hits.
  • the twelfth adiponectin-like polypeptide of SEQ ID NO: 355 is an approximately
  • adiponectin-like polypeptide of SEQ ID NO: 355 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 355 was determined to have following eMATRLX domain hits.
  • the thirteenth adiponectin-like polypeptide of SEQ ED NO: 378 is an approximately 238-amino acid protein with a predicted molecular mass of approximately 27-kDa unglycosylated.
  • the initial methionine starts at position 683 of SEQ ED NO: 377 and the putative stop codon begins at positions 1391 of SEQ ED NO: 377.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol.
  • SEQ ID NO: 378 is homologous to adiponectin.
  • Pfam software program Nonnhammer et al, Nucleic Acids Res., 26:320-322 (1998) herein inco ⁇ orated by reference
  • adiponectin-like polypeptide of SEQ ID NO: 355 revealed its structural homology to Clq and collagen domains. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • adiponectin-like polypeptide of SEQ ED NO: 378 was determined to have following eMATRIX domain hits.
  • a predicted approximately fifteen-residue signal peptide is encoded from approximately residue 1 to residue 15 of SEQ ED NO: 355, or 378 (SEQ ID NO: 357).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst.
  • SEQ ED NO: 358 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 355.
  • SEQ ID NO: 380 is the resulting peptide when the signal peptide is removed from SEQ ED NO: 378.
  • the adiponectin-like polypeptides and polynucleotides of the invention may be used to treat obesity, diabetes, lipoatrophy, coronary artery diseases, atherosclerosis, and other obesity and diabetes-related cardiovascular pathologies. Adiponectin-like polypeptides and polynucleotides of the invention may also be used in treatment of autoimmune diseases and inflammation, to modulate immune responses, and to treat transplant patients.
  • signaling proteins e.g. methionine enkaphalin
  • hormones e.g. methionine enkaphalin
  • Proteinases are classified based on the central amino acid residue in the active
  • Se ⁇ ins se ⁇ ins (serine proteinase inhibitors) are a superfamily of more than 500 proteins, about 350-500 amino acids in size, that fold into a conserved structure and employ a unique suicide substrate-like inhibitory strategy (Silverman et al, J. Biol. Chem. 276:33293-33296 (2001), herein inco ⁇ orated by reference in its entirety).
  • the se ⁇ in superfamily has evolved over 500 million years with representatives found in viruses, plants, protozoa, insects, and higher vertebrates (Schich et al, J. Biol. Chem. 272:1849-1855 (1997), herein inco ⁇ orated by reference in its entirety).
  • the tertiary structures of se ⁇ ins demonstrate 3 ⁇ -sheets, ⁇ 9 ⁇ - helices, and several loops that are arranged into a metastable conformation (Askew et al, J. Biol. Chem. 276:49320-49330 (2001), herein inco ⁇ orated by reference in its entirety).
  • the mobile reactive site loop (RSL) is displayed on the surface, and serves as pseudo-substrate to bind to proteinase. Upon binding to proteinase and cleavage of the RSL loop the se ⁇ in molecule undergoes a conformational change that traps the proteinase in a covalent acyl- enzyme intermediate. Se ⁇ ins regulate serine proteinases involved in coagulation, fibrinolysis, inflammation, cell migration, and extracellular matrix remodeling.
  • a subclass of se ⁇ ins exhibits strong sequence similarity to chicken ovalbumin.
  • the se ⁇ in-like molecule of present invention which has strong homology to SERPINB12, belong to this subclass of se ⁇ ins.
  • These ov-se ⁇ ins lack both the N-terminal signal peptides and C-terminal extensions of other se ⁇ ins. They also exhibit a variable length loop between C and D helices that may harbor functional motifs.
  • the ov-se ⁇ ins are proposed to be either cytoplasmic or nucleocytoplasmic proteins. However, many of them (maspin, megsin, and SCCAs) may function extracellularly as they are released from cells under certain conditions.
  • the ov-se ⁇ ins are functional inhibitors of serine or cysteine proteinases. Many of them inhibit more than one class of proteinases. Many of the ov-se ⁇ ins are present in the same cells that secrete the proteinases and thus may have regulatory functions. They may also help protect the secreting cell from the proteinases.
  • Se ⁇ in-like polypeptides and polynucleotides of the invention may be used to treat emphysema, arthritis, blood clotting disorders, and cardiovascular disease.
  • Se ⁇ in- like polypeptides and polynucleotides of the invention may also be used in treatment of immune disorders and inflammation, to modulate immune responses, and to treat transplant patients.
  • Se ⁇ in-like polypeptides may also be useful as marker in diagnosis and prognosis of certain cancers.
  • the Se ⁇ in-like polypeptide of SEQ ID NO: 408 is an approximately 425-amino acid protein with a predicted molecular mass of approximately 48-kDa unglycosylated.
  • the initial methionine starts at position 78 of SEQ ED NO: 407 and the putative stop codon begins at positions 1353 of SEQ LD NO: 407.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol Biol.
  • SEQ ED NO: 408 is homologous to SERPINB12 and squamous cell carcinoma antigen 2 (SCCA2).
  • SCCA2 squamous cell carcinoma antigen 2
  • Figure 28 shows the BLASTP amino acid sequence alignment of the first high scoring pair (HSP) between Se ⁇ in-like polypeptide SEQ ED NO: 408 and SERPENB12 SEQ ED NO: 416 (Askew et al, J. Biol. Chem.
  • Figure 29 shows the BLASTP amino acid sequence alignment of the second high scoring pair (HSP) between Se ⁇ in-like polypeptide SEQ ED NO: 408 and SERPENB12 SEQ ED NO: 416 (Askew et al, J. Biol. Chem. 276:49320-49330 (2001), herein inco ⁇ orated by reference in its entirety), indicating that the two sequences share 100% similarity over 81 amino acid residues and 100% identity over the same 81 amino acid residues, wherein
  • HSP second high scoring pair
  • I Isoleucine
  • K Lisine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • Gaps are presented as dashes.
  • Se ⁇ in- like polypeptide of SEQ ED NO: 408 was determined to have following eMATRIX domain hits.
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine.
  • Se ⁇ ins undergo a conformational change upon binding of the proteinase substrate thereby trapping the proteinase in a covalent acyl-enzyme intermediate (Huntington et al, Nature 407:923-926 (2000), herein inco ⁇ orated by reference). Se ⁇ ins utilize this mechanism to regulate proteinase cascades involved in blood clotting, fibrinolysis, complement activation, cell motility, inflammation, and cell death (Silverman et al, J. Biol. Chem. 276:33293-33296 (2001); Carrell et al, Mol Biol Med. 6:35-42 (1989); Potempa et al, J. Biol. Chem.
  • SERPENB12 is a potent inhibitor of trypsin-like serine proteinases, including trypsin and plasmin (Askew et al, J. Biol Chem. 276:49320-49330 (2001), herein inco ⁇ orated by reference).
  • polypeptides of the invention are expected to have similar functions as se ⁇ ins, specifically the ov-se ⁇ ins such as SERPENB12, acting as an inhibitor of serine and cysteine proteinases.
  • the polypeptides, polynucleotides, antibodies, and other compositions of the invention are expected to be useful in treating the following disorders: emphysema, arthritis, blood clotting disorders and cardiovascular diseases.
  • Se ⁇ in-like polypeptides and polynucleotides of the invention may also be used in the treatment of immune disorders and inflammation, to modulate immune responses, and to treat transplant patients. Se ⁇ in-like polypeptides may also be useful as markers in diagnosis and prognosis of certain cancers.
  • NOGO-RECEPTOR-LIKE NgRHy
  • a key aspect of this process is the regulation of axon growth, which is mediated by a variety of chemotropic factors (Skaper, et al, Prog. Neurobiol. 56:593-608 (2001), herein inco ⁇ orated by reference).
  • Chemotropism which determines the direction of axonal growth, results from the concerted action of chemoattractant and chemorepellent cues (Yu and Bargmann, Nat. Neurosci. 4(Suppl.):l 169-1176 (2001), herein inco ⁇ orated by reference).
  • Nogo-A is the full-length protein of 1192 amino acids and is expressed primarily in the brain and optic nerve.
  • Nogo-B 373 amino acids, may correspond to the NI-35 fraction of myelin preparation and is located in small amounts in the optic nerve.
  • Nogo-C 199 amino acids long, is found primarily in the brain.
  • Nogo-A and -B share the same common N-terminus of 172 amino acids, while all three Nogo isoforms share a common C-terminal region which shows approximately 70% similarity to the C-terminus of the reticulon (Rtn) family of proteins (GrandPre et al, supra).
  • the C-termini contain two hydrophobic transmembrane domains separated by a 66 amino acid hydrophilic loop that protrudes from the cell surface.
  • Nogo neuronal growth inhibitory domains demonstrates that two distinct sites play a role in preventing neurite outgrowth.
  • the Nogo-A protein was shown to inhibit axonal growth in dorsal root ganglion (DRG) explants in vitro.
  • Fine mapping of Nogo-A by Chen et al, (supra) demonstrates that the amino terminal portion, known as Amino-Nogo, inhibits neurite outgrowth in culture.
  • the 66 amino acid linker of Nogo-C has inhibitory properties as well, inhibiting growth cone formation and inducing growth cone collapse in chick DRG neurons in vitro (GrandPre et al supra). Further mapping of Nogo-66 revealed that residues 33-55 of the extracellular sequence are responsible for the growth cone inhibition (GrandPre et al supra).
  • the receptor for the Nogo-66 peptide was identified by Fournier et al. by using a Nogo-66-alkaline phosphatase fusion protein (Nogo-AP) which was shown to bind with high affinity to chick DRG axons (Fournier, et al, supra).
  • the Nogo-66 receptor (NgR) is 473 amino acids, contains a signal sequence followed by eight leucine rich repeat (LRR) domains, an LRR flanking carboxy-terminal (LRRCT) domain that is cysteine-rich, a unique region, and a C-terminal glycophosphtidyl inositol (GPI) anchoring sequence (Fournier, et al, supra).
  • the NgR mRNA is primarily expressed in the brain. Cleavage of NgR from the axonal cell surface renders neurons insensitive to Nogo-66. Furthermore, neurons that do not express NgR are insensitive to Nogo-66-induced growth cone collapse. However, expression of recombinant NgR in these cells renders axonal growth cones sensitive to Nogo-66-induced collapse, indicating that NgR facilitates Nogo activity in neurons (Fournier, et al, supra).
  • the NgRHy polypeptide of SEQ ID NO: 420 is an approximately 420 amino acid transmembrane protein with a predicted molecular mass of approximately 46 kDa unglycosylated.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21:403-10 (1990), herein inco ⁇ orated by reference
  • SEQ ED NO: 420 is homologous to human NgR.
  • Figure 32 shows a schematic diagram illustrating the major structural features of the Nogo-receptor, NgR, and the Nogo-receptor homolog, NgRHy.
  • a predicted approximately 16 residue signal peptide is encoded from approximately residue 1 through residue 30 of SEQ ED NO: 420 (SEQ ED NO: 422).
  • the extracellular portion (SEQ ED NO: 439) is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (from Center for Biological Sequence Analysis, The Technical University of Denmark).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • NgRHy is expected to have five leucine-rich repeat (LRR) domains at residues 130-144 of SEQ ID NO: 420 (SEQ ED NO: 423), residues 154-168 of SEQ LD NO: 420 (SEQ ED NO: 424), residues 157-171 of SEQ ED NO: 420 (SEQ ID NO: 425), residues 178-192 of SEQ ED NO: 420 (SEQ ED NO:
  • LRR leucine-rich repeat
  • NgRHy polypeptides and polynucleotides of the invention may be used in the treatment of spinal cord injury, cranial or cerebral trauma, stroke, and demyelinating diseases.
  • an NgRHy polypeptide of the invention may manifest as modulating neural growth activity, such as stimulation of neurite outgrowth, stimulation of neural cell proliferation, regeneration of nerve and brain tissue, a soluble form of NgRHy can act as a competitive inhibitor to block NgRHy thereby stimulating axonal growth, alternatively, NgRHy can act as a decoy receptor to modulate, i.e. stimulate or inhibit, axonal growth.
  • the mechanism underlying the particular condition or pathology will dictate whether NgRHy polypeptides, binding partners thereof, or inhibitors thereof would be beneficial to the subject in need of treatment.
  • the present invention provides methods for modifying, such as inducing or inhibiting, proliferation of neural cells and for regeneration of nerve and brain tissue, which comprise administering a composition of NgRHy polypeptides, disclosed in the present invention.
  • proteins of the present invention may be used to treat central and peripheral nervous system disorders, neuropathies, and lesions, 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 disease, 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 injuries, head trauma, and cerebrovascular diseases including stroke.
  • Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • NgRHy polypeptides are used to produce antibodies that will bind to NgRHy and/or NgR, thereby inhibiting NgRHy and/or NgR activity. Inhibition of either receptor will block
  • Nogo-induced neurite growth inhibition and can be an effective therapeutic to restore axonal function after injury or disease.
  • the soluble ectodomain of NgRHy is used as a competitive inhibitor to bind to and/or block the activity of NgRHy or NgR thereby rendering cells insenstitive to Nogo protein inhibition of axonal growth.
  • NgRHy inhibits Nogo-dependent signaling by acting as a decoy receptor. Binding of Nogo proteins and or other ligands for NgR and NgRHy to ectopically expressed NgRHy can result in decreased binding of said ligands to NgR thereby reducing the effect of Nogo signaling on axonal growth.
  • Antibodies raised agains the NgRHy polypeptide or fragment thereof, can be used as a therapeutic for treatment of neurological conditions such as spinal cord injury, cranial or cerebral trauma, stroke, and demyelinating diseases.
  • Anti-NgRHy antibodies can inhibit the activity of either NgRHy or NgR by blocking access, either by sterically inhibiting binding of the ligand or by changing the conformation of the receptor such that ligand binding does not occur or that the receptor is unable to activate downstream signaling molecules even if the ligand is bound
  • Macrophages actively uptake a wide range of molecules including proteins, bacteria and viral particles, apoptotic cells and red blood cells, and low density lipoproteins (LDLs)
  • the scavenger receptors were first reported as receptors for oxidized and acetyl-LDLs. From cross-competition experiments it has become clear that macrophages and other cells express several classes of scavenger receptors. These receptors include type I and type II class A receptors, CD36 and SR-B1 class B receptors and CD68 and LOX-1 class C receptors that are distinct from the receptors for plasma LDLs.
  • Atherosclerosis begins when lipoproteins accumulate in the arterial intima and become chemically modified thus initiating local vessel wall inflammation. This brings in monocytes-derived macrophages which avidly take up the modified lipids, becoming fat-laden "foam" cells which reside in the vessel wall and exacerbate the local inflammation.
  • Class A type I and II macrophage scavenger receptors are trimeric proteins of about 220-250 kDa with an amino-terminal collagenous domain that is essential for ligand binding.
  • Type I receptors have a scavenger receptor Cysteine-rich domain (SRCR) while type II receptors do not.
  • Receptors containing the SRCR domain bind immunoglobulin domain containing proteins and may serve as adhesion receptors.
  • the collagen domains of these receptors have Gly-X-Y repeats and form a triple helical structure.
  • the modified LDL binding site resides at the carboxy terminus of the collagen domain in a stretch of basic amino acid residues.
  • the cytoplasmic domain is essential for cell surface expression and receptor endocytosis.
  • Type I and II receptors are expressed on all tissue macrophages. They are also expressed in brain in the perivascular macrophages called MATO cells, and endothelial cells of the liver, the adrenal gland and lymph nodes. Cytokines and other growth factors are known to modulate scavenger receptor expression. Type I and type II receptors bind and endocytose multiple ligands including acetyl-LDL, advanced glycation end products (AGE), and apoptotic cells. They also bind bacterial endotoxins, gram-positive bacteria and recognize lipoteichoic acid. The binding of endotoxins does not lead to endotoxin signaling and thus may be a way of getting rid of excess endotoxins.
  • Type I and type II scavenger receptors also mediate cell adhesion and may assist in developing robust immune response.
  • accumulation of the scavenged materials results in the formation of foam cells similar to that found with atherosclerosis and contributes to narrowing of the lumen of the arterioles in the cortex.
  • the scavenger receptor- like polypeptides and polynucleotides of the invention may be used in the treatment of atherosclerosis, disorders caused by the accumulation of denatured materials and cellular debris, bacterial and viral infections, inflammation, strengthening of immune response, and Alzheimer's disease.
  • the scavenger receptor-like polypeptide of SEQ ID NO: 444 is an approximately 495-amino acid protein with a predicted molecular mass of approximately 54 kDa unglycosylated.
  • Protein database searches with the BLASTX algorithm (Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21:403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ID NO: 444 is homologous to macrophage scavenger receptors.
  • Figure 34 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ED NO: 443 (i.e. SEQ ED NO: 444) scavenger receptor-like polypeptide and mouse macrophage scavenger receptor type I (SEQ ED NO: 481), indicating that the two sequences share 57% similarity over a 335 amino acid residue region of SEQ ED NO: 444 and 40% identity over the same 335 amino acid residues of SEQ ID NO: 444.
  • SEQ ED NO: 443 i.e. SEQ ED NO: 444
  • SEQ ED NO: 481 mouse macrophage scavenger receptor type I
  • SEQ ED NO: 444 was examined for domains with homology to certain peptide domains.
  • scavenger receptor-like polypeptide (SEQ ID NO: 444) is also expected to have sixteensperact receptor repeat proteins domain proteins signatures as shown in Table 19.
  • eMATRIX software package (Stanford University, Stanford, CA) (Wu et al, J. Comp. Biol.
  • SEQ ED NO: 4444 scavenger receptor-like polypeptide
  • Table 19 The domains corresponding to SEQ ED NO: 445-479 are as follows:
  • the scavenger receptor-like polypeptides and polynucleotides of the invention may be used in the treatment of atherosclerosis, disorders caused by the accumulation of denatured materials and cellular debris, bacterial and viral infections, inflammation, strengthening of the immune response, and Alzheimer's disease.
  • NEURAL IMMUNOGLOBULIN CELL ADHESION MOLECULE-LIKE NEURAL IgCAM POLYPEPTIDES
  • a key aspect of this process is the regulation of axon growth, which is mediated by a variety of chemotropic factors (Skaper, et al, Prog. Neurobiol. 56:593-608 (2001), inco ⁇ orated herein by reference).
  • Chemotropism which determines the direction of axonal growth, results from the concerted action of chemoattractant and chemorepellent cues (Yu and Bargmann, Nat. Neurosci. 4 (Suppl.):l 169-1176 (2001), inco ⁇ orated herein by reference).
  • Subfamilies of neural IgCAMs are categorized according to the number of immunoglobulin (Ig) domains and fibronectin repeats, as well as the mode of attachment to the cell surface (either a transmembrane domain or a glycophosphatidyl inositol linkage), and the presence of a catalytic cytoplasmic domain (reviewed in Crossin and Krushel, Dev. Dyn. 218:260-279
  • the fibroblast growth factor (FGF) receptor has been shown to be stimulated by interactions with neural IgCAMs via a "CAM homology domain" in the FGF receptor (Williams et al, Neuron 13:583-594 (1994); Williams et al, J. Cell Sci. 108:3523-3530 (1995), herein inco ⁇ orated by reference).
  • FGF fibroblast growth factor
  • nonreceptor tyrosine kinases such as ERK1 and ERK2 have been implicated in signaling pathways associated with neural IgCAM in neurite outgrowth (Schmid et al, J. Neurobiol. 38:542-558 (1999), herein inco ⁇ orated by reference).
  • amino acid sequence SEQ ED NO: 487 (and encoding nucleotide sequence SEQ ED NO: 486)
  • amino acid SEQ ED NO: 505 and encoding nucleotide sequence SEQ ED NO: 504
  • amino acid sequence SEQ ED NO: 516 (and encoding nucleotide sequence SEQ ED NO: 516
  • amino acid sequence SEQ ID NO: 528 (and encoding nucleotide sequence SEQ ED NO: 527), and amino acid sequence SEQ ID NO: 542 (and encoding nucleotide sequence SEQ ED NO: 541).
  • the first neural IgCAM-like polypeptide of SEQ ID NO: 487 is an approximately 1029-amino acid protein with a predicted molecular mass of approximately 113 -kDa unglycosylated.
  • the initial methionine starts at position 178 of SEQ ED NO: 486 and the putative stop codon begins at position 3262 of SEQ ED NO: 486.
  • a signal peptide of 18 residues is predicted from approximately residue 1 to residue 18 of SEQ ID NO: 487 (i.e. SEQ ED NO: 489).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 487 is predicted to have a transmembrane domain at approximately residue 904 to residue 920. Removal of the transmembrane domain renders soluble fragments that can be used to inhibit receptor activity.
  • An exemplary extracellular domain spans approximately residue 19 to residue 903 of SEQ ED NO: 487 (i.e. SEQ ED NO: 501).
  • SEQ ED NO: 487 is homologous to murine PANG, a neuronal CAM (SEQ ED NO: 502).
  • the second neural IgCAM-like polypeptide of SEQ ED NO: 505 is an approximately 231 -amino acid protein with a predicted molecular mass of approximately 25-kDa unglycosylated.
  • the initial methionine starts at position 17 of SEQ ED NO: 504 and the putative stop codon begins at position 707 of SEQ ED NO: 504.
  • a signal peptide of 20 residues is predicted from approximately residue 1 to residue 20 of SEQ ID NO: 505 (i.e. SEQ ID NO: 507).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 505 is predicted to have a transmembrane domain at approximately residue 213 to residue 230. Removal of the transmembrane domain renders soluble fragments that can be used to inhibit receptor activity.
  • An exemplary extracellular domain spans approximately residue 21 to residue 212 of SEQ ED NO: 505 (i.e. SEQ ID NO: 512).
  • Figure 36 shows the BLASTP amino acid sequence alignment between the protein derived from SEQ ED NO: 504 (i.e. SEQ ED NO: 505) and bovine NCAM-140 precursor amino acids 343-528 of SEQ ED NO: 513, indicating that the two sequences share 45% similarity over 191 amino acid residues of SEQ ID NO: 505 and 29% identity over the same
  • the third neural IgCAM-like polypeptide is a variant of SEQ ID NO: 504.
  • SEQ ID NO: 541 contains a 10 bp insertion between nucleotides 701 and 702 of
  • the neural IgCAM-like polypeptide of SEQ ED NO: 541 (i.e. SEQ ED NO: 542) is an approximately 256 amino acid protein with a prediceted molecular mass of approximately 28 kDa unglycosylated.
  • the initial methionine starts at position 17 of SEQ ED NO: 541 and the putative stop codon begins at position 788 of SEQ ED NO: 541.
  • a signal peptide of 20 residues is predicted from approximately residue 1 to residue 20 of SEQ
  • ED NO: 542 i.e. SEQ ED NO: 507.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 542 is predicted to have a transmembrane domain at approximately residue 217 to residue 236 (i.e. SEQ ED NO: 545). Removal of the transmembrane domain renders soluble fragments that can be used to inhibit receptor activity.
  • An exemplary extracellular domain spans approximately 21 to residue 216 of SEQ ID NO: 542 (i.e. SEQ LD NO: 546).
  • SEQ ED NO: 542 is homologous to bovine NCAM- 140 precursor (SEQ ID NO: 513).
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • Gaps are represented as dashes (-), asterisks (*) represent identical amino acids, colons (:) represent conservative substitutions, and periods (.) represent semi- conservative substitutions.
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • the fourth neural IgCAM-like polypeptide of SEQ ED NO: 516 is an approximately 674-amino acid protein with a predicted molecular mass of approximately 74-kDa unglycosylated.
  • the initial methionine starts at position 1 of SEQ ED NO: 516 and the putative stop codon begins at position 2000 of SEQ ED NO: 515.
  • a signal peptide of 32 residues is predicted from approximately residue 1 to residue 32 of SEQ ID NO: 516 (i.e. SEQ LD NO: 518).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J.
  • DDM36 (SEQ LD NO: 525).
  • the fifth neural IgCAM-like polypeptide of SEQ ID NO: 528 is an approximately 1045-amino acid protein with a predicted molecular mass of approximately 115-kDa unglycosylated.
  • the initial methionine starts at position 117 of SEQ ID NO: 527 and the putative stop codon begins at position 3249 of SEQ ED NO: 527.
  • a signal peptide of 18 residues is predicted from approximately residue 1 to residue 18 of SEQ ID NO: 528 (i.e. SEQ ED NO: 530).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ID NO: 528 is predicted to have a transmembrane domain at approximately residue 1023 to residue 1040. Removal of the transmembrane domain renders soluble fragments that can be used to inhibit receptor activity.
  • An exemplary extracellular domain spans approximately residue 19 to residue 1022 of SEQ ID NO: 528 (i.e. SEQ ID NO: 539).
  • Neural IgCAMs such as BIG-2, PANG, and NCAM-140 mediate the formation, maintenance, and plasticity of functional neuronal networks (Yoshihara, et al, J. Neurobiol, 28:51-69 (1995), herein inco ⁇ orated by reference). These neural IgCAMs facilitate neurite extension promoting axon growth and guidance (Connelly, et al, Proc. Natl. Acad. Sci.
  • Neural IgCAMs mediate interactions with the extracellular environment by binding to extracellular matrix proteins, such as NCAM-140 binding to heparan sulfate proteoglycans (Prag, et al, J. Cell. Sci., 115:283-292 (2002), herein inco ⁇ orated by reference).
  • Neural IgCAMs are found predominantly on neural cells, but are also found on muscle cells, NK cells, T cells, and transiently expressed on a variety of cells during embryogenesis.
  • PANG is a neural glycoprotein that is found primarily in neuronal cells, but is also ectopically expressed on plasmacytoma cells indicating that it may play a role in tumor metastasis as well as in axon guidance (Connelly, et al, 2001. supra).
  • the polypeptides of the invention are expected to have similar activities as those listed above, and therefore would be involved in neural development, specifically neurite outgrowth, neural cell proliferation, as well as in learning, behavior, and memory.
  • polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to provide potential treatments for disorders involving, but not limited to cognition, memory and learning, mood, dementia (including without limitation
  • Alzheimer's disease dementia associated with Parkinson's disease, multi-infarct dementia and others
  • depression anxiety (including without limitation manic-depressive illness, obsessive-compulsive disorders, generalized anxiety and others), different forms of epilepsy, schizophrenia and schizophrenaform disorders (including without limitation schizoaffecto disorder), cerebral palsy and hypertension (see, e.g. U.S. Patent No. 5,861,283, inco ⁇ orated herein by reference).
  • the polypeptides, polynucleotides, antibodies and other compositions of the invention may provide therapeutic compositions and methods of treatment for neurological conditions such as spinal cord injury, cranial or cerebral trauma, stroke, demyelinating diseases, and other neurodegenerative disorders including amyotrophic lateral sclerosis, progressive spinal muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and post polio syndrome, and hereditary motor sensory neuropathy (Charcot-Marie-Tooth Disease).
  • neurological conditions such as spinal cord injury, cranial or cerebral trauma, stroke, demyelinating diseases, and other neurodegenerative disorders including amyotrophic lateral sclerosis, progressive spinal muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and post polio syndrome, and hereditary motor sensory neuropathy (Charcot-Marie-Tooth Disease).
  • hGH Human growth hormone
  • somatotropin is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants of growth hormone (Nichol et al, Endocrine Reviews, 7:169 (1986), inco ⁇ orated herein by reference).
  • the hGH gene cluster is located on chromosome 17 and consists of five highly conserved genes, hGH-N, hGH-V, hCS-L, hCS-A, and hCS-B.
  • Human growth hormone-N is a 22,000-dalton hormone expressed in the somatotrope and lactosomatotrope cells of the anterior pituitary. Human growth hormone-N exhibits a multitude of biological effects, including linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic effects, among others (Chawla, Annu. Rev. Med., 34:519 (1983), inco ⁇ orated herein by reference; Edwards et al, Science, 39:769 (1988), inco ⁇ orated herein by reference; Isaksson et al, Annu. Rev. Physiol, 47:483 (1985), inco ⁇ orated herein by reference; Thomer and Vance, J. Clin.
  • the remaining four genes of the growth hormone family are expressed in the syncytiotrophoblastic layer of the mid- to late gestational placenta (Su et al, J. Biol. Chem., 275;11 (2000), inco ⁇ orated herein by reference).
  • the hGH-V gene also known as growth hormone-2, is a natural analog of hGH-N and is also potent somatogen. Like hGH-N, it binds growth hormone binding protein, increases glucose oxidation, induces refractoriness to insulin-like stimulation and lipolysis in the presence of glucocorticoids.
  • hGH The biological effects of hGH derive from the interaction between hGH and specific cellular receptors. These interactions activate signaling pathways which contribute to growth hormone-induced changes in enzymatic activity, transport function, and gene expression that ultimately culminate in changes in growth and metabolism (Carter-Su et al, Annu. Rev. Physiol, 5:187 (1996), inco ⁇ orated herein by reference).
  • amino acid sequence SEQ ED NO: 548 (and encoding nucleotide sequence SEQ ED NO: 549) and amino acid sequence SEQ ED NO: 557 (and encoding nucleotide sequence SEQ ED NO: 556).
  • the growth hormone-like polypeptide of SEQ ID NO: 548 is an approximately 173-amino acid protein with a predicted molecular mass of approximately 19 kDa unglycosylated.
  • the initial methionine starts at position 58 of SEQ ED NO: 547 and the putative stop codon begins at position 577 of SEQ ED NO: 547.
  • a signal peptide of twenty- six residues is predicted from approximately residue 1 to residue 26 of SEQ ED NO: 548.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl .l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program. Protein database searches with the BLASTP algorithm (Altschul S.F. et al, J. Mol.
  • SEQ ED NO: 548 is homologous to somatotropin/prolactin hormones.
  • SEQ ED NO: 548 was examined for domains with homology to known conserved peptide domains.
  • Table 25 shows the SEQ ED NO: of the Pfam domain, the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain within SEQ ED NO: 548 for the identified model within the sequence as follows:
  • the growth hormone-like polypeptide of SEQ ID NO: 548 was determined to have following the eMATRIX domain hits.
  • the second growth hormone-like polypeptide of SEQ ID NO: 557 is an approximately 256-amino acid protein with a predicted molecular mass of approximately 28 kDa unglycosylated.
  • the initial methionine starts at position 58 of SEQ ID NO: 556 and the putative stop codon begins at position 826 of SEQ ED NO: 556.
  • a signal peptide of twenty- six residues is predicted from approximately residue 1 to residue 26 of SEQ ED NO: 557.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst.
  • Protein database searches with the BLASTP algorithm (Altschul S.F. et al, J Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21:403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 557 is homologous to somatotropin/prolactin hormones.
  • Gaps are presented as dashes.
  • SEQ ED NO: 557 was examined for domains with homology to known conserved peptide domains.
  • Table 27 shows the SEQ ED NO: of the Pfam domain, the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain within SEQ ED NO: 558 for the identified model within the sequence as follows:
  • the growth hormone-like polypeptide of SEQ ED NO: 557 was determined to have following the eMATRIX domain hits.
  • the growth hormone-like polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to be useful in treating disorders where the growth of limbs and internal organs are effected, such as dwarfism, giantism, and acromegaly.
  • Growth hormone-like polypeptides, polynucleotides, antibodies and other compositions of the invention may be used to treat metabolic disorders, including diabetes and obesity.
  • Growth hormone-like polypeptides, polynucleotides, antibodies and other compositions of the invention may be used to treat inflammation, autoimmune diseases, and to modulate immune response.
  • Lipocalins are a diverse family of proteins that are typically small (160-180 residues in length), extracellular proteins that bind small lipophilic molecules (such as retinol), cell surface receptors, and form covalent and non-covalent complexes with other soluble macromolecules (reviewed in Flower et al, Biochim. Biophys. Ada 1482:9-24 (2000), herein inco ⁇ orated by reference). Proteins in the lipocalin family share a characteristic conserved lipocalin sequence motif as well as a common three-dimensional structure forming a /3-barrel. Lipocalins have been shown to be overexpressed in a variety of diseases including cancer and inflammatory diseases.
  • Neutrophil gelatinase associated lipocalin a constituent of neutrophils granules, is a member of the lipocalin family.
  • NGAL is highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease (Goetz et al, Biochemistry 39:1935- 1941 (2000), herein inco ⁇ orated by reference).
  • NGAL is proposed to mediate inflammatory responses by sequestering neutrophils chemoattractants, particularly N-formylated tripeptides as well as leukotriene B4 and platelet activating factor.
  • Lipocalins are mainly extracellular carriers of lipophilic molecules, although exceptions with properties like prostaglandin synthesis and protease inhibition are observed for specific lipocalins. Study of lipocalins in cancer has so far been focused on the variations in concentration and the modification of their expression in distinct cancer forms. In addition, lipocalins have been assigned a role in cell regulation. Lipocalins have also been used extensively as biochemical markers of disease (see Xu and Venge, Biochim. Biophys. Acta 1482:298-307 (2000), herein inco ⁇ orated by reference). The clinical indications relate to almost any field of medicine, such as inflammatory disease, cancer, lipid disorders, liver and kidney function.
  • NGALHy 1 and NGALHy2 Two exemplary NGAL-like sequences of the invention (NGALHy 1 and NGALHy2) are described below: amino acid sequence SEQ ID NO: 572 (and encoding nucleotide sequence SEQ ED NO: 571), and amino acid SEQ ED NO: 579 (and encoding nucleotide sequence SEQ ED NO: 578).
  • the NGALHyl polypeptide of SEQ ED NO: 572 is an approximately 157-amino acid protein with a predicted molecular mass of approximately 17-kDa unglycosylated.
  • the initial methionine starts at position 192 of SEQ ID NO: 571 and the putative stop codon begins at position 660 of SEQ ED NO: 571.
  • a signal peptide of 19 residues is predicted from approximately residue 1 to residue 19 of SEQ ED NO: 572.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 572 is homologous to mouse lipocalin (SEQ ID NO: 585) and human NGAL precursor (SEQ ED NO: 586).
  • Y Tyrosine and asterisks (*) indicate identical residues, colons (:) indicate conserved substitutions, and periods (.) indicate distant substitutions.
  • NGALHyl polypeptide of SEQ ED NO: 572 was determined to have following the eMATRIX domain hits.
  • the NGALHy2 polypeptide of SEQ ED NO: 579 is an approximately 200-amino acid protein with a predicted molecular mass of approximately 22-kDa unglycosylated.
  • the initial methionine starts at position 128 of SEQ ED NO: 578 and the putative stop codon begins at position 725 of SEQ ED NO: 578.
  • a signal peptide of 19 residues is predicted from approximately residue 1 to residue 19 of SEQ ED NO: 579.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 579 is homologous to mouse lipocalin (SEQ LD NO: 585) and human NGAL precursor (SEQ ED NO: 586).
  • Y Tyrosine. Gaps are presented as dashes and asterisks (*) represent identical residues, colons (:) represent conservative substitutions, periods (.) represent semi-conservative substitutions.
  • NGALHy2 polypeptide of SEQ ED NO: 579 was determined to have following the eMATRIX domain hits.
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V V aline
  • W Tryptophan
  • Y Tyrosine.
  • NGAL forms a heterodimeric complex with matrix metalloproteinase 9 (MMP9) which protects MMP9 from degradation and allows MMP9 to degrade the extracellular matrix thereby enhancing tumor cell metastasis (Yan et al, J. Biol. Chem. 276:37258-37265
  • MMP9/NGAL complex is induced in several cancers and is used as a marker for metastatic cancer.
  • NGAL also modulates the immune response during the acute phase response during inflammation to enhance non-specific host defenses by binding to and neutralizing pro-infectious bacterial products, such as the chemoattractant N-formyl-Met-Leu-Phe (Goetz et al, 2000. supra; Logdberg and Wester,
  • Circulating NGAL levels are used as a marker for inflammatory conditions, such as cystic fibrosis and acute peritonitis, and are capable of distinguishing between bacterial and viral acute infections.
  • NGAL and lipocalins in general, also play a role in cell regulation, cell differentiation, and cell proliferation.
  • polypeptides of the invention are expected to have similar functions as NGAL as a marker for diseases including cancer and inflammatory diseases, interacting with matrix metalloproteases to modulate cell proliferation, modulation of inflammation by enhancing non-specific host defenses, via activities such as binding to bacterial pro-inflammatory proteins.
  • polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to be useful in treating the following disorders: inflammatory diseases, including bacterial and viral infections, acute peritonitis, cystic fibrosis, asthma, chronic obstructive pulmonary disease, pulmonary emphysema, Sjogren's syndrome, rheumatoid arthritis; neoplastic colorectal disease, colitis, and other disorders in which the barrier of the colorectal mucosa is disrupted; wound healing; cancer, including breast, colorectal, pancreatic, prostate, bladder, renal cancers, colorectal and hepatic tumors, adenocarcinomas, including lung, colon, pancreas; lipid disorders, and modulating liver and kidney function.
  • inflammatory diseases including bacterial and viral infections, acute peritonitis, cystic fibrosis, asthma, chronic obstructive pulmonary disease, pulmonary emphysema, Sjogren's
  • Mucolipidosis TV is an autosomal recessive neurodegenerative lysosomal storage disorder characterized clinically by psychomotor retardation and ophthalmologic abnormalities including corneal opacitiy, retinal degeneration, and strabismus. Maximal development of the patient is between 12 and 15 months and age of the patients with this disease ranges from 1 to 40 years. Life expectancy of the patients is not known. Over 80% of the patients diagnosed with MLIV showing severe or mild symptoms are the Ashknazi Jews. The patients excrete chondroitin sulphate in their urine.
  • the disease is characterized by massive engorgement of superficial and intermediate epithelial cells of both the cornea and conjunctiva with fine granular material consistent with mucopolysaccharide and concentric lamellar bodies.
  • the storage materials have been identified as sphingolipids, phospholipids and acid mucopolysaccharides. In this disease, excessive storage of these materials is also observed in macrophages, plasma cells, ciliary epithelial cells, Schwann cells, retinal ganglion cells and vascular endothelial cells.
  • MLEV is not associated with a lack of lysosomal hydrolases. Instead the MLIV cells display abnormal endocytosis of lipids and accumulate large vesicles indicating that a defect in endocytosis may underlie the disease as shown by Chen, et al. (Chen, et al, Proc. Natl. Acad. Sci. USA. 98:6373-6378 (1998), herein inco ⁇ orated by reference). Bassi, et al (Bassi, et al, Human Genet.
  • mucolipin 1 plays an important role in endocytosis, a fact that has been borne out by the studies of Fares and Greenwald using C. elegans as an animal model (Fares and Greenwald. Nature Genet. 28:64-68, (2001), herein inco ⁇ orated by reference). They showed that a loss-of- function mutation in the C. elegans mucolipinl homolog, Cup-5 results in increased rate of uptake of fluid-phase markers, decreased degradation of the endocytosed protein and and accumulation of large vacuoles. Overexpression of cup-5 causes the opposite phenotype and rescue with human mucolipinl results in normalizing the endocytosis.
  • Cup-5 is also essential for the viability and regulates the lysosomes in multiple cell types in C. elegans (Hersh et al. Proc Natl Acad Sci USA. 99:4355-4360, (2002), herein inco ⁇ orated by reference).
  • the metabolic defect causing this accumulation has recently been identified as dysfunctional endocytosis and the gene responsible had been named mucolipinl (Bargal, et al, Nature Genet. 26:20-123, (2000), Bassi, et al, Human Genet. 67:1110-1120, (2000), Sun, et al Hum. Molec. Genet. 9:2471-2478, (2000), all of which are herein inco ⁇ orated by reference) and it is a transcript of the gene MCOLN1 shown to be located on chromosome
  • the MLEV gene consists of 14 exons spanning approximately 14 kb of genomic DNA and encoding a protein of 580 amino acid in length (Bargal, et al. Nature Genet. 26: 120-123, (2000), herein inco ⁇ orated by reference).
  • the mucolipin protein appears to contain one transmembrane helix in the N-terminal region and at least 5 transmembrane domains ion the C-terminal half of the protein. This protein localizes on the plasma membrane and in the C-terminal region shows homology to polycistin-2, the product of the polycystic kidnay disease (PKD2) gene (Bassi, et al, Human Genet.
  • the gene also belongs to a family of transient receptor potential calcium ion channels (Sun, et al, Hum. Molec. Genet. 9:2471-2478, (2000), herein inco ⁇ orated by reference) and may play a role in calcium ion transport.
  • mucolipinl also known as mucolipidin
  • mucolipidin a C. elegans cup-5 protein homologous to the mucolipinl
  • studies on mucolipinl and cup-5 have shown the impact these proteins can have on cell viability, normal cellular transport, lysosomal storage and resulting in mental retardation, ophthalmic abnormalities such as corneal opacity, retinal degeneration and strabismus, there clearly exists a need for identifying further members of this family of proteins. Identification of such proteins and their methods of use to modulate cellular lysosomal transport provide therapeutic compositions and methods of treatments for the above-mentioned conditions.
  • the mucolipin-like polypeptide of SEQ ID NO: 588 is an approximately 542-amino acid protein with a predicted molecular mass of approximately 59.6-kDa unglycosylated.
  • the initial methionine starts at position 1 of SEQ ID NO: 587 and the putative stop codon begins at position 1629 of SEQ ED NO: 587:
  • Gaps are presented as dashes.
  • the mucolipin-like polypetide is not predicted to have a secretion signal peptide.
  • the absence of signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997), herein inco ⁇ orated by reference).
  • the transmembrane regions of the polypeptide were determined.
  • the TMpred program makes a prediction of membrane-spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occuring transmembrane proteins. The prediction is made using a combination of several weight-matrices for scoring. (K.
  • Protein database searches with the BLASTP algorithm (Altschul, et al, J. Mol. Evol. 36:290-300 (1993); Altschul et al, J. Mol Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 588 is best homologous to mouse mucolipin 2.
  • a multiple sequence alignment of SEQ ID NO: 588 with other homologous sequences showing conserved regions is shown in Figure 46.
  • Figure 46 shows a multiple sequence alignment between mucolipin-like polypeptide (SEQ ED NO: 588) and other members of the family: mouse mucolipin 2 (SEQ ED NO:
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine. Gaps are presented as dashes.
  • mucolipin-like polypeptide of SEQ ED NO: 588 revealed its sequence homology to calcium ion transport pfam domain. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • Mucolipin-like polypeptide contains a conserved serine lipase site spanning amino acid residues 74 to 90 of SEQ ED NO: 588 that is found in mucolipin 1 and other lipolytic enzymes.
  • Figure 47 shows an alignment of the conserved serine lipase active site between mucolipin-like polypeptide (SEQ ED NO: 596) and mucolipin 1 (SEQ ED NO: 597) as well as other lipolytic enzymes: H. liph. triacylglycerol lipase hepatic precursor (SEQ ED NO:
  • H. liph. lipoprotein lipase precursor SEQ ID NO: 599
  • H lcat. phosphatidylcholine-sterol acyltransferase precursor SEQ ID NO: 600.
  • Homologous family members SEQ ED NO: 592 and 595 have the following activities: endocytosis, calcium ion transport, apoptosis induction and lipolysis through a conserved serine lipase domain.
  • the polypeptides of the invention are expected to have the following activities: based on homology and analysis of predicted pfam domains, the mucolipin-like polypeptide is expected to function as not only a calcium ion transport molecule but also as a serine lipase and play a role in apoptosis induction, endocytosis and lipid metabolism.
  • polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to be useful in treating the following disorders: cholesterol storage diseases such as MLEV, cardiovascular, ophthalmic and neurologic diseases as well as diseases associated with apoptosis such as follicular lymphoma, autoimmune diseases and retinal degeneration.
  • Peroxidasin was first identified and characterized in Drosophila as a novel enzyme- matrix protein based on its hybrid structure which combines an enzymatically active peroxidase motif with domains that usually occur as parts of interacting extracellular proteins (e.g. cell adhesion molecules) (Nelson et al, The EMBO Journal; 13:3438- 3447(1994), inco ⁇ orated herein by reference).
  • Peroxidasin is a 1535 amino acid protein, wherein the amino acid sequence of the peroxidase domain is quite similar to the vertebrate peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO). MPO, EPO, and LPO play key roles in human oxidative defense (Everse et al, Peroxidases in Chemistry and Biology; (1990), inco ⁇ orated herein by reference). Since the expression of peroxidasin is accompanied by phagocytosis in the Drosophila embryo, peroxidasin may also function in phagocytosis.
  • MPO myeloperoxidase
  • EPO eosinophil peroxidase
  • LPO lactoperoxidase
  • TPO thyroid peroxidase
  • peroxidasin In addition to its peroxidase domain, peroxidasin possesses six leucine rich repeats (LRR) and four immunoglobulin (Ig) repeats. LRR's and Ig loops are involved in protein-protein interactions and indicate a role for peroxidasin in extracellular matrix consolidation and cell adhesion (Nelson et al, The EMBO Journal; 13:3438-3447(1994), inco ⁇ orated herein by reference). Overexpression of p53, a tumor suppressor protein whose inactivation has been observed in a large number of human cancers, leads to either programmed cell death (apoptosis) or growth arrest.
  • LRR leucine rich repeats
  • Ig immunoglobulin
  • a human homologue of Drosophila peroxidasin was shown to be differentially expressed in a human colon cancer cell line undergoing p53-dependent apoptosis (Horikoshi et al, Biochem. Biophys. Res. Commun.; 261:864-869(1999), inco ⁇ orated herein by reference).
  • MG50 novel melanoma gene
  • mitchell et al, Cancer Research; 60:6448-6456(2000), inco ⁇ orated herein by reference There is evidence that suggests MG50 is relatively restricted to tumors such as melanoma, breast cancer, ovarian cancer, and glioblastoma.
  • MG50 appears to be absent from archived specimens of normal tissues, with the exception of skin (Mitchell et al, Cancer Research; 60:6448-6456(2000), inco ⁇ orated herein by reference). Since MG50 seems to be relatively tumor associated, it was hypothesized that MG50 could be a potentially useful immunogen and target for immunotherapy. There exists a need for identifying further members of this family of proteins.
  • amino acid sequence SEQ ED NO: 602 (and encoding nucleotide sequence SEQ ED NO: 601), amino acid sequence SEQ ED NO: 618 (and encoding nucleotide sequence SEQ ED NO: 617), amino acid sequence SEQ ED NO: 622 (and encoding nucleotide sequence SEQ ED NO: 621), amino acid sequence SEQ ED NO: 626 (and encoding nucleotide sequence SEQ ED NO: 625), amino acid sequence SEQ ED NO: 607 (and encoding nucleotide sequence SEQ ED NO: 606), amino acid sequence SEQ ID NO: 612 (and encoding nucleotide sequence SEQ ED NO: 611 ).
  • the peroxidasin-like polypeptide of SEQ ED NO: 603 is an approximately 1507- amino acid protein with a predicted molecular mass of approximately 166 kDa unglycosylated.
  • the initial methionine starts at position 261 of SEQ ED NO: 601 and the putative stop codon begins at position 4782 of SEQ ED NO: 601.
  • a signal peptide of twenty three residues is predicted from approximately residue 1 to residue 23 of
  • SEQ ED NO: 602. The extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program
  • SEQ ED NO: 602 is predicted to have transmembrane domains at approximately residue 5 to residue 26, residue 505 to residue 518, residue 593 to residue 608, and residue 1086 to residue 1104. Removal of one or more transmembrane domains renders fragments that can be useful on their own.
  • One example is a fragment from residue 24 to residue 504 of SEQ ED NO: 602.
  • One of skill in the art will recognize that the actual transmembrane domains may be different than that predicted by the computer program.
  • Protein database searches with the BLASTP algorithm (Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 602 is homologous to peroxidasin-like proteins.
  • SEQ ED NO: 602 was examined for domains with homology to known conserved peptide domains.
  • Table 31 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ID NO: 602 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ED NO: 602 was determined to have following the eMATRIX domain hits.
  • the results in Table 32 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 603:
  • a first variant of SEQ ED NO: 602 is SEQ ED NO: 618.
  • the variant is an approximately 1538 amino acid protein with a predicted molecular mass of approximately 169 kDa unglycosylated.
  • the initial methionine starts at position 12 of SEQ ED NO: 617, and the putative stop codon begins at position 4626 of SEQ ED NO: 617.
  • SEQ ED NO: 618 differs from SEQ ED NO: 602 at the N-terminus where it contains an additional 31 amino acids.
  • the remainder of SEQ ID NO: 618 is identical to SEQ ED NO: 602. Therefore, SEQ ED NO: 618 comprises SEQ ED NO: 602.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 618 is predicted to have a transmembrane domain at approximately residue 525 to residue 550. Removal of the transmembrane domain renders fragments that can be useful on their own.
  • the actual transmembrane domain may be different than that predicted by the computer program.
  • SEQ ID NO: 618 was examined for domains with homology to known conserved peptide domains.
  • Table 33 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ED NO: 618 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ED NO: 618 was determined to have following the eMATRIX domain hits.
  • the results in Table 34 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 618:
  • a second variant of SEQ LD NO: 602 is SEQ ED NO: 622.
  • the splice site occurs after nucleotide 329 of SEQ ID NO: 601.
  • the variant is an approximately 1400 amino acid protein with a predicted molecular mass of approximately 154 kDa unglycosylated.
  • the initial methionine starts at position 103 of SEQ ED NO: 621, and the putative stop codon begins at position 4303 of SEQ ED NO: 621.
  • a signal peptide of 23 residues is predicted from approximately residue 1 to residue 23 of SEQ ID NO: 622.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 622 was examined for domains with homology to known conserved peptide domains.
  • Table 35 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ED NO: 622 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ID NO: 622 was determined to have following the eMATRIX domain hits.
  • the results in Table 36 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 622:
  • a third variant of SEQ ID NO: 602 is SEQ ED NO: 626.
  • the splice site occurs after nucleotide 329 of SEQ ID NO: 601.
  • the variant is an approximately 1439 amino acid protein with a predicted molecular mass of approximately 158 kDa unglycosylated.
  • the initial methionine starts at position 261 of SEQ ED NO: 625, and the putative stop codon begins at position 4578 of SEQ ED NO: 625.
  • a signal peptide of 23 residues is predicted from approximately residue 1 to residue 23 of SEQ ED NO: 626.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581 -599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581 -599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 626 was examined for domains with homology to known conserved peptide domains.
  • Table 37 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ED NO: 626 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ED NO: 626 was determined to have following the eMATRIX domain hits.
  • the results in Table 38 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 626:
  • Protein database searches with the BLASTP algorithm (Altschul S.F. et al, J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21:403-10 (1990), herein inco ⁇ orated by reference) indicate that the variant sequences SEQ ED NO: 619, 623 and 627 are homologous to the human peroxidasin-like protein (accession number B AA13219.1) that is also known as the melanoma-associated antigen MG50 (Accession number AF200349_1) (SEQ ED NO: 617).
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • W Tryptophan
  • Y Tyrosine.
  • Gaps are presented as dashes, asterisks (*) represent identical residues, colons (:) represent conservative substitutions, and periods (.) represent semi- conservative substitutions.
  • the peroxidasin-like polypeptide of SEQ ED NO: 607 is an approximately 1463- amino acid protein with a predicted molecular mass of approximately 161 kDa unglycosylated.
  • the initial methionine starts at position 145 of SEQ ED NO: 606 and the putative stop codon begins at position 4534 of SEQ ED NO: 606.
  • a signal peptide of twenty three residues (SEQ ED NO: 609) is predicted from approximately residue 1 to residue 23 of SEQ ED NO: 607.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J.
  • SEQ ID NO: 607 is predicted to have transmembrane domains at approximately residue 6 to residue 20, residue 585 to residue 600, and residue 1042 to residue 1060. Removal of one or more transmembrane domains renders fragments that can be useful on their own. One example is a fragment from residue 24 to residue 584 of SEQ ED NO: 607.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al, J. Mol.
  • SEQ ID NO: 608 is homologous to peroxidasin-like proteins.
  • Y Tyrosine. Gaps are presented as dashes.
  • SEQ ED NO: 607 was examined for domains with homology to known conserved peptide domains.
  • Table 39 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ED NO: 607 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ED NO: 607 was determined to have following the eMATRIX domain hits.
  • the results in Table 40 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 607:
  • the peroxidasin-like polypeptide of SEQ ED NO: 612 is an approximately 1439- amino acid protein with a predicted molecular mass of approximately 158 kDa unglycosylated.
  • the initial methionine starts at position 145 of SEQ ED NO: 611 and the putative stop codon begins at position 4462 of SEQ ED NO: 611.
  • a signal peptide of twenty-three residues (SEQ ED NO: 614) is predicted from approximately residue 1 to residue 23 of SEQ ED NO: 612.
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • Neural Network SignalP Vl.l program Neural Network SignalP Vl.l program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)).
  • the actual cleavage site may be different than that predicted by the computer program.
  • SEQ ED NO: 612 is predicted to have transmembrane domains at approximately residue 6 to residue 20, residue 561 to residue 576, and residue 1018 to residue 1036. Removal of one or more transmembrane domains renders fragments that can be useful on their own. One example is a fragment from residue 24 to residue 560 of SEQ ED NO: 612.
  • One of skill in the art will recognize that the actual transmembrane domains may be different than that predicted by the computer program.
  • Protein database searches with the BLASTP algorithm (Altschul S.F. et al, J. Mol Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403-10 (1990), herein inco ⁇ orated by reference) indicate that SEQ ED NO: 612 is homologous to peroxidasin-like proteins.
  • SEQ ED NO: 612 was examined for domains with homology to known conserved peptide domains.
  • Table 41 shows the name of the Pfam model found, the description, the e-value, Pfam score, number of repeats, and position of the domain(s) within SEQ ED NO: 612 for the identified model within the sequence as follows:
  • the peroxidasin-like polypeptide of SEQ ED NO: 612 was determined to have following the eMATRIX domain hits.
  • the results in Table 42 describe: the eMATRIX domain name, the corresponding p- value, Signature ED number, and the corresponding position of the domain within SEQ ED NO: 613:
  • Peroxidasin-like polypeptides are expected to play roles in phagocytosis and cell adhesion and possess peroxidase-like enzymatic activity. Additionally, peroxidasin-like polypeptides may serve as tumor markers and tumor-specific antigens for immunotherapy.
  • Immunotherapy provides a method of harnessing the immune system to treat various pathological states, including cancer, autoimmune disease, transplant rejection, hype ⁇ roliferative conditions, and allergic reactions.
  • Antibody therapy for cancer involves the use of antibodies, or antibody fragments, against a tumor antigen to target antigen-expressing cells.
  • Antibodies, or antibody fragments may have direct or indirect cytotoxic effects or may be conjugated or fused to cytotoxic moieties.
  • Direct effects include the induction of apoptosis, the blocking of growth factor receptors, and anti-idiotype antibody formation.
  • Indirect effects include antibody- dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cellular cytotoxicity (CMCC).
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • CMCC complement-mediated cellular cytotoxicity
  • the antibodies, or fragments thereof provide a method of targeting the cytotoxicity towards the tumor antigen expressing cells.
  • Rituximab is a chimeric antibody directed against CD20, a B cell-specific surface molecule found on >95% of B-cell non-Hodgkin's lymphoma (Press, et al, Blood 69:584-591 (1987), inco ⁇ orated herein by reference; Malony, et al, Blood
  • Rituximab induces ADCC and inhibits cell proliferation through apoptosis in malignant B cells in vitro (Maloney, et al, Blood 88:637a (1996), inco ⁇ orated herein by reference).
  • Rituximab is currently used as a therapy for advanced stage or relapsed low-grade non-Hodgkin's lymphoma, which has not responded to conventional therapy.
  • Active immunotherapy whereby the host is induced to initiate an immune response against its own tumor cells can be achieved using therapeutic vaccines.
  • tumor-specific vaccine uses purified idiotype protein isolated from tumor cells, coupled to keyhole limpet hemocyanin (KLH) and mixed with adjuvant for injection into patients with low- grade follicular lymphoma (Hsu, et al, Blood 89:3129-3135 (1997), inco ⁇ orated herein by reference).
  • KLH keyhole limpet hemocyanin
  • Another type of vaccine uses antigen-presenting cells (APCs), which present antigen to naive T cells during the recognition and effector phases of the immune response.
  • APCs antigen-presenting cells
  • Dendritic cells one type of APC, can be used in a cellular vaccine in which the dendritic cells are isolated from the patient, co-cultured with tumor antigen and then reinfused as a cellular vaccine (Hsu, et al, Nat. Med. 2:52-58 (1996), inco ⁇ orated herein by reference).
  • Immune responses can also be induced by injection of naked DNA. Plasmid DNA that expresses bicistronic mRNA encoding both the light and heavy chains of tumor idiotype proteins, such as those from B cell lymphoma, when injected into mice, are able to generate a protective, anti-tumor response (Singh, et al, Vaccine 20:1400-1411 (2002), inco ⁇ orated herein by reference).
  • peroxidasin-like polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to be useful in providing therapeutic compositions and diagnostic methods for treating and identifying cancer, hype ⁇ roliferative disorders, auto-immune diseases, and organ transplant rejection.
  • Synaptic associated protein 90/ postsynaptic density protein 95 kDa-associated proteins (SAPAPs) (Takeuchi et al, JBiol Chem. 272:11943-11951 (1997), herein inco ⁇ orated by reference), also called GKAPs (Guanylate kinase-associated proteins) (Kim et al, J Cell Biol. 136:669-678 (1997) (Naisbitt et al, J Neurosci. 17:5687-5696 (1997), both herein inco ⁇ orated by reference) or DAPs (hDLG-associated proteins) (Satoh et al, Genes Cells.
  • SAPAPs postsynaptic density protein 95 kDa-associated proteins
  • SAPAPs are associated with the postsynaptic density protein 95 kDa/synaptic associated protein 90 (PSD-95/SAP90) which belongs to the large family of synaptic membrane-associated guanylate kinases (MAGUKs). This class of proteins contains characteristic domains, which mediate protein/protein interactions, including PDZ,
  • MAGUKs SH3, and guanylate kinase domains. These domains enable the MAGUKs to build scaffolds of synaptic components that include: a) ion channels and neurotransmitter receptors via their NH2-terminal PDZ domains (for example NMDA receptors and potassium channels) (Kim et al, J Cell Biol. 136:669-678 (1997), herein inco ⁇ orated by reference); b) intracellular signaling molecules; and c) cytoskeletal proteins (Naisbitt et al, J Neurosci. 17:5687-5696
  • PSD-95 family proteins function as molecular anchors for coupling synaptic receptors and ion channels to downstream signaling molecules and cytoskeleton.
  • SAPAPs play a role in the molecular organization of synapses and neuronal cell signaling is suggested by the following observations: SAPAPs bind directly to a) the guanylate kinase domain of the postsynaptic density protein 95 (PSD-95) family, b) members of the dynein light chain family (Naisbitt et al, J Neurosci.
  • SAPAPs may orchestrate functional interactions between metabotropic and ionotropic systems. This is relevant in the context of synaptic transmission and stabilization since SAPAPs also modulate NMDA channel conductance (Yamada et al, FEBSLett.
  • SAPAPs may be involved in the molecular organization of synapses and neuronal cell signaling.
  • SAP proteins are expressed not only in the synapse, but also in epithelial cells (Fujita and Kurachi, Biochem Biophys Res Commun. 269: 1-6 (2000), herein inco ⁇ orated by reference).
  • various SAPAP proteins help SAPs perform specific functions in different tissues. Therefore, it is important to identify other members of this family of proteins.
  • the SAPAP-like polypeptide of SEQ ED NO: 630 is an approximately 979-amino acid protein with a predicted molecular mass of approximately 107.7-kDa unglycosylated.
  • the initial methionine starts at position 1 of SEQ ID NO: 629 and the putative stop codon begins at position 2938 of SEQ LD NO: 629.
  • SAPAP-like polypeptide of SEQ ED NO: 630 revealed its structural homology to Guanylate-kinase-associated protein (GKAP) corresponding to amino acids of 621-979 of the full length protein of SEQ ID NO: 630 that correspond to the Pfam domain and nucleotides of 1858-2937 the open reading frame of SEQ ED NO: 631 and is shown in Table 43. Further description of the Pfam models can be found at http://pfam.wustl.edu/.
  • SAPAP- like polypeptide of SEQ ED NO: 630 was determined to have following eMATRIX domain hits.
  • polypeptides of the invention may play a role in the formation and function of the nervous system, by regulating the molecular organization of synapses and neuronal cell signaling.
  • they could function as adapter proteins linking ion channels and other synaptic proteins to the subsynaptic cytoskeleton which is important for the localization and concentration of synaptic molecules to the postsynaptic membrane.
  • polypeptides, polynucleotides, antibodies and other compositions of the invention are expected to be useful in treating the following disorders: Alzheimer's disease, anxiety, autism, brain injury, depression, epilepsy, Huntington's disease, mania, pain, Parkinsonism, Parkinson's disease, Schizophrenia, Tardive dyskinesia, myasthenia gravis, amyotrophic lateral sclerosis, episodic ataxia/myokymia, hyperkalemix periodic paralysis, hypokalemic periodic paralysis, Lamber-Eaton syndrome, paramyotonia congenita, Rasmussen's encephalitis, Startle disease, and seizure disorders, including neonatal seizure disorders and generally, learning and memory disorders.
  • biological activity refers to those forms of the polypeptide that retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
  • biologically active refers to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.
  • biologically active or biological activity refers to the capability of the natural, recombinant or synthetic polypeptide of the invention, or any peptide thereof, to induce a specific biological response in appropriate animals or cells and to bind with specific antibodies.
  • activated cells are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.
  • complementary or “complementarity” refer to the natural binding of polynucleotides by base pairing.
  • sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'.
  • Complementarity between two single-stranded molecules may be "partial” such that only some of the nucleic acids bind or it may be "complete” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
  • Embryonic stem cells refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.
  • GSCs germ line stem cells
  • primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.
  • primordial germ cells PGCs
  • PGCs primary germ cells
  • PGCs are the source from which GSCs and ES cells are derived. The PGCs, the GSCs and the ES cells are capable of self-renewal.
  • totipotent refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
  • pluripotent refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
  • EMF expression modulating fragment
  • a sequence is said to "modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.
  • EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).
  • One class of EMFs is nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.
  • nucleotide sequence or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • A is adenine
  • C cytosine
  • G guanine
  • T thymine
  • N is A, T, G, or C.
  • nucleic acid segments may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
  • oligonucleotide fragment or a "polynucleotide fragment", “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.
  • the fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.
  • the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.
  • the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
  • a fragment or segment may uniquely identify each polynucleotide sequence of the present invention.
  • the fragment comprises a sequence substantially similar to a portion of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-
  • Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P.S. et al, PCR Methods Appl 1 :241-250 (1992)). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al, 1989, Molecular Cloning: A Laboratory
  • nucleic acid sequences of the present invention also include the sequence information from any of the nucleic acid sequences of SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29,
  • the sequence information can be a segment of SEQ LD NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183- 185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,
  • One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one
  • a segment when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.
  • the probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1 ⁇ 4 25 ) times the increased probability for mismatch at each nucleotide position (3 * 25).
  • the probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.
  • the probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
  • ORF open reading frame
  • open reading frame means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.
  • operably linked or “operably associated” refer to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.
  • operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
  • pluripotent refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.
  • a pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
  • polypeptide or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.
  • a polypeptide "fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about
  • the peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids. Preferably the peptide is from about 5 to about 200 amino acids.
  • any polypeptide must have sufficient length to display biological and/or immunological activity.
  • naturally occurring polypeptide refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • translated protein coding portion means a sequence which encodes for the full length protein which may include any leader sequence or a processing sequence.
  • mature protein coding sequence refers to a sequence which encodes a peptide or protein without any leader/signal sequence.
  • the "mature protein portion” refers to that portion of the protein without the leader/signal sequence.
  • the peptide may have the leader sequences removed during processing in the cell or the protein may have been produced synthetically or using a polynucleotide only encoding for the mature protein coding sequence. It is contemplated that the mature protein portion may or may not include an initial methionine residue. The initial methionine is often removed during processing of the peptide.
  • derivative refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
  • variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.
  • Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
  • recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
  • Various codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
  • Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.
  • conservative amino acid replacements may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.
  • the variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
  • insertions, deletions or non- conservative alterations can be engineered to produce altered polypeptides.
  • Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.
  • such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation turnover rate.
  • Such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • purified or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.
  • the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present
  • isolated refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.
  • the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other components normally present in a solution of the same.
  • isolated and purified do not encompass nucleic acids or polypeptides present in their natural source.
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • recombinant microbial defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
  • recombinant expression vehicle or vector refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.
  • An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • recombinant expression system means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.
  • Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
  • Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous
  • the cells can be prokaryotic or eukaryotic.
  • secreted includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.
  • proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.
  • proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.
  • “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P.A. and Young, P.R. Cytokine 4:134 -143 (1992)) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W.P. et. al. Annu.
  • an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
  • a signal or leader sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
  • stringent is used to refer to conditions that are commonly understood in the art as stringent.
  • Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2x SSC/0.1% SDS at 42°C).
  • highly stringent conditions i.e., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C
  • moderately stringent conditions i.e., washing in 0.2x SSC/0.1% SDS at 42°C.
  • Other exemplary hybridization conditions are described herein in the examples.
  • additional exemplary stringent hybridization conditions include washing in 6x SSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20- base oligonucleotides), and 60°C (for 23-base oligonucleotides).
  • substantially equivalent can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.
  • a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).
  • Such a sequence is said to have 65% sequence identity to the listed sequence.
  • a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).
  • Substantially equivalent, e.g. , mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 90% sequence identity.
  • nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.
  • nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, and most preferably at least about 95% identity.
  • sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.
  • sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J. Methods Enzymol. 183:626-645 (1990)). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.
  • totipotent refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
  • transformation means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.
  • transfection refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.
  • infection refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.
  • an "uptake modulating fragment,” UMF means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.
  • UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer- based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.
  • the isolated polynucleotides of the invention include, but are not limited to a polynucleotide comprising any of the nucleotide sequences of SEQ ED NO: 1-4, 6, 14, 16,
  • a polynucleotide comprising the full length protein coding sequence of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631; a polynucleotide comprising the full length protein coding sequence of SEQ ED NO: 1-4, 6, 14, 16, 25-27
  • the polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485- 486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,
  • Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.
  • the polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.
  • the polynucleotides may include the entire coding region of the cDNA or may represent a portion of the coding region of the cDNA.
  • the present invention also provides genes corresponding to the cDNA sequences disclosed herein.
  • 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. Further 5' and 3' sequence can be obtained using methods known in the art.
  • full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-
  • 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570- 571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
  • the nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.
  • the EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.
  • polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.
  • Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide recited above.
  • nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ LD NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617,
  • fragments 619, 621, 623, 625, 627, 629, or 631, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to any one of the polynucleotides of the invention) are contemplated.
  • Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.
  • sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ LD NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570- 571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631, a representative fragment thereof,
  • the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.
  • the nearest neighbor result for the nucleic acids of the present invention including SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,
  • BLAST stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S.F., JMol Evol. 36 290-300 (1993) and Altschul S.F., et al. J. Mol. Biol. 21:403-410 (1990)).
  • Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encodes proteins which are identical, homologous or related to that encoded by the polynucleotides.
  • the nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.
  • These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.
  • Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
  • conservative choices e.g., hydrophobic amino acid to a different hydrophobic amino acid
  • more distant choices e.g., hydrophobic amino acid to a charged amino acid
  • Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.
  • Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
  • terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.
  • polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.
  • This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site being changed.
  • site-directed mutagenesis is well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al, DNA 2:183 (1983).
  • a versatile and efficient method for producing site- specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic
  • PCR may also be used to create amino acid sequence variants of the novel nucleic acids.
  • primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.
  • PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.
  • the product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.
  • a further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells, et al, Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook, et al, supra, and Current Protocols in Molecular Biology, Ausubel, et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions. Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
  • the polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
  • the polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.
  • polynucleotide sequences comprising the mature protein coding sequences, coding for any one of SEQ ED NO: 5, 15, 28, 160, 186, 215, 241, 272, 302, 323, 348, 355, 378, 408, 420, 444, 487, 505, 516, 528, 542, 548, 557, 572, 579, 588, 602, 607, 612, 618, 622, 626, or 630, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.
  • a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
  • Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
  • the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
  • Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
  • the present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157- 159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 or a fragment thereof or any other polynucleotides of the invention.
  • the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345- 347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503,
  • a vector such as a plasmid or viral vector
  • the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, ⁇ NH8a, pNHl ⁇ a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
  • 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.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, and trc.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), a- factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3- phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA).
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Polynucleotides of the invention can also be used to induce immune responses.
  • nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.
  • the nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that can hybridize to or are complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631, or fragments, analogs or derivatives thereof
  • sense nucleic acid encoding a protein e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence of the invention.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "conceding region" of the coding strand of a nucleotide sequence of the invention.
  • conceding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • coding strand sequences e.g. SEQ ED NO: 1-4, 6, 14, 16, 25-27, 29, 157- 159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA of the invention, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of an mRNA of the invention.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an mRNA of the invention.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following section).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule.
  • An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual alpha-units, the strands run parallel to each other. See, e.g., Gaultier, et al, Nucl Acids Res. 15:6625-6641 (1987).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (see, e.g., Inoue, et al. Nucl. Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al, FEBSLett. 215:327-330 (1987).
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they can be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, Nature 334: 585-591 (1988)
  • a ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein (e.g. SEQ ED NO: 1-4, 6, 14, 16, 25-27,
  • a derivative of a Tetrahymena L-l 9 EVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of the invention.
  • mRNA of the invention See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al.
  • Stem cell growth factor-like mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, et ⁇ /., Science 261:1411-1418 (1993).
  • gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., the promoter and/or enhancers of the gene relating to the invention) to form triple helical structures that prevent transcription of the gene in target cells.
  • the regulatory region e.g., the promoter and/or enhancers of the gene relating to the invention
  • triple helical structures that prevent transcription of the gene in target cells.
  • the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, Bioorg. Med. Chem. 4:5-23 (1996).
  • peptide nucleic acids refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, Proc. Natl. Acad. Sci. USA 93:14670-14675 (1996).
  • PNAs of the invention can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of the invention can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (see, Hyrup, et al, ⁇ 996. supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of the invention can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al, 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. Supra, et al, Nucl Acids Res 24:3357-3363 (1996). For example, a
  • DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy- thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, Nucl Acid Res 17:5973-5988 (1989). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, Bioorg. Med. Chem. Lett. 5:1119-11124 (1975).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, Proc. Natl. Acad. Sci. U.S.A. 86:6553- 6556 (1989); Lemaitre, et al, Proc. Natl. Acad. Sci. USA 84:648-652 (1987); PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger, et al, Proc. Natl. Acad. Sci. U.S.A. 86:6553- 6556 (1989); Lemaitre, et al
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol, et al, BioTechniques 6:958-976 (1988)) or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539-549 (1988)).
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • the present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.
  • host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.
  • the present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.
  • the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • the host cells containing one of polynucleotides of the invention can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.
  • Any host/vector system can be used to express one or more of the ORFs of the present invention.
  • These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.
  • the most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. A number of types of cells may act as suitable host cells for expression of the protein.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo205 cells human Colo205 cells
  • CV-1 cells other transformed primate cell lines
  • normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast eukaryotes
  • prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida albicans, 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.
  • cells and tissues maybe engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
  • gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
  • regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
  • sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell- type specificity than the naturally occurring elements.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous
  • Markers useful for this pu ⁇ ose include the He ⁇ es Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
  • TK He ⁇ es Simplex Virus thymidine kinase
  • gpt bacterial xanthine-guanine phosphoribosyl-transferase
  • a "chimeric protein" or “fusion protein” of the invention comprises a polypeptide of the invention operatively linked to another polypeptide.
  • the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.
  • a fusion protein comprises at least one biologically active portion of a protein according to the invention.
  • a fusion protein comprises at least two biologically active portions of a protein according to the invention.
  • a fusion protein comprises at least three biologically active portions of a protein according to the invention.
  • fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.
  • the fusion protein is a GST- fusion protein in which the polypeptide sequences according to the invention are fused to the C-terminus of the GST (glutathione S-transferase) sequences.
  • GST- fusion protein in which the polypeptide sequences according to the invention are fused to the C-terminus of the GST (glutathione S-transferase) sequences.
  • the fusion protein is a protein according to the invention containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of the polypeptide according to the invention can be increased through use of a heterologous signal sequence.
  • the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences of the invention are fused to sequences derived from a member of the immunoglobulin protein family.
  • the immunoglobulin fusion proteins of the invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein according to the invention on the surface of a cell, to thereby suppress signal transduction mediated by the protein according to the invention in vivo.
  • the immunoglobulin fusion proteins can be used to affect the bioavailabihty of a cognate ligand.
  • Inhibition of the ligand/protein interaction can be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival.
  • the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide according to the invention with a ligand.
  • a chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamphfied to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamphfied to generate a chimeric gene sequence
  • a fusion moiety e.g., a fusion moiety
  • a nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.
  • the isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequence set forth as any one of SEQ ID NO: 5, 7-
  • Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ED NO: 1-4, 6,
  • the invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ED NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188- 213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487, 489- 501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618,
  • substantially equivalents thereof (e.g., with at least about 65%), at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or 94% and even more typically at least about 95%, 96%
  • Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516, 518-524,
  • 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 inco ⁇ orated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many pu ⁇ oses, including increasing the valency of protein binding sites.
  • the present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.
  • the protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.
  • the mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.
  • the sequence of the mature form of the protein is also determinable from the amino acid sequence of the full- length form.
  • proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which it is expressed.
  • Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • an acceptable carrier such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • the present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.
  • degenerate variant is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.
  • Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
  • the amino acid sequence can be synthesized using commercially available peptide synthesizers.
  • 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. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. 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.
  • polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.
  • a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.
  • One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.
  • the invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.
  • the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.
  • the polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.
  • Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.
  • the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.
  • One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer- Verlag (1994); Sambrook, et al, in
  • Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.
  • the purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.
  • the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal cells.
  • the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.
  • the toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ED NO: 5, 7-13, 15, 17-24, 28, 30- 156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622,
  • 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 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 sequence 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.
  • Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acid(s) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.
  • 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, Calif, U.S.A. (the MaxBatTM kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), inco ⁇ orated 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.
  • Purification of the protein of the invention may also include an affinity column containing agents which will bind to the protein of the invention; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaff ⁇ nity chromatography.
  • affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM
  • hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
  • immunoaff ⁇ nity chromatography immunoaff ⁇ nity 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), or as a His tag.
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.
  • the protein of the invention can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • FLAG® is commercially available from Kodak (New Haven, Conn.).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • RP- HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • 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 polypeptides of the invention include analogs (variants).
  • polypeptides of the invention which comprise one or more amino acids deleted, inserted, or substituted.
  • analogs of the polypeptides of the invention embrace fusions of the polypeptides of the invention or modifications of the polypeptides of the invention, wherein the polypeptide or analog of the invention is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability.
  • moieties which may be fused to the polypeptide or an analog of the invention include, for example, targeting moieties which provide for the delivery of polypeptides of the invention to neurons, e.g., antibodies to central nervous system, or antibodies to receptor and ligands expressed on neuronal cells.
  • moieties which may be fused to polypeptides of the invention include therapeutic agents which are used for treatment, for example anti- depressant drugs or other medications for neurological disorders.
  • polypeptides of the invention may be fused to neuron growth modulators, and other chemokines for targeted delivery.
  • Preferred identity and/or similarity are designed to give the largest match between the sequences tested.
  • Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP
  • Mutations in the gene encoding the polypeptide of the invention may result in loss of normal function of the encoded protein.
  • the invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.
  • Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retro virus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
  • Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
  • preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.
  • antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.
  • Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated R ⁇ A sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.
  • the present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.
  • DNA sequences allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
  • Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
  • cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
  • gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
  • Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
  • sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • Markers useful for this pu ⁇ ose include the He ⁇ es Simplex Virus thymidine kinase (TK) gene or the bacterial xanfhine-guanine phosphoribosyl-transferase (gpt) gene.
  • one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination (Capecchi, Science 244:1288-1292 (1989)).
  • Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.
  • Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals.
  • Knockout animals preferably non-human mammals, can be prepared as described in U.S. Patent No. 5,557,032, inco ⁇ orated herein by reference.
  • Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.
  • Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.
  • Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Patent No
  • Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.
  • the homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
  • polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express functional polypeptides of the invention or that express a variant of the polypeptides of the invention. Such animals are useful as models for studying the in vivo activities of polypeptides of the invention as well as for studying modulators of the polypeptides of the 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 herein.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.
  • compositions of the invention include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.
  • modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.
  • polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.
  • the polynucleotides provided by the present invention can be used by the research community for various pu ⁇ oses.
  • 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 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 finge ⁇ rinting; 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
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al, Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • polypeptides provided by the present invention can similarly be used in assays 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 polypeptide 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. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • polypeptides of the invention are also useful for making antibody substances that are specifically immunoreactive with proteins according to the invention.
  • Antibodies and portions thereof e. ., Fab fragments
  • Fab fragments which bind to the polypeptides of the invention can be used to identify the presence of such polypeptides in a sample. Such determinations are carried out using any suitable immunoassay format, and any polypeptide of the invention that is specifically bound by the antibody can be employed as a positive control.
  • a polypeptide of the present invention may exhibit activity relating to 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.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
  • the activity of therapeutic compositions 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, CMK, HUVEC, and Caco.
  • Therapeutic compositions of the invention can be used in the following:
  • 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-
  • Assays for cytokine production and or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interferon- ⁇ , Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and
  • 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. USA 77:6091-6095 (1980); Weinberger, et al, Eur. J. Immun. 11 :405-411
  • a polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.
  • Administration of the polypeptide of the invention to stem cells in vivo or ex vivo may maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, and manufacture of bio-pharmaceuticals and the development of bio-sensors.
  • the ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases
  • tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others
  • organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LEF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL- 6, macrophage inflammatory protein 1 -alpha (MIP-1 -alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).
  • SCF stem cell factor
  • LEF leukemia inhibitory factor
  • Flt-3L Flt-3 ligand
  • MIP-1 -alpha macrophage inflammatory protein 1 -alpha
  • G-CSF G-CSF
  • GM-CSF thrombopo
  • stem cells Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.
  • Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.
  • stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.
  • Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Patent No. 5,690,926).
  • Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.
  • polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.
  • the polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the 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.
  • these cells can be cultured in vitro to form other differentiated cells, such as skin tissue that can be used for transplantation.
  • the expanded stem cell populations can also be genetically altered for gene therapy pu ⁇ oses and to decrease host rejection of replacement tissues after grafting or implantation.
  • Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.
  • a broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell- type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive.
  • stem cells can be induced to differentiate into cardiomyocytes (Wobus et al, Differentiation, 48:173-182 (1991); Klug, et al, J. Clin.
  • stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • a differentiation factor such as retinoic acid
  • an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.
  • Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson, et al. Proc. Natl. Acad. Sci, U.S.A., 92:7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.
  • the ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein, et al, Blood, 11: 2316-2321 (1991).
  • a polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional colony stimulating factor 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
  • compositions of the invention can be used in the following:
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson, et al. Cellular Biology 15:141-15 (1995); Keller, et al, Mol. Cell. Biol. 13:473-486 (1993); McClanahan, et al, Blood 81:2903-2915 (1993).
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al.
  • a polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of bums, incisions and ulcers.
  • a polypeptide 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.
  • Compositions of a polypeptide, antibody, binding partner, or other modulator 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 polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone- forming cells.
  • Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, 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 may also be possible using the composition of the invention.
  • Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.
  • 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.
  • 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.
  • compositions of the present invention may provide 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, ca ⁇ al 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.
  • compositions 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 composition of the invention 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 composition of the invention.
  • compositions 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.
  • compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, and endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, and endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • a polypeptide of the present invention may also exhibit angiogenic activity.
  • a composition 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 composition 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.
  • compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in:
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pp. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year
  • a polypeptide 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 polynucleotide of the invention can encode a polypeptide exhibiting such activities.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCED)), 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.
  • SCED severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HEV) 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 HEV, hepatitis viruses, he ⁇ es viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • proteins 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 (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens- Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.
  • allergic reactions and conditions e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema,
  • a protein (or antagonists thereof) of the present invention may also be treatable using a protein (or antagonists thereof) of the present invention.
  • the therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom, et al, Toxicology 125: 59-66 (1998)), skin prick test (Hoffmann, et al, Allergy 54: 446-54 (1999)), guinea pig skin sensitization test (Vohr, et al, Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber, et al,
  • Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non- responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
  • tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as, for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • the administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow, et al, Science 257:789-792 (1992) and Turka, et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed.,
  • 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 stimulation of T cells 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 erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840- 856).
  • Upregulation of an antigen function 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 may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.
  • 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.
  • a polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts 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 alpha chain protein and ⁇ 2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta 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.
  • 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.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al, J. Immunol. 134:536-544 (1995); Inaba et al, J. Exp. Med. 173:549-559 (1991); Macatonia, et al, J. Immunol. 154:5071-5079 (1995); Porgador, et al, J. Exp. Med. 182:255-260 (1995); Nair, et al, J.
  • 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 Res. 53:1945-1951 (1993); Itoh, et al, Cell 66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai, et al, Cytometry 14:891-897 (1993); Gorczyca, et al, Int. J. Oncol. 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, Cell. Immunol. 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 polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
  • Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic compositions e.g. proteins, antibodies, binding partners, or modulators of the invention
  • 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.
  • Therapeutic compositions of the invention can be used in the following: 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. Marguiles, 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 alEur. J. Immunol. 25:1744-1748; Gruber, et al. J.
  • a polypeptide of the present invention may also exhibit activin- or inhibin-related activities.
  • a polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.
  • 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 polypeptide 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.
  • polypeptide 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,
  • a polypeptide 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, but not limited to, cows, sheep and pigs.
  • the activity of a polypeptide 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 polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
  • Compositions may 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 composition 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).
  • compositions of the invention can be used in the following: 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,
  • Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymo ⁇ hisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.
  • compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian
  • compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.
  • composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.
  • An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.
  • anti-cancer cocktails as a cancer treatment is routine.
  • Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HC1
  • Semustine, Teniposide, and Vindesine sulfate Semustine, Teniposide, and Vindesine sulfate.
  • therapeutic compositions of the invention may be used for prophylactic treatment of cancer.
  • hereditary conditions and/or environmental situations e.g. exposure to carcinogens
  • In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987)
  • Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.
  • a polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.
  • 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.
  • polypeptide 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-
  • the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).
  • Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art. Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.
  • the polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods. ("Guide to Protein Purification" Murray P.
  • radioisotopes include, but are not limited to, tritium and carbon- 14.
  • colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
  • toxins include, but are not limited, to ricin.
  • This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.
  • the polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.
  • Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.
  • the sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.
  • Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.
  • peptide and oligonucleotide combinatorial libraries are peptide and oligonucleotide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see Myers, Curr. Opin. Biotechnol 8:701-707 (1997).
  • For reviews and examples of peptidomimetic libraries see Al-Obeidi et al, Mol. Biotechnol, 9:205-23 (1998); Hruby, et al, Curr Opin Chem Biol, 1 : 114-19 (1997); Dorner, et al, Bioorg Med Chem, 4:709-15
  • Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit” (or “lead") to optimize the capacity of the "hit” to bind a polypeptide of the invention.
  • the molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.
  • the binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.
  • toxins e.g., ricin or cholera
  • the toxin- binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.
  • the binding molecules may be complexed with imaging agents for targeting and imaging pu ⁇ oses.
  • the invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor.
  • the invention also provides methods to detect specific binding of a polypeptide of the invention to a binding partner polypeptide, and in particular a ligand polypeptide.
  • Ligands useful in binding assays of this type include, for example Nogo-A,
  • Nogo-B, Nogo-C, and Nogo-66 or related protein for NgRHy, and other binding partner/receptors for other polypeptides of the invention identified using assays well known and routinely practiced in the art.
  • receptor activity of the polypeptides of the invention is determined using a method that involves (1) forming a mixture comprising a polypeptide of the invention, and/or its agonists and antagonists (or agonist or antagonist drug candidates) and/or antibodies specific for the polypeptides of the invention; (2) incubating the mixture under conditions whereby, but for the presence of said polypeptide of the invention and/or agonists and antagonists (or agonist or antagonist drug candidates) and/or antibodies specific for the polypeptides of the invention, the ligand binds to the receptor; and (3) detecting the presence or absence of specific binding of the polypeptide of the invention to its ligand.
  • the art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.
  • expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.
  • affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.
  • libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.
  • Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) is then compared.
  • an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).
  • BIAcore assays can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
  • downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.
  • a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified is produced in a host cell.
  • the cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.
  • Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e. phosphorylation.
  • Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.
  • Leukemia and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.
  • leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman, et al, 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). 4.17.15 NERVOUS SYSTEM DISORDERS
  • Nervous system disorders involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:
  • traumatic lesions including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
  • ischemic lesions in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, he ⁇ es zoster, or he ⁇ es simplex virus or with Lyme disease, tuberculosis, syphilis;
  • degenerative lesions in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
  • lesions associated with nutritional diseases or disorders in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the co ⁇ us callosum), and alcoholic cerebellar degeneration;
  • neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
  • lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and
  • demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, monophasic demyelination, encephalomyelitis, panencephalaitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy, Krabbe's disease, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, Guillain-Barre Syndrome, and central pontine myelinolysis.
  • a demyelinating disease including but not limited to multiple sclerosis, monophasic demyelination, encephalomyelitis, panencephalaitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodys
  • Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.
  • therapeutics which elicit any of the following effects may be useful according to the invention:
  • a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or
  • increased survival of neurons may be measured by the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods set forth in Pestronk, et al. (Exp. Neurol. 70:65-82 (1980)) or Brown, et al. (Ann. Rev. Neurosci.
  • neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio- Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
  • disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary
  • a polypeptide 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 circadian 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, co-factors 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
  • polymo ⁇ hisms makes possible the identification of such polymo ⁇ hisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.
  • Such polymo ⁇ hisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.
  • the existence of a polymo ⁇ hism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymo ⁇ hism.
  • Polymo ⁇ hisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymo ⁇ hism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.
  • the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymo ⁇ hism is extended with one or more labeled nucleotides).
  • allele-specific oligonucleotide hybridization in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch
  • a single nucleotide extension assay in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymo ⁇ hism is extended with one or more labeled nucleotides.
  • traditional restriction fragment length polymo ⁇ hism analysis using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymo ⁇ hism
  • the array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.
  • any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.
  • polymo ⁇ hism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.
  • the immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.
  • the experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et al, Science, 219:56 (1983), or by B. Waksman, et al, Int. Arch. Allergy Appl Immunol, 23: 129 (1963).
  • Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • the route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.
  • the polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.
  • the control consists of administering PBS only.
  • the procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.
  • compositions of the present invention may also exhibit other 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.
  • compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation 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.
  • Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, or in the prevention of premature labor secondary to intrauterine infections.
  • conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, or in the prevention of premature labor secondary to intrauterine infections.
  • Polynucleotides and polypeptides 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 polypeptide 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 polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. Additionally, the polypeptides of the invention can be used as markers, and as a food supplement. Protein food supplements are well known and the formulation of suitable food supplements including polypeptides of the invention is within the level of skill in the food preparation art.
  • a polynucleotide and polypeptide of the invention may also be involved in the prevention, diagnosis and management of metabolic disorders involving carbohydrates, lipids, amino acids, vitamins etc., including but not limited to diabetes mellitus, obesity, aspartylglusomarinuria, carbohydrate deficient glycoprotein syndrome (CDGS), cystinosis, diabetes insipidus, Fabry, fatty acid metabolism disorders, galactosemia, Gaucher, glucose- 6-phosphate dehydrogenase (G6PD), glutaric aciduria, Hurler, Hurler-Scheie, Hunter, hypophosphatemia, I-cell, Krabbe, lactic acidosis, long chain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), lysosomal storage diseases, mannosidosis, maple syrup urine, , Maroteaux-Lamy, metachromatic leukodystrophy, mitochondrial Morquio, mucopolysaccharidosis, neuro-metabolic, Niemann-Pick, organic acidemia
  • Hereditary and/or environmental factors known in the art can predispose an individual to developing metabolic disorders and conditions resulting therefrom. Under these circumstances, it maybe beneficial to treat these individual with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing the disorder. Examples of such disorders include diabetes mellitus, obesity and cardiovascular disease.
  • polynucleotide sequences encoding the invention may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELIS A or chip assays utilizing fluids or tissues from patient biopsies to detect altered expression of the polynucleotides of the invention. Such qualitative or quantitative methods are well known in the art.
  • Polypeptides and polynucleotides of the invention may also be involved in the prevention, diagnosis and management of cardiovascular disorders such as coronary artery disease, atherosclerosis and hyper- and hypolipoproteinemia, hypertension, angina pectoris, myocardial infarction, congestive heart failure, cardiac arrythmias including paroxysmal arrythmias, restenosis after angioplasty, aortic aneurysm and related complications involving various organs including but not limited to kidney, eye, brain, heart etc.
  • cardiovascular disorders such as coronary artery disease, atherosclerosis and hyper- and hypolipoproteinemia, hypertension, angina pectoris, myocardial infarction, congestive heart failure, cardiac arrythmias including paroxysmal arrythmias, restenosis after angioplasty, aortic aneurysm and related complications involving various organs including but not limited to kidney, eye, brain, heart etc.
  • Polypeptides of the invention may also have direct and indirect effects on myocardial contractility, electrical activity of the heart, atrial fibrillation, atrial fluter, anomalous atrio-ventricular pathways, sino-atrial dysfunction, vascular insufficiency and arterial embolism.
  • Hereditary and/or environmental factors known in the art can predispose an individual to developing metabolic disorders and conditions resulting therefrom. Under these circumstances, it maybe beneficial to treat these individual with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing the disorder.
  • disorders include but are not limited to coronary artery disease, atherosclerosis, hyper- and hypolipoproteinemia, hypertension, angina pectoris, myocardial infarction, cardiac arrythmias including paroxysmal arrythmias, diabetes mellitus, inflammatory glomerulonephritis, ischemic renal failure, extracellular matrix accumulation, fibrosis, hypertension, coronary vasoconstriction, ischemic heart disease, and lesions occurring in brain disorders such as stroke, trauma, infarcts, aneurysms.
  • polynucleotide sequences encoding the invention may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies" to detect altered expression of the polynucleotides of the invention. Such qualitative or quantitative methods are well known in the art.
  • compositions including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides
  • therapeutic applications include, but are not limited to, those exemplified herein.
  • One embodiment of the invention is the administration of an effective amount of the polypeptides of the invention or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention. While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus.
  • the dosage of polypeptides of the invention or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.
  • polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
  • the vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.
  • a protein or other composition of the present invention may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.
  • a composition may optionally contain (in addition to protein or other active ingredient 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, EFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and TGF- ⁇ ), insulin-like growth factor (IGF), as well as cytokines described herein.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement 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 or other active ingredient of the invention, or to minimize side effects.
  • protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent (such as IL-lRa, IL-1 Hyl, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).
  • a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., 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 therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.
  • Protein or other active ingredient 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 or other active ingredient 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.
  • cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors are administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • Administration of protein or other active ingredient 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.
  • the compounds may be administered topically, for example, as eye drops.
  • one may administer the drug in a targeted drug delivery system for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.
  • the liposomes will be targeted to and taken up selectively by the afflicted tissue.
  • the polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.
  • a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.
  • Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.
  • Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
  • compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
  • protein or other active ingredient of the present invention When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient 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 or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient 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 or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.
  • protein or other active ingredient of the present invention When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyro gen- free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein or other active ingredient 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 or other active ingredient 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 agents of the invention maybe formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a co- solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the co-solvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein or other active ingredient stabilization may be employed.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.

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Abstract

L'invention concerne des nouveaux polynucléotides et des polypeptides codés par lesdits polynucléotides ainsi que leurs mutants ou variants qui correspondent auxdits nouveaux polynucléotides et polypeptides. Dans d'autres modes de réalisation, l'invention concerne des procédés contenant des vecteurs pour produire des nouveaux polypeptides et des anticorps spécifiques pour lesdits polypeptides.
PCT/US2002/038526 2000-01-21 2002-12-02 Procedes et matieres associes a de nouveaux polypeptides et polynucleotides Ceased WO2003048326A2 (fr)

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US10/496,905 US20050192215A1 (en) 2000-01-21 2002-12-02 Methods and materials relating to novel polypeptides and polynucleotides
US10/758,846 US20040248156A1 (en) 2001-12-03 2004-01-16 Methods and materials relating to novel C1q domain-containing polypeptides and polynucleotides

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1492558A4 (fr) * 2002-03-28 2006-07-19 Lilly Co Eli Nouvelles proteines secretees et leurs utilisations
EP2128623A1 (fr) * 2006-02-22 2009-12-02 Philogen S.p.A. Marqueurs de tumeurs vasculaires
US8603971B2 (en) 2005-09-16 2013-12-10 Cornell Research Foundation, Inc. Methods for reducing CD36 expression
EP4004024A4 (fr) * 2019-07-22 2023-09-20 University of Florida Research Foundation, Incorporated Domaines protéiques multimères pour la multifonctionnalité et la sécrétion améliorée de protéines thérapeutiques

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2399776A1 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Acides nucleiques et polypeptides
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1492558A4 (fr) * 2002-03-28 2006-07-19 Lilly Co Eli Nouvelles proteines secretees et leurs utilisations
US8603971B2 (en) 2005-09-16 2013-12-10 Cornell Research Foundation, Inc. Methods for reducing CD36 expression
CN102552874B (zh) * 2005-09-16 2015-04-22 科内尔研究基金会 用于减少cd36表达的方法
US9150614B2 (en) 2005-09-16 2015-10-06 Cornell Research Foundation, Inc. Methods for reducing CD36 expression
US11447523B2 (en) 2005-09-16 2022-09-20 Cornell Research Foundation, Inc. Methods for reducing CD36 expression
EP2128623A1 (fr) * 2006-02-22 2009-12-02 Philogen S.p.A. Marqueurs de tumeurs vasculaires
EP4004024A4 (fr) * 2019-07-22 2023-09-20 University of Florida Research Foundation, Incorporated Domaines protéiques multimères pour la multifonctionnalité et la sécrétion améliorée de protéines thérapeutiques

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AU2002360460A1 (en) 2003-06-17
WO2003048326A3 (fr) 2004-12-02

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