EP1141285A2 - Polypeptides secretes et transmembranaires et acides nucleiques codant pour ceux-ci - Google Patents

Polypeptides secretes et transmembranaires et acides nucleiques codant pour ceux-ci

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Publication number
EP1141285A2
EP1141285A2 EP99962992A EP99962992A EP1141285A2 EP 1141285 A2 EP1141285 A2 EP 1141285A2 EP 99962992 A EP99962992 A EP 99962992A EP 99962992 A EP99962992 A EP 99962992A EP 1141285 A2 EP1141285 A2 EP 1141285A2
Authority
EP
European Patent Office
Prior art keywords
seq
polypeptide
pro
sequence identity
nucleic acid
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.)
Withdrawn
Application number
EP99962992A
Other languages
German (de)
English (en)
Inventor
David Botstein
Luc Desnoyers
Napoleone Ferrara
Sherman Fong
Wei-Qiang Gao
Audrey Goddard
Austin L. Gurney
James Pan
Margaret Ann Roy
Timothy A. Stewart
Daniel Tumas
Colin K. Watanabe
William I. Wood
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.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1999/012252 external-priority patent/WO1999063088A2/fr
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to DK02016081T priority Critical patent/DK1300417T3/da
Priority to EP02016080A priority patent/EP1283215B1/fr
Priority to EP02016081A priority patent/EP1300417B1/fr
Priority to EP02016082A priority patent/EP1251173B1/fr
Priority to DK02016082T priority patent/DK1251173T3/da
Priority to EP02016083A priority patent/EP1277833B1/fr
Priority to DK02016083T priority patent/DK1277833T3/da
Publication of EP1141285A2 publication Critical patent/EP1141285A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides.
  • Extracellular proteins play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms.
  • secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.
  • Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors.
  • Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins.
  • Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents.
  • Efforts are being undertaken by both industry and proficient to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Patent No. 5,536,637)].
  • Membrane-bound proteins and receptors can play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms.
  • membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins . Protein tyrosine kinases , enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
  • Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-hgand interactions
  • Receptor immunoadhesins for instance, can be employed as therapeutic agents to block receptor-hgand interactions
  • the membrane -bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/hgand interaction
  • Hep27 protein is synthesized and accumulated in the nucleus of human hepatoblastoma cells (HepG2 cells) following growth arrest induced by butyrate treatment (Gabrielli et al , Eur J Biochem 232 473-477 (1995)) The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis
  • the Hep27 protein sequence shows significant homology to the known short-chain alcohol dehydrogenase (SCAD) family of proteins and it has been suggested that Hep27 is a new member of the SCAD family of proteins In agreement with its nuclear localization, Hep27 has a region similar to the bipartite nuclear- targeting sequence and Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transc ⁇ ptional level
  • TGF- ⁇ transforming growth factor-beta
  • Wnt fibroblast growth factors
  • hedgehog families have been associated with patterning activity of different cells and structures in Drosophila as well as m vertebrates.
  • Costal-2 is a novel kinesin-related protein in the Hedgehog signaling pathway Hedgehog (Hh) was first identified as a segment-polarity gene by a genetic screen in Drosophila melanogaster, Nusslem-Volhard et al , Roux. Arch. Dev.
  • Hh also plays a role in the development of limbs (Krauss et al Cell 75 1431 44 (1993) Laufer et al Cell 79 993-1003 (1994)), somites (Fan and Tessier-Lavigne CeU 29, 1 175-86 (1994), Johnson et al , Cell 79 1165 73 (1994)), lungs (Bellusci et al , Develop J124 53-63 ( 1997) and sk (Oro et al , Science 276 817 21 (1997) Likewise, IHh and DHh are involved in bone, gut and germinal cell development, Apelqvist et al , Cttrr Biol 7 801-4 (1997), Bellusci et al , Dev Suppl Y2 ⁇
  • the nei gene has been described to encode a protein that is expressed in the neural tissues of chicken (Watanabe et al. , Genomics 38(3):273-276 (1996)).
  • NELL1 and NELL2 two novel human cDNAs (designated NELL1 and NELL2) have been isolated and characterized which encode polypeptides having homology to that encoded by the chicken nei gene, wherein those human polypeptides contain six EGF-like repeats (Watanabe et al. , supra). Given the neural-specific expression of these genes, it is suggested that they may play a role in neural development. There is, therefore, significant interest in identifying and characterizing novel polypeptides having homology to nei, NELL1 and NELL2.
  • PRO 1434 polypeptides having homology to the nei protein, designated herein as PRO 1434 polypeptides.
  • inositol phosphatases The characterization of inositol phosphatases is of interest because it is fundamental to the understanding of signaling activities that stimulate the release of Ca 2+ from the endoplasmic reticulum. Molecular cloning allowed the identification of a multiple inositol polyphosphate phosphatase which is highly expressed in kidney and liver (Craxton et al. (1997) Biochem J. 328:75-81).
  • PRQ1868 The inflammatory response is complex and is mediated by a variety of signaling molecules produced locally by mast cells, nerve endings, platelets, leucocytes and complement activation. Certain of these signaling molecules cause the endothelial cell lining to become more porous and/or even to express selectins which act as cell surface molecules which recognize and attract leucocytes through specific carbohydrate recognition. Stronger leucocyte binding is mediated by integrins, which mediate leukocyte movement through the endothelium. Additional signaling molecules act as chemoattractants, causing the bound leucocytes to crawl towards the source of the attractant. Other signaling molecules produced in the course of an inflammatory response escape into the blood and stimulate the bone marrow to produce more leucocytes and release them into the blood stream
  • Inflammation is typically initiated by an antigen, which can be virtually an> molecule capable of initiating an immune response Under normal physiological conditions these are foreign molecules, but molecules generated by the organism itself can serve as the catalyst as is known to occur in various disease states
  • T-cell proliferation is a mixed lymphocyte culture or mixed lymphocyte reaction (MLR) is an established indication of the ability of a compound to stimulate the immune system
  • MLR mixed lymphocyte reaction
  • the responding leucocytes can be neutrophihc, eosinophihc, monocytic or lymphocytic Histological examination of the affected tissues provides evidence of an immune stimulating or inhibiting response
  • IBD Inflammatory bowel disease
  • IBD is a term used to collectively describe gut disorders including both ulcerative colitis (UC) and Crohn's disease, both of which are classified as distinct disorders, but share common features and likely share pathology
  • IBD Inflammatory bowel disease
  • IBD is a term used to collectively describe gut disorders including both ulcerative colitis (UC) and Crohn'
  • JAM Junctional Adhesion Molecule
  • the compounds of the invention also bear significant homology to A33 antigen, a known colorectal cancer-associated marker
  • A33 antigen is expressed in more than 90% of primary or metastatic colon cancers as well as normal colon epithelium In carcinomas originating from the colonic mucosa, the A33 antigen is expressed homogeneously in more than 95 % of all cases
  • the A33 antigen has not been detected m a wide range of other normal issues, I e its expression appears to be organ specific Therefore, the A33 antigen appears to play an important role in the induction of colorectal cancer
  • mAbs monoclonal antibodies
  • Radioactive gene, toxins and/or drug tagged mAbs can be used for treatment in situ with minimal patient description
  • mAbs can also be used to diagnose during the diagnosis and treatment of colon cancers
  • serum levels of the A33 antigen are elevated in a patient
  • a drop of the levels after surgery would indicate the tumor resection was successful
  • serum A33 antigen levels after surgery would indicate that metastases of the original tumor may have formed or that new primary tumors may have appeared
  • monoclonal antibodies can be used in lieu of, or in conjunction with surgery and/or other chemotherapies
  • preclimcal analysis and localization studies in patients infected with colorectal carcinoma with a mAb to A33 are described in Welt et al , J Chn Oncol 8 1894-1906 (19
  • PRO 1868 polypeptides having homology to A33 antigen protein, designated herein as PRO 1868 polypeptides
  • PR03434 polypeptides are novel secreted polypeptides.
  • Proteins are glycosylated by a complex set of reactions which are mediated by membrane bound glycosyltransferases
  • glycosyltransferases that account for the array of carbohydrate structures synthesized N-acetylglucosaminyltransferase proteins comprise a family of glycosyltransferases that provide for a variety of important biological functions in the mammalian organism
  • UDP-N-acetylglucosamine alpha-3-D-mannos ⁇ de beta-l ,2-N-acetylglucosammyltransferase I is an glycosyltransferase that catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans (Sarkar et al , Proc Natl Acad Sci USA 88 234-238 (1991) UPD-N- acetylglucosamine alpha 1 3 D-mannoside
  • a cDNA clone (DN A35672-2508) has been identified, having homology to nucleic acid encoding Hep27 protem, that encodes a novel polypeptide, designated m the present application as "PRO1800"
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1800 polypeptide
  • the invention concerns an isolated nucleic acid molecule encoding a PRO1800 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides 36 or about 81 and about 869, inclusive, of Figure 1 (SEQ ID NO 1)
  • hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203538 (DNA35672-2508) or (b) the complement of the nucleic acid molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203538 (DNA35672-2508)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO 2), or (b) the complement of the DNA of (a)
  • the invention concerns an isolated nucleic acid molecule having at least 230 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO1800 polypeptide having the sequence of ammo acid residues from 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO 2), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80 % sequence identity, prefereably at least about an 85 % sequence identity, more preferably at least about a 90 % sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), isolating the test DNA molecule.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1800 polypeptide, with or without the N-terminal signal sequence and/or the initiating methionine,or is complementary to such encoding nucleic acid molecule.
  • the signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 15 in the sequence of Figure 2 (SEQ ID NO: 1
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85 % positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO:2), or (b) the complement of the DNA of (a).
  • nucleic acid fragments may be from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length and most preferably from about 20 to about 40 nucleotides in length and may be derived from the nucleotide sequence shown in Figure 1 (SEQ ID NO: l).
  • the invention provides isolated PRO 1800 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention provides isolated native sequence PRO1800 polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues 1 or about 16 to about 278 of Figure 2 (SEQ ID NO:2).
  • the invention concerns an isolated PRO 1800 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO:2).
  • the invention concerns an isolated PRO 1800 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85 % positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO:2).
  • the invention concerns an isolated PRO1800 polypeptide, comprising the sequence of amino acid residues 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO:2), or a fragment thereof sufficient to provide a binding site for an anti-PRO1800 antibody.
  • the PRO 1800 fragment retains a qualitative biological activity of a native PRO 1800 polypeptide.
  • the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1800 polypeptide having the sequence of amino acid residues from about 1 or about 16 to about 278, inclusive of Figure 2 (SEQ ID NO: 3), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferabl ⁇ at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), (n) cultu ⁇ ng a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (in) recovering the polypeptide from the cell culture
  • the invention concerns agonists and antagonists of a native PRO 1800 polypeptide
  • the agonist or antagonist is an anti PRO 1800 antibody
  • the invention concerns a method of identifying agonists or antagonists of a native PRO 1800 polypeptide by contacting the native PRO 1800 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide
  • the mvention concerns a composition comprising a PRO 1800 polypeptide, or an agonist or antagonist as hereinabove defined in combination with a pharmaceutically acceptable carrier
  • a cDNA clone (DNA47465-1561) has been identified, having homology to nucleic acid encoding Costal-2 protein, that encodes a novel polypeptide, designated in the present application as 'PR0539"
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0539 polypeptide
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding a PR0539 polypeptide having the sequence of ammo acid residues from about 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or (b) the complement of the DNA molecule of (a)
  • the invention concerns an isolated nucleic acid molecule encoding a PR0539 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides 186 and about 2675, inclusive, of Figure 3 (SEQ ID NO 6)
  • hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203661 (DNA47465-1561) or (b) the complement of the nucleic acid molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203661 (DNA47465-1561)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or (b) the complement of the DNA of (a)
  • the invention concerns an isolated nucleic acid molecule having at least 100 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PR0539 polypeptide having the sequence of ammo acid residues from 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80 % sequence identity, prefereably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0539 polypeptide, with or without the initiating methiomne, or is complementary to such encoding nucleic acid molecule
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or (b) the complement of the DNA of (a) Another embodiment is directed to fragments of a PR0539 polypeptide coding sequence that may find use as hybridization probes Such nucleic acid fragments are from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length and most preferably from
  • the invention provides isolated native sequence PR0539 polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues 1 to about 830 of Figure 4 (SEQ ID NO 7)
  • the invention concerns an isolated PR0539 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of amino acid residues 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7)
  • the invention concerns an isolated PR0539 polypeptide, comprising an ammo acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the amino acid sequence of residues 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7)
  • the invention concerns an isolated PR0539 polypeptide, comprising the sequence of amino acid residues 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or a fragment thereof sufficient to provide a binding site for an ant ⁇ -PR0539 antibody
  • the PR0539 fragment retains a qualitative biological activity of a native PR0539 polypeptide
  • the invention provides a polypeptide produced by (1) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PR0539 polypeptide having the sequence of amino acid residues from about 1 to about 830, inclusive of Figure 4 (SEQ ID NO 7), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), ( ⁇ ) culturmg a
  • the invention concerns agonists and antagonists of a native PR0539 polypeptide
  • the agonist or antagonist is an anti PR0539 antibody
  • the invention concerns a method of identifying agonists or antagonists of a native PR0539 polypeptide by contacting the native PR0539 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide
  • the biological activity is either binding to microtubiles or the ability to complex with fused and cubitus mterruptus
  • the invention concerns a composition
  • a composition comprising a PR0539 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier
  • the invention provides for compounds and methods for developing antagonists against and agonist promoting PR0539 modulation of Hedgehog signaling
  • an antagonist of vertebrate PR0539 which blocks, prevents, inhibits and/or neutralized the normal functioning of PR0539 in SH signaling pathway, including both small bioorgamc molecules and antisense nucleotides
  • the invention provides for alternatively spliced variants of human PR0539
  • the invention provides a method of screening or assaying for identifying molecules that alter the PR0539 modulation of hedgehog signaling
  • the molecules either prevent interaction of PR0539 with its associative complexmg proteins (such as fused or cubitus mterruptus) or prevent or inhibit dissociation of complexes
  • the assay comprises the incubation of a mixture comprising PR0539 and a substrate with a candidate molecule and detection of the ability of the candidate molecule to modulate PR0539 hedgehog signaling
  • the screened molecules preferably are small molecule drug candidates
  • the method relates to a technique of diagnosing to determine whether a particular disorder is modulated by hedgehog signaling, comprising
  • a cDNA clone (DNA57700-1408) has been identified that encodes a novel polypeptide, designated in the present application as "PR0982
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0982 polypeptide
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding a PR0982 polypeptide having the sequence of amino acid residues from 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9), or
  • the invention concerns an isolated nucleic acid molecule encoding a PR0982 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about residues 89 and about 400, inclusive, of Figure 5 (SEQ ID NO 8)
  • hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA m ATCC Deposit No 203583 (DNA57700-1408), or (b) the complement of the DNA molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203583 (DNA57700-1408)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues from 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO: 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO:
  • the invention concerns an isolated nucleic acid molecule having at least about 50 nucleotides, and preferably at least about 100 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PR0982 polypeptide having the sequence of amino acid residues from 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), isolating the test DNA molecule
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0982 polypeptide, with or without the N-terminal signal sequence and/or the initiating methiomne, or is complementary to such encoding nucleic acid molecule
  • the signal peptide has been tentatively identified as extending from ammo acid position 1 through about amino acid position 21 m the sequence of Figure 6 (SEQ ID NO-9)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the ammo acid sequence of residues 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9), or (b) the complement of the DNA of (a)
  • nucleic acid fragments are from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides m length, more preferably from about 20 to about 50 nucleotides in length, and most preferably from about 20 to about 40 nucleotides in length
  • the invention provides isolated PR0982 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove defined
  • the invention provides isolated native sequence PR0982 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 or about 22 to 125 of Figure 6 (SEQ ID NO 9)
  • the invention concerns an isolated PR0982 polypeptide, comprising an ammo acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of amino acid residues 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9)
  • the invention concerns an isolated PR0982 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 22 to 125 of Figure 6 (SEQ ID NO 9)
  • the invention concerns an isolated PR0982 polypeptide, comprising the sequence of amino acid residues 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9), or a fragment thereof sufficient to provide a binding site for an ant ⁇ -PR0982 antibody
  • the PR0982 fragment retains a qualitative biological activity of a native PR0982 polypeptide
  • the invention provides a polypeptide produced by (l) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PR0982 polypeptide having the sequence of amino acid residues from 1 or about 22 to about 125, inclusive of Figure 6 (SEQ ID NO 9), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), (u) culturmg a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (in) recovering the polypeptide from the cell culture
  • a cDNA clone (DNA68818-2536) has been identified, having homology to nucleic acid encoding nei protein, that encodes a novel polypeptide, designated in the present application as "PR01434"
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1434 polypeptide
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity preferably at least about 85 % sequence identi more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding a PRO 1434 polypeptide having the sequence of ammo acid residues from about 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO 11), or (b) the complement of the DNA molecule of (a)
  • the invention concerns an isolated nucleic acid molecule encoding a PR01434 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides 581 or about 662 and about 1555, inclusive, of Figure 7 (SEQ ID NO 10) Preferably, hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferabl) at least about 95 % sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protem cDNA in ATCC Deposit No 203657 (DNA68818-2536) or (b) the complement of the nucleic acid molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203657 (DNA68818-2536)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of ammo acid residues 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO 11), or (b) the complement of the DNA of (a)
  • the invention concerns an isolated nucleic acid molecule having at least 65 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1434 polypeptide having the sequence of amino acid residues from 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO 11), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, prefereably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), isolating the test DNA molecule
  • the invention provides an isolated nucleic acid molecule comprising DNA encodmg a PR01434 polypeptide, with or without the N-terminal signal sequence and/or the initiating methiomne, and its soluble, I e , transmembrane domain deleted or inactivated variants, or is complementary to such encoding nucleic acid molecule
  • the signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 27 in the sequence of Figure 8 (SEQ ID NO 11)
  • the transmembrane domain has been tentatively identified as extending from about amino acid position 11 to about amino acid position 30 in the PR01434 amino acid sequence ( Figure 8, SEQ ID NO 11)
  • the mvention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the
  • nucleic acid fragments are from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length and most preferably from about 20 to about 40 nucleotides in length and may be derived from the nucleotide sequence shown in Figure 7 (SEQ ID NO: 10).
  • the invention provides isolated PR01434 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention provides isolated native sequence PR01434 polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues 1 or about 28 to about 325 of Figure 8 (SEQ ID NO: 11).
  • the invention concerns an isolated PRO 1434 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of amino acid residues 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO: 11).
  • the invention concerns an isolated PRO 1434 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the amino acid sequence of residues 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO: 11).
  • the invention concerns an isolated PRO 1434 polypeptide, comprising the sequence of amino acid residues 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO: 11), or a fragment thereof sufficient to provide a binding site for an anti-PR01434 antibody.
  • the PR01434 fragment retains a qualitative biological activity of a native PRO 1434 polypeptide.
  • the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1434 polypeptide having the sequence of amino acid residues from about 1 or about 28 to about 325, inclusive of Figure 8 (SEQ ID NO: 11), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO 1434 polypeptide.
  • the agonist or antagonist is an anti-PR01434 antibody.
  • the invention concerns a method of identifying agonists or antagonists of a native PR01434 polypeptide by contacting the native PR01434 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.
  • the invention concerns a composition
  • a composition comprising a PR01434 polypeptide, or an agonist or antagonist as hereinabove defined in combination with a pharmaceutically acceptable carrier
  • a cDNA clone (DNA59847-2510) has been identified that encodes a novel transmembrane polypeptide, designated in the present application as "PRO 1863
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1863 polypeptide
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding a PRO 1863 polypeptide having the sequence of amino acid residues from about 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO: 1)
  • the invention concerns an isolated nucleic acid molecule encoding a PRO 1863 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides 17 or about 62 and about 1327, inclusive, of Figure 9 (SEQ ID NO 15) Preferably, hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203576 (DNA59847-2510) or (b) the complement of the nucleic acid molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203576 (DNA59847 2510)
  • the invention concerns an isolated nucleic acid molecule having at least 345 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1863 polypeptide having the sequence of amino acid residues from 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, prefereably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), isolating the test DNA molecule
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1863 polypeptide, with or without the N-terminal signal sequence and/or the initiating methiomne, and its soluble, I e , transmembrane domain deleted or inactivated variants
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85 % positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the amino acid sequence of residues 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16), or (b) the complement of the DNA of (a)
  • nucleic acid fragments are from about 20 to about 80 nucleotides m length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length and most preferably from about 20 to about 40 nucleotides in length and may be derived from the nucleotide sequence shown m Figure 9 (SEQ ID NO 15)
  • the invention provides isolated PRO 1863 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
  • the invention provides isolated native sequence PRO 1863 polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues 1 or about 16 to about 437 of Figure 10 (SEQ ID NO 16)
  • the invention concerns an isolated PRO 1863 polypeptide, comprising an ammo acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of ammo acid residues 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16)
  • the invention concerns an isolated PRO 1863 polypeptide, comprising an ammo acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the amino acid sequence of residues 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16)
  • the invention concerns an isolated PR01863 polypeptide, comprising the sequence of ammo acid residues 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16), or a fragment thereof sufficient to provide a binding site for an ant ⁇ -PR01863 antibody
  • the PRO 1863 fragment retains a qualitative biological activity of a native PRO 1863 polypeptide
  • the invention provides a polypeptide produced by (l) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1863 polypeptide having the sequence of ammo acid residues from about 1 or about 16 to about 437, inclusive of Figure 10 (SEQ ID NO 16), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), ( ⁇ ) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (in) recovering the polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO 1863 polypeptide.
  • the agonist or antagonist is an anti-PR01863 antibody.
  • the invention concerns a method of identifying agonists or antagonists of a native PRO 1863 polypeptide by contacting the native PRO 1863 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.
  • the invention concerns a composition
  • a composition comprising a PRO 1863 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.
  • PRQ1917 A cDNA clone (DNA76400-2528) has been identified that encodes a novel polypeptide having homology to inositol phosphatase and designated in the present application as "PR01917” .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR01917 polypeptide.
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding a PRO 1917 polypeptide having the sequence of amino acid residues from 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or (b) the complement of the DNA molecule of (a).
  • the invention concerns an isolated nucleic acid molecule encoding a PR01917 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about residues 96 and about 1466, inclusive, of Figure 11 (SEQ ID NO: 17).
  • hybridization occurs under stringent hybridization and wash conditions.
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203573 (DNA76400-2528), or (b) the complement of the DNA molecule of (a).
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203573 (DNA76400-2528).
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues from 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or the complement of the DNA of (a).
  • the invention concerns an isolated nucleic acid molecule having at least about 50 nucleotides, and preferably at least about 100 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1917 polypeptide having the sequence of amino acid residues from 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), isolating the test DNA molecule.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR01917 polypeptide, with or without the N-terminal signal sequence and/or the initiating methionine, or is complementary to such encoding nucleic acid molecule.
  • the signal peptide has been tentatively identified as extending from amino acid position 1 through about amino acid position 30 in the sequence of Figure 12 (SEQ ID NO: 1
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95 % positives when compared with the amino acid sequence of residues 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or (b) the complement of the DNA of (a).
  • nucleic acid fragments are from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length, and most preferably from about 20 to about 40 nucleotides in length.
  • the invention provides isolated PR01917 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove defined.
  • the invention provides isolated native sequence PR01917 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 or about 31 to 487 of Figure 12 (SEQ ID NO: 18).
  • the invention concerns an isolated PRO 1917 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18).
  • the invention concerns an isolated PR01917 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 31 to 487 of Figure 12 (SEQ ID NO: 18).
  • the invention concerns an isolated PRO 1917 polypeptide, comprising the sequence of amino acid residues 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or a fragment thereof sufficient to provide a binding site for an anti-PR01917 antibody.
  • the PRO 1917 fragment retains a qualitative biological activity of a native PRO 1917 polypeptide.
  • the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1917 polypeptide having the sequence of amino acid residues from 1 or about 31 to about 487, inclusive of Figure 12 (SEQ ID NO: 18), or (b) the complement of the DNA molecule of (a) and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), ( ⁇ ) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (in) recovering the polypeptide from the cell culture
  • the invention concerns agonists and antagonists of a native PR01917 polypeptide
  • the agonist or antagonist is an anti PRO 1917 antibody
  • the invention concerns a method of identifying agonists or antagonists of a native PRO 1917 polypeptide, by contacting the native PRO 1917 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide
  • the invention concerns a composition comprising a PRO 1917 polypeptide, or an agonist or antagonist as hereinabove defined, m combination with a pharmaceutically acceptable carrier
  • the present invention concerns compositions and methods for the diagnosis and treatment of inflammatory diseases in mammals, including humans
  • the present invention is based on the identification of proteins (including agonist and antagonist antibodies) which either stimulate or inhibit the immune response m mammals
  • Inflammatory diseases can be treated by suppressing the inflammatory response Molecules that enhance an inflammatory response stimulate or potentiate the immune response to an antigen Molecules which stimulate an inflammatory response can be inhibited where suppression of the inflammatory response would be beneficial Molecules which stimulate the inflammatory response can be used therapeutically where enhancement of the inflammatory response would be beneficial
  • Such stimulatory molecules can also be inhibited where suppression of the inflammatory response would be of value
  • Neutralizing antibodies are examples of molecules that inhibit molecules having immune stimulatory activity and which would be beneficial in the treatment of inflammatory diseases Molecules which inhibit the inflammatory response can also be utilized (proteins directly or via the use of antibody agonists) to inhibit the inflammatory response and thus ameliorate inflammatory diseases
  • the proteins of the invention are useful for the diagnosis and/or treatment (including prevention) of immune related diseases
  • Antibodies which bind to stimulatory proteins are useful to suppress the inflammatory response
  • Antibodies which bind to inhibitory proteins are useful to stimulate inflammatory response and the immune system
  • the proteins and antibodies of the invention are also useful to prepare medicines and medicaments for the treatment of inflammatory and immune related diseases
  • the invention concerns antagonists and agonists of a PRO 1868 polypeptide that inhibits one or more of the functions or activities of a PRO 1868 polypeptide
  • the invention concerns a method for determining the presence of a PRO 1868 polypeptide comprising exposing a cell suspected of containing the polypeptide to an anti-PRO 1868 antibody and determining binding of the antibody to the cell
  • the present invention relates to a method of diagnosing an inflammatory related disease in a mammal, comprising detecting the level of expression of a gene encoding a PRO 1868 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of an inflammatory disease in the mammal.
  • the present invention relates to method of diagnosing an inflammatory disease in a mammal, comprising (a) contacting an anti-PRO 1868 antibody with a test sample of tissue culture cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO 1868 polypeptide.
  • the detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type. A larger quantity of complexes formed in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art.
  • the test sample is usually obtained from an individual suspected of having a deficiency or abnormality relating to the inflammatory response.
  • the present invention relates to a diagnostic kit, containing an anti-PRO 1868 antibody and a carrier (e.g., a buffer) in suitable packaging.
  • a carrier e.g., a buffer
  • the kit preferably contains instructions for using the antibody to detect the PRO 1868 polypeptide.
  • the invention concerns an article of manufacture, comprising: a container; a label on the container; and a composition comprising an active agent contained within the container; wherein the composition is effective for stimulating or inhibiting an inflammatory response in a mammal, the label on the container indicates that the composition can be used to treat an inflammatory disease, and the active agent in the composition is an agent stimulating or inhibiting the expression and/or activity of the PRO 1868 polypeptide .
  • the active agent is a PRO 1868 polypeptide or an anti-PRO 1868 antibody.
  • a further embodiment is a method for identifying a compound capable of inhibiting the expression and/or activity of a PRO 1868 polypeptide by contacting a candidate compound with a PRO 1868 polypeptide under conditions and for time sufficient to allow these two compounds to interact.
  • either the candidate compound or the PRO 1868 polypeptide is immobilized on a solid support.
  • the non-immobilized component carries a detectable label.
  • the invention relates to a method of treating an inflammatory disease, by administration of an effective therapeutic amount of a PRO 1868 antagonist to a patient in need thereof for the treatment of a disease selected from: inflammatory bowel disease, systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),
  • a disease selected
  • the invention provides a method for inhibiting the growth of tumor cells comprising exposing a cell which overexpresses a PRO 1868 polypeptide to an effective amount of an agent inhibiting the expression and/or activity of the PRO 1868 polypeptide
  • the agent preferably is an anti PRO 1868 polypeptide, a small organic and inorganic peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule
  • the agent, e g , anti-PRO 1868 antibody induces cell death
  • the tumor cells are further exposed to radiation treatment and/or a cytotoxic or chemotherapeutic agent
  • the invention concerns an article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained within the contamer, wherein the composition is effective for inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO 1868 poly
  • a cDNA clone (DNA77624-2515) has been identified, having homology to nucleic acid encoding A33 antigen, that encodes a novel polypeptide, designated in the present application as "PRO 1868"
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1868 polypeptide
  • the invention concerns an isolated nucleic acid molecule encoding a PRO 1868 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides 51 or about 141 and about 980, inclusive, of Figure 13 (SEQ ID NO 19) Preferably, hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203553 (DNA77624-2515) or (b) the complement of the nucleic acid molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203553 (DNA77624-2515)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 31 to about 310 inclusive of Figure 14 (SEQ ID NO: 1
  • the invention concerns an isolated nucleic acid molecule having at least 390 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1868 polypeptide having the sequence of amino acid residues from 1 or about 31 to about 310, inclusive of Figure 14 (SEQ ID NO 20), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, prefereably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), isolating the test DNA molecule
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1868 polypeptide, with or without the N-terminal signal sequence and/or the initiating methiomne, and its soluble, 1 e , transmembrane domain deleted or inactivated variants, or is complementary to such encoding nucleic acid molecule
  • the signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 30 in the sequence of Figure 14 (SEQ ID NO 20)
  • the transmembrane domain has been tentatively identified as extending from about amino acid position 243 to about amino acid position 263 in the PR01868 amino acid sequence ( Figure 14, SEQ ID NO 20)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of
  • the invention provides isolated native sequence PRO 1868 polypeptide, which in certain embodiments, includes an ammo acid sequence comprising residues 1 or about 31 to about 310 of Figure 14 (SEQ ID NO 20)
  • the invention concerns an isolated PRO 1868 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 31 to about 310, inclusive of Figure 14 (SEQ ID NO 20)
  • the invention concerns an isolated PRO 1868 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the ammo acid sequence of residues 1 or about 31 to about 310, inclusive of Figure 14 (SEQ ID NO 20)
  • the invention concerns an isolated PRO 1868 polypeptide, comprising the sequence of amino acid residues 1 or about 31 to about 310, inclusive of Figure 14 (SEQ ID NO 20), or a fragment thereof sufficient to provide a binding site for an anti-PRO 1868 antibody
  • the PRO 1868 fragment retains a qualitative biological activity of a native PRO 1868 polypeptide
  • the mvention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1868 polypeptide having the sequence of amino acid residues from about 1 or about 31 to about 310, inclusive of Figure 14 (SEQ ID NO 20), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95 % sequence identity to (a) or (b), (n) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO 1868 polypeptide.
  • the agonist or antagonist is an anti-PR01868 antibody.
  • the invention concerns a method of identifying agonists or antagonists of a native PRO 1868 polypeptide by contacting the native PRO 1868 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.
  • the invention concerns a composition
  • a composition comprising a PRO 1868 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.
  • the invention provides a composition containing a PRO 1868 polypeptide or an agonist or antagonist antibody in admixture with a carrier or excipient.
  • the composition contains a therapeutically affective amount of the peptide or antibody.
  • the composition when the composition contains an inflammation stimulating molecule, the composition is useful for: (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) stimulating or enhancing an immune response in a mammal in need thereof, or (c) increasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
  • the composition when the composition contains an inflammatory inhibiting molecule, the composition is useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an inflammatory response in a mammal in need thereof, or (c) decreasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
  • the composition contains a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent.
  • the composition is sterile.
  • the invention concerns nucleic acid encoding an anti-PRO 1868 antibody, and vectors and recombinant host cells comprising such nucleic acid.
  • the invention concerns a method for producing such an antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture.
  • a cDNA clone (DNA77631-2537) has been identified that encodes a novel polypeptide, designated in the present application as "PR03434. "
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR03434 polypeptide.
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding a PR03434 polypeptide having the sequence of amino acid residues from 1 or about 17 to about 1029, inclusive of Figure 16 (SEQ ID NO:22), or (b) the complement of the DNA molecule of (a).
  • the invention concerns an isolated nucleic acid molecule encoding a PR03434 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about residues 46 or about 94 and about 3132, inclusive, of Figure 15 (SEQ ID NO 21)
  • hybridization occurs under stringent hybridization and wash conditions
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203651 (DNA77631-2537), or (b) the complement of the DNA molecule of (a)
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No 203651 (DNA77631-2537)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95 % sequence identity to the sequence of ammo acid residues from 1 or about 17 to
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR03434 polypeptide, with or without the N terminal signal sequence and/or the initiating methiomne, or is complementary to such encoding nucleic acid molecule
  • the signal peptide has been tentatively identified as extending from amino acid position 1 through about amino acid position 16 in the sequence of Figure 16 (SEQ ID NO 22)
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 17 to about 1029, inclusive of Figure 16 (SEQ ID NO 22), or (b) the complement of the DNA of (a)
  • nucleic acid fragments are from about 20 to about 80 nucleotides m length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length, and most preferably from about 20 to about 40 nucleotides m length
  • the mvention provides isolated PR03434 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove defined
  • the invention provides isolated native sequence PR03434 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 or about 17 to 1029 of Figure 16 (SEQ ID NO:22).
  • the invention concerns an isolated PR03434 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 17 to about 1029, inclusive of Figure 16 (SEQ ID NO:22).
  • the invention concerns an isolated PR03434 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 17 to 1029 of Figure 16 (SEQ ID NO:22).
  • the invention concerns an isolated PR03434 polypeptide, comprising the sequence of amino acid residues 1 or about 17 to about 1029, inclusive of Figure 16 (SEQ ID NO:22), or a fragment thereof sufficient to provide a binding site for an anti-PR03434tibody .
  • the PR0982 fragment retains a qualitative biological activity of a native PR03434 polypeptide.
  • the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PR03434 polypeptide having the sequence of amino acid residues from 1 or about 17 to about 1029, inclusive of Figure 16 (SEQ ID NO:22), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85 % sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PR03434 polypeptide.
  • the agonist or antagonist is an anti-PR03434 antibody.
  • the invention concerns a method of identifying agonists or antagonists of a native PR03434 polypeptide, by contacting the native PR03434 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.
  • the invention concerns a composition
  • a composition comprising a PR03434 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.
  • a cDNA clone (DNA82307-2531 )has been identified that encodes a novel polypeptide having homology to glycosyltransferases, and is designated in the present application as "PR01927”.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1927 polypeptide .
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding a PRO 1927 polypeptide having the sequence of amino acid residues from 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a).
  • the invention concerns an isolated nucleic acid molecule encoding a PR01927 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about residues 120 and about 1694, inclusive, of Figure 17 (SEQ ID NO:23).
  • hybridization occurs under stringent hybridization and wash conditions.
  • the invention concerns an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity, preferably at least about 85 % sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203537 (DNA82307-2531), or (b) the complement of the DNA molecule of (a).
  • the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203537 (DNA82307-2531).
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues from 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or the complement of the DNA of (a).
  • the invention concerns an isolated nucleic acid molecule having at least about 50 nucleotides, and preferably at least about 100 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1927 polypeptide having the sequence of amino acid residues from 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a), and, if the DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), isolating the test DNA molecule.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO 1927 polypeptide, with or without the N-terminal signal sequence and/or the initiating methionine, and its soluble variants (i.e. transmembrane domain deleted or inactivated), or is complementary to such encoding nucleic acid molecule.
  • the signal peptide has been tentatively identified as extending from amino acid position 1 through about amino acid position 23 in the sequence of Figure 18 (SEQ ID NO:24).
  • a type II transmembrane domain has been tentatively identified as extending from about amino acid position 6 to about amino acid position 25 in the PR01927 amino acid sequence ( Figure 18, SEQ ID NO:24).
  • the invention concerns an isolated nucleic acid molecule comprising (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 85% positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or (b) the complement of the DNA of (a).
  • Another embodiment is directed to fragments of a PRO 1927 polypeptide coding sequence that may find use as hybridization probes.
  • nucleic acid fragments are from about 20 to about 80 nucleotides in length, preferably from about 20 to about 60 nucleotides in length, more preferably from about 20 to about 50 nucleotides in length, and most preferably from about 20 to about 40 nucleotides in length.
  • the invention provides isolated PRO 1927 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove defined.
  • the invention provides isolated native sequence PRO 1927 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 or about 24 to 548 of Figure 18 (SEQ ID NO:24).
  • the invention concerns an isolated PRO 1927 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to the sequence of amino acid residues 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24).
  • the invention concerns an isolated PR01927 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85 % positives, more preferably at least about 90% positives, most preferably at least about 95% positives when compared with the amino acid sequence of residues 1 or about 24 to 548 of Figure 18 (SEQ ID NO:24).
  • the invention concerns an isolated PRO 1927 polypeptide, comprising the sequence of amino acid residues 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or a fragment thereof sufficient to provide a binding site for an anti-PR01927 antibody.
  • the PR01927 fragment retains a qualitative biological activity of a native PRO 1927 polypeptide.
  • the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a PRO 1927 polypeptide having the sequence of amino acid residues from 1 or about 24 to about 548, inclusive of Figure 18 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% sequence identity, preferably at least about an 85% sequence identity, more preferably at least about a 90% sequence identity, most preferably at least about a 95% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO 1927 polypeptide.
  • the agonist or antagonist is an anti-PR01927 antibody.
  • the invention concerns a method of identifying agonists or antagonists of a native PRO 1927 polypeptide, by contacting the native PRO 1927 polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.
  • the invention concerns a composition comprising a PRO 1927 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier 10.
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides
  • Host cell comprising any such vector are also provided
  • the host cells may be CHO cells, E coli, or yeast
  • a process for producing any of the herein described polypeptides is further provided and comprises culturmg host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85 % sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93 % sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least at least
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83 % sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85 % sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93 % sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97 % sequence identity, yet more preferably at least about 98% sequence identity and yet more
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83 % sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85 % sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95 % sequence identity , yet more preferably at least about 96 % sequence identity , yet more preferably at least about 97% sequence identity, yet more preferably at least about
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain- inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides m length yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length yet more preferably at least about 150 nucleotides m length, yet more preferably at least about 160 nucleotides m length, yet more preferably at least about 170 nucle
  • the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
  • the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85 % sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positives, yet more preferably at least about 83 % positives, yet more preferably at least about 84% positives, yet more preferably at least about 85 % positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91 % positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more preferably at least at least
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein.
  • the agonist or antagonist is an anti-PRO antibody or a small molecule.
  • the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide.
  • the PRO polypeptide is a native PRO polypeptide.
  • the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
  • Figure 1 shows a nucleotide sequence (SEQ ID NO: 1) of a native sequence PRO1800 cDNA, wherein SEQ ID NO: l is a clone designated herein as "DNA35672-2508".
  • Figure 2 shows the amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: l shown in Figure 1.
  • Figure 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequence PR0539 cDNA, wherein
  • SEQ ID NO:6 is a clone designated herein as "DNA47465-1561 ".
  • Figure 4 shows the amino acid sequence (SEQ ID NO:7) derived from the coding sequence of SEQ ID NO:6 shown in Figure 3.
  • Figure 5 shows a nucleotide sequence (SEQ ID NO:8) of a native sequence PR0982 cDNA, wherein SEQ ID NO:8 is a clone designated herein as "DNA57700-1408" .
  • Figure 6 shows the amino acid sequence (SEQ ID NO:9) derived from the coding sequence of SEQ ID NO: 8 shown in Figure 5.
  • Figure 7 shows a nucleotide sequence (SEQ ID NO: 12) of a native sequence PRO 1434 cDNA, wherein SEQ ID NO: 12 is a clone designated herein as "DNA68818-2536" .
  • Figure 8 shows the amino acid sequence (SEQ ID NO: 13) derived from the coding sequence of SEQ
  • Figure 9 shows a nucleotide sequence (SEQ ID NO: 17) of a native sequence PR01863 cDNA, wherein SEQ ID NO: 17 is a clone designated herein as "DNA59847-2510".
  • Figure 10 shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 9.
  • Figure 11 shows a nucleotide sequence (SEQ ID NO : 19) of a native sequence PRO 1917 cDN A , wherein SEQ ID NO: 19 is a clone designated herein as "DNA76400-2528".
  • Figure 12 shows the ammo acid sequence (SEQ ID NO 20) derived from the coding sequence of SEQ ID NO 19 shown in Figure 1 1
  • Figure 13 shows a nucleotide sequence (SEQ ID NO 21 ) of a native sequence PRO 1868 cDN A, wherein SEQ ID NO 21 is a clone designated herein as "DNA77624-2515 '
  • Figure 14 shows the amino acid sequence (SEQ ID NO 22) derived from the coding sequence of SEQ ID NO 21 shown in Figure 13
  • Figure 15 shows a nucleotide sequence (SEQ ID NO 23) of a native sequence PR03434 cDNA, wherein SEQ ID NO 23 is a clone designated herein as "DNA77631-2537"
  • Figure 16 shows the amino acid sequence (SEQ ID NO 24) derived from the coding sequence of SEQ ID NO 23 shown in Figure 15
  • Figure 17 shows a nucleotide sequence (SEQ ID NO 25) ofa native sequence PRO 1927 cDNA, wherein
  • SEQ ID NO 25 is a clone designated herein as "DNA82307-2531"
  • Figure 18 shows the amino acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO 25 shown in Figure 17
  • PRO polypeptide and "PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (1 e , PRO/number) refers to specific polypeptide sequences as described herein
  • PRO/number polypeptide and “PRO/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein)
  • the PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
  • a “native sequence PRO polypeptide comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence PRO polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g.
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length ammo acids sequences shown in the accompanying figures Start and stop codons are shown m bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 m the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the ammo acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides.
  • the PRO polypeptide "extracellular domain” or “ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and c ⁇ toplasmic domains
  • a PRO polypeptide ECD will have less than 1 % of such transmembrane and/or c> toplasmic domains and preferably, will have less than 0 5 % of such domains
  • am transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain
  • the exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein
  • an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified m the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucle
  • PRO polypeptide variant means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein
  • Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C- termmus of the full-length native amino acid sequence
  • a PRO polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81 % ammo acid sequence identity, more preferably at least about 82% ammo acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% ammo acid sequence identity, more
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the ammo acid residues m the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc and the source
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given ammo acid sequence B is calculated as follows
  • NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997))
  • the NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • PRO variant polynucleotide or "PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein.
  • a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence
  • PRO variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.
  • Percent (%) nucleic acid sequence identity with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessarv , to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in v arious ways that are withm the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST 2, ALIGN or Megahgn (DNASTAR) software For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN 2 wherein the complete source code for the ALIGN 2 program is provided in Table 1 below The ALIGN 2 sequence comparison computer program was authored by Genentech, Inc and the source code shown in Table 1 below has been filed with user documentation in the U S Copyright Office, Washington D C , 20559,
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
  • % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN 2 computer program
  • % nucleic acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzymology 266 460-480 (1996))
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide encoding nucleic acid and the comparison nucleic acid molecule
  • Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to,
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • the % value of positives is determined by dividing (a) the number of amino acid residues scoring a positive value between the PRO polypeptide ammo acid sequence of interest having a sequence derived from the native PRO polypeptide sequence and the comparison ammo acid sequence of interest (I e , the amino acid sequence against which the PRO polypeptide sequence is being compared) as determined in the BLOSUM62 matrix of WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest
  • % value of positives is calculated as described in the immediately preceding paragraph
  • Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the ammo acid residue of interest or are a preferred substitution (as defined in Table 6 below) of the amino acid residue of interest
  • % value of positives of a given amino acid sequence A to, with, or against a given ammo acid sequence B is calculated as follows
  • Isolated, when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-protemaceous solutes
  • the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step
  • an "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide encoding nucleic acid
  • An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide- encoding nucleic acid molecule as it exists in natural cells
  • an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • antibody is used in the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al. , Current Protocols in Molecular Biology. Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 %Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1 % SDS
  • Moderately stringent conditions ma ⁇ be identified as described by Sambrook et al , Molecular Cloning A Laboratory Manual. New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and %SDS) less stringent that those described above
  • An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 °C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necerney to accommodate factors such as probe length and the like
  • 'epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide "
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes
  • Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
  • the term "immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an adhesin") with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an ammo acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (l e , is "heterologous"), and an immunoglobulin constant domain sequence
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the immunoglobulin constant domam sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1 , IgG-2, IgG 3, or IgG-4 subtypes, IgA (including IgA-1 and IgA 2), IgE, IgD or IgM "Active” or "
  • agonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or ammo acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc
  • Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented
  • “Chronic administration” refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time
  • “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc
  • the mammal is human
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, protems, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids such as glycine, glutamine, asparagme, argimne or lysme, monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dextrms, chelating agents such as EDTA, sugar alcohols such as mannitol or
  • Papain digestion of antibodies produces two identical antigen-bmding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc' fragment, a designation reflecting the ability to crystallize readily Pepsin treatment yields an F(ab') ⁇ fragment that has two antigen-combining sites and is still capable of cross linking antigen
  • Fv is the minimum antibody fragment which contains a complete antigen recognition and -binding site This region consists of a dimer of one heavy- and one light chain variable domain m tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen- bmding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affimtv than the entire binding sue
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH I) of the heavy chain Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab' SH is the designation herein for Fab in which the cysteme res ⁇ due(s) of the constant domains bear a free thiol group F(ab'), antibody fragments originally were produced as pairs of Fab fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • the "light chains' of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains
  • immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgGl , IgG2, IgG3, IgG4, IgA, and IgA2 "Single-chain Fv" or "sFv' antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present m a single polypeptide chain
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
  • sFv see Pluckthun m The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds , Springer- Verlag, New York,
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or , preferably , silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g. , controlled pore glass), polysaccharides (e.g. , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275, 149.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal.
  • a drug such as a PRO polypeptide or antibody thereto
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • tilel and t ⁇ le2 are two dna or two protein sequences
  • sequences can be in uppei- or lower-case an may contain ambiguity
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more ot its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /tmp to hold info about traceback.
  • OxFFFFFFF 1 ⁇ ⁇ 10, 1 ⁇ 11, 1 ⁇ 12, 1 ⁇ 13, 1 ⁇ 14,
  • dumpblockO * numsO -- put out a number line- dumpblockO * puthneO - put out a iine (name, [num] , seq, [num]): dumpblockO
  • static nm, /* matches in core — for checking */ static lmax, /* lengths of stripped file names */ static ⁇ j[2], /* jmp index for a path */ static nc[2]; /* number at start of current line */ static m[2], /* current elem number -- for gapping */ static s ⁇ z[2], static char *ps[2]; /* ptr to current element */ static char *po[2], /* ptr to next output char slot */ static char oouutt[[22]][[IP_LINE], /* output line */ static char star[P ] I], I* set by stars() */
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + ; ps[ ⁇ ] + + ,
  • *py++ *p ⁇ else if ( ⁇ slower(*px))
  • *py++ toupper(*px), if ( ⁇ ndex("ATGCU *(p ⁇ -l))) natgc + + ⁇
  • endgaps)? -siz • MAXGAP; ⁇ l + + ; ⁇ else if (siz > 0) ⁇ /* gap in first seq */ gapx+ + ; ngapx + siz;
  • the present invention provides newly identifiedand isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides
  • PRO polypeptides cDNAs encodmg various PRO polypeptides have been identified and isolated, as disclosed further detail in the Examples below
  • proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protem, and will not be changed
  • the prote encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO will be referred to as "PRO/number", regardless of their origin or mode of preparation
  • various cDNA clones have been deposited with the ATCC The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods m the art The predicted amino acid sequence can be determined from the nucleotide sequence
  • PRO1800 disclosed in the present application is a newly identified Hep27 homolog and possesses activity typical of that protein
  • PR0539 discloses a portion of the full-length native sequence PR0539 (shown in Figure 4 and SEQ ID NO 7) has certain amino acid sequence identity with a portion of a kinesin-related protein from Drosophila melanogaster (AF019250_1) Accordingly, it is presently believed that PR0539 disclosed in the present application is a newly identified member of the Hedgehog signaling pathway protein family and possesses activity typical of the Drosophila Costal 2 protein
  • DNA57700-1408 sequence encodes a novel secreted factor designated herein as PR0982.
  • PR0982 a novel secreted factor
  • PRO 1434 disclosed in the present application is a newly identified nei homolog and may possess activity typical of the nei protein family
  • the DNA59847-2510 clone was isolated from a human prostate tissue library As far as is known, the DNA59847-2510 sequence encodes a novel factor designated herein as PR01863, using the WU-BLAST2 sequence alignment computer program, no significant sequence identities to any known proteins were revealed
  • PR01917 (shown in Figure 14 and SEQ ID NO 20) has certain amino acid sequence identity with an inositol phosphatase designated in the Dayhoff database as " AF012714 1 " Accordingly, it is presently believed that PR01917 disclosed in the present application is a newly identified member of mositol phosphatase family and may possess enzymatic activity typical of mositol phosphatases
  • PRO 1868 polypeptide may find use in the therapeutic treatment of inflammatory diseases as described above and colorectal cancer
  • the DNA77631-2537 clone was isolated from a human aortic tissue library using a trapping technique that selects for nucleotide sequences encoding secreted proteins As far as is known, the DNA77631-2537 sequence encodes a novel factor designated herein as PR03434, using the WU-BLAST2 sequence alignment computer program, no significant sequence identities to any known proteins were revealed
  • PRO 1927 disclosed in the present application is a newly identified member of the glycosyltransferase family of proteins and may possess glycosylation activity B.
  • PRO variants can be prepared.
  • PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide.
  • amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PRO or in various domains of the PRO described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934.
  • Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO.
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide.
  • PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
  • PCR polymerase chain reaction
  • Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • neutral hydrophilic cys, ser, thr
  • Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al. , Nucl. Acids Res., .13:4331 (1986); Zoller et al. , Nucl. Acids Res.. 10:6487 (1987)]
  • cassette mutagenesis [Wells et al., Gene. 34:315 (1985)]
  • restriction selection mutagenesis [Wells et al. , Philos. Trans. R. Soc. London Ser A, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA
  • Scanning ammo acid analysis can also be employed to identify one or more ammo acids along a contiguous sequence
  • preferred scanning amino acids are relatively small, neutral amino acids
  • amino acids include alanine, glycine, se ⁇ ne, and cysteme
  • Alanme is typically a preferred scanning amino acid among this group because it eliminates the side chain beyond the beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science, 244 1081-1085 (1989)]
  • Alanine is also typically preferred because it is the most common ammo acid Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W H Freeman & Co , N Y ), Chothia, J Mol Biol , 150 1 (1976)] If alanine substitution does not yield adequate amounts of variant, an lsote ⁇ c amino acid can be used
  • Covalent modifications of PRO are included within the scope of this invention
  • One type of covalent modification includes reacting targeted ammo acid residues of a PRO polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N- or C terminal residues of the PRO De ⁇ vatization with bifunctional agents is useful, for instance, for crosslinking PRO to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa
  • Commonly used crosslinking agents include, e g , l , l-b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional lmidoesters, including disuccimmidyl esters such as 3,3'-d ⁇ th ⁇ ob ⁇ s(succm ⁇ m ⁇ dylprop ⁇ onate), bifunctional male
  • Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the ammo acid sequence
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threomne residues to the native sequence PRO (for O-linked glycosylation sites)
  • the PRO ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encodmg the PRO polypeptide at preselected bases such that codons are generated that will translate mto the desired amino acids
  • Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , m WO 87/05330 published 1 1 September 1987 and m Aplin and W ⁇ ston, CRC C ⁇ t Rev Biochem . pp 259 306 (1981)
  • Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem B ⁇ oph ⁇ s , 259 52 (1987) and by Edge et al , Anal Biochem .
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzymol , 138 350 (1987)
  • Another type of covalent modification of PRO comprises linking the PRO polypeptide to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301, 144, 4,670,417, 4,791 , 192 or4, 179,337
  • the PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence
  • a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO
  • the presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine (poly his) or poly-histidine-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol
  • Tag polypeptides include the Flag-peptide [Hopp et al , BioTechnologv.
  • the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin
  • an immunoglobulin also referred to as an "immunoadhesin”
  • a fusion could be to the Fc region of an IgG molecule
  • the Ig fusions preferably mclude the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide m place of at least one variable region within an Ig molecule
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgG 1 molecule
  • US Patent No 5 ,428 , 130 issued June 27, 1995 See also US Patent No 5 ,428 , 130 issued June 27, 1995.
  • PRO sequence or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g. , Stewart et l. , Solid-Phase Peptide Synthesis, W.H. Freeman Co. , San Francisco. CA (1969): Merrifield. J. Am. Chem. Soc , 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation.
  • Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions.
  • Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO.
  • DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples.
  • the PRO- encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
  • Probes such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al. , supra. Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. 2 Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured m conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation
  • principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach, M Butler, ed (IRL Press, 1991) and Sambrook et al , supra
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as E coh
  • E cob strains are publicly available, such as E cob K12 strain MM294 (ATCC 31,446), E cob X1776 (ATCC 31,537), E cob stram W3110 (ATCC 27,325) and K5 772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such as Eschertchia, e g , E cob, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimunum, Serratia, e g , Serratia marcesc
  • colt W3110 strain 9E4 which has the complete genotype tonA ptr3, E cob W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT kan r , E cob W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7
  • E cob W31 10 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation, and an E cob strain having mutant pe ⁇ plasmic protease disclosed in U S Patent No 4,946,783 issued 7 August 1990 Alternatively, //; vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schizosaccharomy cespombe (Beach and Nurse, Nature. 290 140 [1981], EP 139,383 published 2 May 1985), Kluweromyces hosts (U S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968-975 (1991)) such as, e g , K lactis (MW98-8C, CBS683, CBS4574, Louvencourt et al , J Bacte ⁇ ol .
  • Schizosaccharomy cespombe Beach and Nurse, Nature. 290 140 [1981], EP 139,383 published 2 May 1985
  • Kluweromyces hosts U S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968
  • K fragibs (ATCC 12,424), K bulgancus (ATCC 16,045), K wickeramu (ATCC 24, 178), K waltu (ATCC 56,500), K drosoph ⁇ arum (ATCC 36,906, Van den Berg et al , Bio/Technology, 8 135 (1990)), K thermotolerans , and K marxianus, yarrowia (EP 402,226), Pichia pastons (EP 183,070, Sreekrishna et al , J Basic Microbiol , 28 265-278 [1988]), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Case et al , Proc Natl Acad Sci USA.
  • Schwanniomyces such as Schwanniomyces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g., Neurospora, Pentcilbum, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A nidulans (Ballance et al , Biochem Biophys Res Commun . 112 284 289 [1983], Tilburn et al , Gene. 26 205-221 [1983], Yelton et al , Proc Natl Acad Sci USA.
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Biochemistry of Methylotrophs. 269 (1982)
  • Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV1 line transformed by SV40 (COS 7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J_ Gen Virol . 36 59 (1977)), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci. USA.
  • mice sertoh cells TM4, Mather, Biol Reprod , 23 243-251 (1980)
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PRO may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression
  • a replicable vector for cloning (amplification of the DNA) or for expression
  • the vector may, for example, be m the form of a plasmid, cosmid, viral particle, or phage
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to one or more of a signal sequence an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard hgation techniques which are known to the skilled artisan
  • the PRO may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicilhnase, lpp, or heat-stable enterotoxm II leaders
  • the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders, the latter described m U S Patent No 5,010, 182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362,
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors m mammalian cells
  • Selection genes will typically contain a selection gene, also termed a selectable marker
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxms, e g , ampicillin, neomycm, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e g , the gene encodmg D-alamne racemase for Bacilli
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA.
  • a suitable selection gene for use in yeast is the trpl gene present m the yeast plasmid YRp7 [Stinchcomb et al , Nature, 282.39 (1979), Kingsman et al , Gene, 7 141 (1979), Tschemper et al , Gene, 10 157 (1980)]
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 [Jones, Genetics. 85 12 (1977)]
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis
  • Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al., Nature, 275 615 (1978), Goeddel et al , Nature. 281 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res .
  • Promoters for use in bacterial systems also will contain a Shine Dalgarno (S D ) sequence operably linked to the DNA encoding PRO
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem . 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg , 7 149 (1968), Holland, Biochemistry, 17 4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexok ⁇ nase,pyruvatedecarboxylase,phosphofructok ⁇ nase, glucose- 6-phosphate isomerase, 3-phos ⁇ hoglycerate mutase, pyruvate kinase, t ⁇ osephosphateisomerase.phosphoglucose isomerase, and glucokinase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionem, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use m yeast expression are further described in EP 73,657
  • PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actm promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegal
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3 ' , untranslated regions of eukaryotic or viral DNAs or cDN As These regions contain nucleotide segments transcribed as polyadenylated fragments m the untranslated portion of the mRNA encoding PRO
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Natl Acad Sci USA, 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes
  • the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
  • Antibodies useful for immunohistochemical stammg and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope
  • PRO may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g T ⁇ ton-X 100) or by enzymatic cleavage Cells employed m expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, somcation, mechanical disruption, or cell lysing agents
  • PRO may be desired to purify PRO from recombinant cell proteins or polypeptides
  • the following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, protem A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the PRO
  • Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology. 182 (1990), Scopes, Protein Purification Principles and Practice. Springer- Verlag, New York (1982)
  • the purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO produced
  • PRO Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and m the generation of .anti-sense RNA and DNA PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein
  • the full-length native sequence PRO gene, or portions thereof may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding naturally occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein
  • the length of the probes will be about 20 to about 50 bases
  • the hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and mtrons of native sequence PRO
  • antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequences
  • Antisense or sense oligonucleotides comprise a fragment of the coding region of PRO DNA Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • Stein and Cohen Cancer Res 48 2659, 1988
  • van der Krol et al BioTechniques 6 958, 1988
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means
  • the antisense oligonucleotides thus may be used to block expression of PRO proteins
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases
  • Such oligonucleotides with resistant sugar linkages are stable in vivo (l e , capable of resistmg enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences
  • sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lys ⁇ ne) Further still, intercalating agents, such as ellipticme, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence Antisense or sense oligonucleotides ma be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP0 4 mediated DNA transfection electroporation, or by using gene transfer vectors such as Epstein-Barr virus In a preferred procedure, an antisense or sense oligonucleotide is
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described m WO 91/04753
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase
  • Antisense or sense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases m length, about 20 bases m length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases m length, about 55 bases in length, about 60 bases m length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more
  • the probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO coding sequences
  • Nucleotide sequences encoding a PRO can also be used to construct hybridization probes for mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries
  • the coding sequences for PRO encode a protein which binds to another protein (example, where the PRO is a receptor)
  • the PRO can be used in assays to identify the other proteins or molecules involved m the bindmg interaction
  • inhibitors of the receptor/hgand binding interaction can be identified Protems involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction
  • the receptor PRO can be used to isolate
  • Nucleic acids which encode PRO or its modified forms can also be used to generate either transgenic animals or "knock out animals which, in turn, are useful in the development and screemng of therapeutically useful reagents
  • a transgenic animal e g , a mouse or rat
  • a transgenic animal is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e g , an embryonic stage
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding PRO Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be targeted for
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition
  • non-human homologues of PRO can be used to construct a PRO "knock out" animal which has a defective or altered gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced into an embryonic stem cell of the animal
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques
  • a portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
  • several kilobases of unaltered flanking DNA both at the 5' and
  • Progeny harboring the homologously recombmed DNA in their germ cells can be identified by standard techniques .and used to breed animals in which all cells of the animal contain the homologously recombmed DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.
  • Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.
  • oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al. , Trends in Biotechnology 11. 205-210 [1993]).
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.
  • the PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid sequences may be used for recombinantly expressing those markers.
  • the nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
  • PRO polypeptides and nucleic acid molecules of the present invention may also be used for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another.
  • PRO nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
  • PRO polypeptides described herein may also be employed as therapeutic agents
  • the PRO polypeptides of the present mvention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the PRO product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington s Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)), in the form of lyophihzed formulations or aqueous solutions
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as gly
  • formulations to be used for in v ⁇ o administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophihzation and reconstitution
  • compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • the route of administration is in accord with known methods, e g injection or infusion by intravenous, intraperitoneal, mtracerebral, intramuscular, intraocular, mtraarte ⁇ al or intralesional routes, topical administration, or by sustained release systems
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned The determination of the appropriate dosage or route of administration is well withm the skill of an ordinary physician Animal experiments provide reliable guidance for the determination of effective doses for human therapy Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J and Chappell, W "The use of interspecies scaling in toxicokmetics" In Toxicokinetics and New Drug Development, Yacobi et al , Eds , Pergamon Press, New York 1989, pp 42-96
  • a PRO polypeptide or agonist or antagonist thereof When in vivo administration of a PRO polypeptide or agonist or antagonist thereof is employed, normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day, depending upon the route of administration Guidance as to particular dosages and methods of delivery is provided in the literature, see, for example, U S Pat Nos 4,657,760, 5,206,344, or 5,225,212 It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery m a manner different from that to another organ or tissue Where sustained-release administration of a PRO polypeptide is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the PRO polypeptide, microencapsulation of the PRO polypeptide is contemplated Microencapsulation of recombinant proteins for sustained release has been successfulh
  • the sustained-release formulations of these protems were developed using poly-lactic-coglycohc acid (PLGA) polymer due to its biocompatibihty and wide range of biodegradable properties
  • PLGA poly-lactic-coglycohc acid
  • the degradation products of PLGA, lactic and glycohc acids, can be cleared quickly within the human body
  • the degradabihty of this polymer can be adjusted from months to years depending on its molecular weight and composition Lewis, "Controlled release of bioactive agents from lactide/glycohde polymer, in M Chasin and R Langer (Eds ), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker New York, 1990), pp 1-41
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists)
  • Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art All assays for antagonists are common m that they call for contacting the drug candidate with a PRO polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact
  • the interaction is binding and the complex formed can be isolated or detected in the reaction mixture
  • the PRO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e g , on a microtiter plate, by covalent or non-covalent attachments
  • Non-covalent attachment generally is accomplished bv coating the solid surface with a solution of the PRO polypeptide and drying Alternativel
  • an immobilized antibody, e g a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surface
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e g , the coated surface containing the anchored component
  • the non-reacted components are removed, e g , by washing, and complexes anchored on the solid surface are detected
  • the originally non-immobilized component carries a detect
  • yeast GAL4 89 5789-5793 (1991)
  • Many transcriptional activators such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-bmding domain, the other one functioning as the transcription-activation domain
  • the yeast expression system described in the foregoing publications (generally referred to as the two hybrid system") takes advantage of this property, and employs two hybrid proteins, one m which the target protem is fused to the DNA-bmding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain
  • the expression of a GALl-t ⁇ cZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protein protein interaction Colonies containing interacting polypeptides are detected with a chromogemc substrate for ⁇ galactosidase
  • MATCHMAKERTM complete kit for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech
  • a reaction mixture is prepared containing the product of the gene and the intra or extracellular component under conditions and for a time allowing for the interaction and binding of the two products
  • a placebo may be added to a third reaction mixture, to serve as positive control
  • the binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove
  • the formation of a complex in the control react ⁇ on(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner
  • the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide
  • antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist
  • the gene encodmg the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting Coligan et al , Current Protocols in Immun , 1(2) Chapter 5 (1991)
  • expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide.
  • PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • labeled PRO polypeptide can be photoaffinity- linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
  • potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
  • Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5 ' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al. , Nucl. Acids Res. , 6:3073 (1979); Cooney et al., Science. 241: 456 (1988); Dervan et al., Science. 251 : 1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem..
  • oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide
  • antisense DNA oligodeoxy ⁇ bonucleotidesde ⁇ ved from the translation initiation site, e g , between about -10 and + 10 positions of the target gene nucleotide sequence, are preferred
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide
  • small molecules include, but are not limited to, small peptides or peptide-hke molecules, preferably soluble peptides, and synthetic non peptidyl organic or inorganic compounds
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA
  • Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology. 4 469-471 (1994), and PCT publication No WO 97/33551
  • Nucleic acid molecules in triple -helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition of these oligonucleotides is designed such that it promotes t ⁇ ple-hehx formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex
  • base pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex
  • the present invention further provides anti PRO antibodies
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies
  • the anti-PRO antibodies may comprise polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised m a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected m the mammal by multiple subcutaneous or intraperitoneal injections
  • the immunizing agent may include the PRO polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovme thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycol
  • the anti-PRO antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature. 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PRO polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J, Immunol.. 133:3001 (1984); Brodeur et al. , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc. , New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem.. 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra] .
  • Suitable culture media for this purpose include, for example, Dulbecco' s Modified Eagle ' s Medium and RPMI- 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g. , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) .
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al. , supral or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • the anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g. , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al.. Nature. 321:522-525 (1986): Riechmannet al.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al. , Nature, 332:323-327 (1988); Verhoeyen et al.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol. , 227:381 (1991); Marks et al., J. Mol. Biol.. 222:581 (1991)].
  • the techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al. , Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, p. 77 (1985) and Boerner et al. , J. Immunol.. 147(l):86-95 (1991)] .
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10. 779-783 (1992): Lonberg etal..
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the PRO
  • the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305:537-539 (1983)] .
  • the first heavy-chain constant region (CH I) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan etal. , Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al. , J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. , J. Immunol. 147:60 (1991).
  • bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein.
  • an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide.
  • These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • effector Function Engineering It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g. , the effectiveness of the antibody in treating cancer.
  • cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. , Exp Med. , 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research. 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson et al , Anti Cancer Drug Design, 3 219 230 ( 1989)
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (; e , a radioconjugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (; e , a radioconjugate)
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ⁇ cm A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleuntes fordii proteins, dianthin proteins, Phytolaca americana protems (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaona ⁇ a officinalis inhibitor, gelonm, mitogelhn, rest ⁇ ctocin, phenomycin, enomycin, and the t ⁇ cothecenes
  • radionuchdes are available for the production of radioconjugated antibodies Examples include ,p B ⁇ , 131 I, 131 In, W Y, and
  • the antibody may be conjugated to a "receptor ' (such streptavidm) for utilization in tumor pretargetmg wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e g , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide)
  • a "ligand” e g , avidin
  • cytotoxic agent e g , a radionucleotide
  • the antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al , Proc Natl Acad Sci USA. 82 3688 (1985), Hwang et al , Proc Natl Acad Sci USA. 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed m U S Patent No 5,013,556
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylchohne, cholesterol, and PEG-de ⁇ vat ⁇ zedphosphat ⁇ dylethanolam ⁇ ne(PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al .
  • chemotherapeutic agent such as Doxorubicm
  • a chemotherapeutic agent is optionally contained withm the liposome See Gabizon et al , J National Cancer Inst , 81(19) 1484 (1989)
  • compositions of Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be admmistered for the treatment of various disorders in the form of pharmaceutical compositions
  • the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, hpofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred For example, based upon the variable- region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology See, e s .
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent
  • Such molecules are suitably present m combination in amounts that are effective for the purpose intended
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydro xymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano particles, and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's
  • sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g , films, or microcapsules
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v ⁇ nylalcohol)), polylactides (U.S. Pat. No.
  • anti-PRO antibodies of the invention have various utilities
  • anti-PRO antibodies may be used in diagnostic assays for PRO, e g , detecting its expression in specific cells, tissues, or serum
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases fZola, Monoclonal Antibodies A Manual of Techniques. CRC Press.
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal
  • the detectable moiety may be a radioisotope, such as 3 H, 1 C, 3, P, 35 S, or 12 T, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamme, or lucife ⁇ n, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase Any method known m the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al , Nature.
  • Anti-PRO antibodies also are useful for the affinity purification of PRO from recombinant cell culture or natural sources
  • the antibodies against PRO are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art
  • the immobilized antibody then is contacted with a sample containing the PRO to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the PRO from the antibody
  • EXAMPLES Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA. EXAMPLE 1 Extracellular Domain Homolog ⁇ Screening to Identify Novel Polypeptides and cDNA Encoding Therefor
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss Prot public database were used to search EST databases
  • the EST databases included public databases (e g , Dayhoff, GenBank), and proprietary databases (e g LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST-2 (Altschul et al , Methods m Enzymology 266 460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program ' phrap" (Phil Green, University of Washington, Seattle, WA) Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using phrap In addition
  • oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp m length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5kbp
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation mto a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science. 253- 1278-1280 (1991)) in the unique Xhol and Notl sites
  • EXAMPLE 2 Isolation of cDNA clones by Amylase Screening 1
  • Preparation of oligo dT primed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RNA was used to generate an oligo dT primed cDNA library m the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System)
  • the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl linkered cDNA was cloned into Xhol/Notl cleaved vector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Notl cDNA cloning sites 2
  • a secondary cDNA library was generated in order to preferentially represent the 5 ' ends of the primary cDNA clones
  • Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above)
  • the double stranded cDNA was sized to 500-1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector pSST-AMY 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites
  • DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37°C) Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCl- gradient The purified DNA was then carried on to the yeast protocols below.
  • the yeast methods were divided into three categories (1) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.
  • yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, h ⁇ s3-l l, h ⁇ s3-15, MAL + , SUC + , GAL +
  • yeast mutants can be employed that have deficient post-translational pathways.
  • Such mutants may have translocation deficient alleles in seel I, sec!2, sec62, with truncated secll being most preferred
  • antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other protems implicated in this post translation pathway (e.g , SEC61p, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these protems may also be preferably employed in combination with the amylase- expressing yeast.
  • the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3 ), and resuspended into LiAc/TE (2.5 ml).
  • LiAc/TE 10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3
  • Transformation took place by mixing the prepared cells (100 ⁇ l) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 ⁇ g, vol. ⁇ 10 ⁇ l) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li 2 OOCCH 3 , pH 7.5) was added. This mixture was gently mixed and incubated at 30 °C while agitating for 30 minutes.
  • PEG/TE 600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li 2 OOCCH 3 , pH 7.5
  • the cells were then heat shocked at 42 °C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 ⁇ l) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
  • TE 500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5
  • the transformation was performed using a single , large scale reaction, wherein reagent amounts were scaled up accordingly.
  • the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al. , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208-210 (1994). Transformants were grown at 30°C for 2-3 days.
  • the detection of colonies secreting amylase was performed by including red starch in the selective growth media.
  • Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by
  • the positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies.
  • Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
  • PCR was then performed as follows a Denature 92°C, 5 minutes
  • the first 18 nucleotides of the 5 end of these oligonucleotides contained annealing sites for the sequencing primers
  • the total product of the PCR reaction from an empty vector was 343 bp
  • signal sequence-fused cDNA resulted in considerably longer nucleotide sequences
  • DNA30934 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA30934. Based on the DNA30934 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO 1800.
  • PCR primers forward and reverse were synthesized: forward PCR primer (30934. fl) 5'-GCATAATGGATGTCACTGAGG-3' (SEQ ID NO:3) reverse PCR primer (30934.rl) 5'-AGAACAATCCTGCTGAAAGCTAG-3' (SEQ ID NO:4) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30934 sequence which had the following nucleotide sequence hybridization probe (30934.pl) 5'-GAAACGAGGAGGCGGCTCAGTGGTGATCGTGTCTTCCATAGCAGCC-3' (SEQ ID NO:5)
  • RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PRO 1800 (designated herein as DNA35672-2508 [ Figure 1 , SEQ ID NO: l]; and the derived protein sequence for PRO1800.
  • DNA41882 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1. This consensus sequence is herein designated DNA41882. Based on the DNA41882 consensus sequence shown, oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0539 RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue DNA sequencing of the clones isolated as described abo ⁇ e gave the full length DNA sequence for PR0539 (designated herein as DNA47465-1561 [ Figure 3, SEQ ID NO 61, and the derived protein sequence for PR0539 The entire nucleotide sequence of DNA47465 1561 is shown m Figure 3 (SEQ ID NO 6) Clone
  • DNA47465-1561 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 186-188 and ending at the stop codon at nucleotide positions 2676-2678 (Figure 3)
  • the predicted polypeptide precursor is 830 amino acids long ( Figure 4)
  • the full length PR0539 protein shown in Figure 4 has an estimated molecular weight of about 95,029 daltons and a pi of about 8 26
  • Analysis of the full-length PR0539 sequence shown in Figure 4 evidences the presence of the following leucine zipper pattern sequences from about amino acid 557 to about amino acid 578 and from about amino acid 794 to about ammo acid 815, potential N glycosylation sites from about ammo acid 133 to about ammo acid 136 and from about amino acid 383 to about amino acid 386 and a kinesin-related protein K ⁇ f-4 coiled coil domain from about amino acid 231 to about amino acid 672 Clone DNA47465 1561 has been deposited with ATCC on February 9, 1999
  • Incyte EST cluster sequence no 43715 This EST sequence was compared to a variety of EST databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies
  • the full length clone shown in Figure 5 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 26-28 and ending at the stop codon found at nucleotide positions 401-403 (SEQ ID NO 8)
  • the predicted polypeptide precursor is 125 ammo acids long, has a calculated molecular weight of approximately 14, 198 daltons and an estimated pi of approximately 9 01
  • Analysis of the full length PR0982 sequence shown m Figure 6 (SEQ ID NO 9) evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 21 and potential anaphylatoxm domain from about ammo acid 50 to about amino acid 59
  • An analysis of the Davhoff database (version 35 45 SwissProt 35) evidenced homology between the PR0982 ammo acid sequence and the following Dayhoff sequences RNTMDCV_1 , A48151 , WAP RAT, S24596, A53640, MT4_HUMAN, U93486 1 , SYNB
  • DNA54187 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described m Example 1 above This consensus sequence is herein designated DNA54187 Based on the DNA54187 consensus sequence, oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full length coding sequence for
  • PCR primers (forward and reverse) were synthesized forward PCR primer 5'-GAGGTGTCGCTGTGAAGCCAACGG 3' (SEQ ID NO 12) reverse PCR primer 5' CGCTCGATTCTCCATGTGCCTTCC-3 (SEQ ID NO 13)
  • oligonucleotide hybridization probe was constructed from the consensus DNA54187 sequence which had the following nucleotide sequence hybridization probe
  • RNA for construction of the cDNA libraries was isolated from human retina tissue (LIB94) DNA sequencing of the clones isolated as described above gave the full length DNA sequence for PRO 1434
  • PRO 1434 sequence shown in Figure 8 (SEQ ID NO 11 ) evidences the presence of a variety of important protem domains as shown m Figure 8 Clone DNA68818-2536 has been deposited with ATCC on February 9, 1999 and is assigned ATCC deposit no 203657
  • Incyte EST cluster sequence no 82468 This EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (Lifeseq ® , Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies
  • EST expressed sequence tag
  • the consensus sequence obtained therefrom is herein designated DNA56029 In light of the sequence homology between the DNA56029 sequence and an EST sequence contained
  • Clone DNA59847-2510 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 17-19 and ending at the stop codon at nucleotide positions 1328-1330 ( Figure 9)
  • the predicted polypeptide precursor is 437 amino acids long ( Figure 10)
  • the full-length PRO 1863 protein shown in Figure 10 has an estimated molecular weight of about 46,363 daltons and a pi of about 6 22
  • Analysis of the full-length PR01863 sequence shown m Figure 10 evidences the presence of the following a signal peptide from about amino acid 1 to about ammo acid 15, a transmembrane domain from about amino acid 243 to about amino acid 260, potential N-glycosylation sites from about ammo acid 46 to about ammo acid 49, from about amino acid 189 to about amino acid 192 and from about amino acid 382 to about ammo acid 385, glycosaminoglycan attachment sites from about amino acid 51 to about amino acid 54 and from about ammo acid 359
  • EST cluster no. 85496 Use of the signal sequence algorithm described in Example 3 above allowed identification of an EST cluster sequence from the LIFESEQ ® database, designated EST cluster no. 85496. This EST cluster sequence was then compared to a the EST databases listed above to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al. , Methods in Enzymology 266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). The consensus sequence obtained therefrom is herein designated DNA56415.
  • PR01917 has a calculated molecular weight of approximately 55,051 daltons and an estimated pi of approximately 8.14. Additional features include: a signal peptide at about amino acids 1-30; potential N-glycosylation sites at about amino acids 242-245 and 481 -484 , protein kinase C phosphorylation sites at about amino acids 95-97, 182-184, and 427-429; N-myristoylation sites at about amino acids 107-112, 113- 118, 117-122, 118-123, and 128-133; and an endoplasmic reticulum targeting sequence at about amino acids 484-487.
  • DNA49803 Based up an observed homology between the DNA49803 consensus sequence and an EST sequence contained withm the Incyte EST clone no 2994689, Incyte EST clone no 2994689 was purchased and its insert obtained and sequenced The sequence of that insert is shown in Figure 13 and is herein designated DNA77624 2515
  • Clone DNA77631-2537 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 46-48 and ending at the stop codon at nucleotide positions 3133-3135 (Figure 15)
  • the predicted polypeptide precursor is 1029 ammo acids long ( Figure 16)
  • the full length PR03434 protem shown in Figure 16 has an estimated molecular w eight of about 114,213 daltons and a pi of about 6 42
  • Analysis of the full-length PR03434 sequence shown in Figure 16 evidences the presence of very important polypeptide domains as shown m Figure 16 Clone DNA77631 2537 has been deposited with ATCC on February 9, 1999 and is assigned ATCC deposit no 203651
  • EST Cluster No 1913 This EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included the databases listed above, including an additional proprietary EST DNA database (Genentech, South San Francisco, CA) to identify existing homologies
  • the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzymology 266 460-480 (1996)) Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap” (Phil Green, University of Washington, Seattle, Washington) The consensus sequence obtained therefrom is herein designated DNA73896
  • the full length clone shown in Figure 17 contained a single open reading frame with an apparent translational initiation site at nucleotide positions 51-53 and ending at the stop codon found at nucleotide positions 1695-1697 ( Figure 17, SEQ ID NO 23)
  • the predicted polypeptide precursor (Figure 18, SEQ ID NO 24) is 548 amino acids long PRO 1927 has a calculated molecular weight of approximately 63, 198 daltons and an estimated pi of approximately 8 10
  • Additional features include a signal peptide at about amino acids 1-23, a putative transmembrane domain at about amino acids 6 25, potential N-glycosylation sites at about ammo acids 5-8, 87-90, 103-106, and 465-469, potential N-my ⁇ stoylation sites at about amino acids 6-11, 136-141 , 370-375, and 509-514.
  • EXAMPLE 13 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay 67)
  • polypeptides of the invention are active as inhibitors of the proliferation of stimulated T-lymphocytes.
  • Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an immune response is beneficial.
  • peripheral blood mononuclear cells are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs). If desired, the cells are frozen in fetal bovine serum and DMSO after isolation.
  • Frozen cells may be thawed overnight in assay media (37°C, 5% C0 2 ) and then washed and resuspended to 3xl0 6 cells/ml of assay media (RPMI; 10% fetal bovine serum, 1 % penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non-essential amino acids, 1 % pyruvate).
  • assay media 37°C, 5% C0 2
  • assay media RPMI; 10% fetal bovine serum, 1 % penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non-essential amino acids, 1 % pyruvate.
  • the stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads).
  • the assay is prepared by plating in triplicate wells a mixture of: 100:1 of test sample diluted to 1 % or to 0.1 % ,
  • irradiated stimulator cells 50 :1 of irradiated stimulator cells, and 50 :1 of responder PBMC cells.
  • 100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control.
  • the wells are then incubated at 37°C, 5% C0 2 for 4 days. On day 5, each well is pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated.
  • PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice.
  • the cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1 % penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non-essential amino acids, 1 % pyruvate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 ⁇ m for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lxlO 7 cells/ml of assay media.
  • Lympholyte M Organon Teknika
  • This assay shows that certain polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at the site of injection of the animal Compounds which stimulate an immune response are useful therapeutically where stimulation of an immune response is beneficial
  • This skin vascular permeability assay is conducted as follows Hairless guinea pigs weighing 350 grams or more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM) A sample of purified polypeptide of the invention or a conditioned media test sample is injected intradermally onto the backs of the test animals with 100 ⁇ l per injection site It is possible to have about 10 30, preferably about 16-24, injection sites per animal One ⁇ l of Evans blue dye (1 % m physiologic buffered saline) is injected intracardially Blemishes at the injection sites are then measured (mm diameter) at 1 hr and 6 hr post
  • This assay shows that certain polypeptides of the invention act as potent mitogens for inner ear supporting cells which are auditory hair cell progenitors and, therefore, are useful for inducing the regeneration of auditory hair cells and treating hearing loss m mammals
  • the assay is performed as follows Rat UEC-4 ut ⁇ cular epithelial cells are ahquoted into 96 well plates with a density of 3000 cells/well in 200 ⁇ l of serum- containmg medium at 33°C The cells are cultured overnight and are then switched to serum-free medium at 37°C Various dilutions of PRO polypeptides (or nothing for a control) are then added to the cultures and the cells are incubated for 24 hours After the 24 hour incubation, 3 H thymidine (1 ⁇ Ci/well) is added and the cells are then cultured for an additional 24 hours The cultures are then washed to remove umnco ⁇ orated radiolabel, the cells harvested and Cpm per well determined Cpm of at least 30 % or greater in the PRO
  • PRO1800 , PR0539-, PR03434- and PR01927-encod ⁇ ng genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines Amplification is associated with overexpression of the gene product, indicating that the polypeptides are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast and other cancers and diagnostic determination of the presence of those cancers
  • Therapeutic agents may take the form of antagonists of PRO1800, PR0539, PR03434 or PR01927 polypeptide, for example, mu ⁇ ne-human chimeric, humanized or human antibodies against a PRO 1800, PR0539, PR03434 or PRO 1927 polypeptide
  • the starting material for the screen was genomic DNA isolated from a variety cancers
  • the DNA is quantitated precisely, e g , fluoromet ⁇ cally
  • DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy m healthy individuals (not shown)
  • the 5' nuclease assay for example, TaqManTM
  • real-time quantitative PCR for example, ABI P ⁇ zm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied Biosystems Division, Foster City, CA)
  • the results were used to determine whether the DNA encoding PRO 1800, PR0539, PR03434 or PRO 1927 is over-represented m any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were screened
  • the primary lung cancers were obtained from individuals with tumors of the type and stage as indicated in Table 6 An explanation of the abbreviations used for the designation of the primary tumors listed m Table 6
  • the results of the TaqManTM are reported in delta ( ⁇ ) Ct units.
  • One unit corresponds to 1 PCR cycle or approximately a 2-fold amplification relative to normal, two units corresponds to 4-fold, 3 units to 8-fold amplification and so on.
  • Quantitation was obtained using primers and a TaqManTM fluorescent probe derived from the PRO1800-, PR0539-, PR03434- or PR01927-encod ⁇ ng gene.
  • Regions of PRO1800, PR0539, PR03434 or PRO 1927 which are most likely to contain unique nucleic acid sequences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e.g , 3 '-untranslated regions
  • the sequences for the primers and probes (forward, reverse and probe) used for the PRO1800, PR0539, PR03434 or PRO 1927 gene amplification analysis were as follows PRO 1800 (DNA35672-2508) forward 5'-ACTCGGGATTCCTGCTGTT-3' (SEQ ID NO: 27) probe 5'-AGGCCTTTACCCAAGGCCACAAC-3' (SEQ ID NO:28) reverse 5'-GGCCTGTCCTGTGTTCTCA-3' (SEQ ID N0.29)
  • PRQ539 (DNA47465-1561) forward 5'-TCCCACCACTTACTTCCATGAA-3' (SEQ ID NO: 30) probe 5 ' -CTGTGGT ACCC AATTGCCGCCTTGT-3 ' (SEQ ID NO .31 ) reverse 5'-ATTGTCCTGAGATTCGAGCAAGA-3' (SEQ ID NO:32) PRQ3434 (DNA77631-2537) forward 5'-GTCCAGCAAGCCCTCATT-3' (SEQ ID NO:33) probe 5'-CTTCTGGGCCACAGCCCTGC-3" (SEQ ID NO:34) reverse 5'-CAGTTCAGGTCGTTTCATTCA-3' (SEQ ID NO:35)
  • PRO 1927 (DNA82307-2531) forward 5'-CCAGTCAGGCCGTTTTAGA-3' (SEQ ID NO:36) probe 5'-CGGGCGCCCAAGTAAAAGCTC-3' (SEQ ID NO:37) reverse 5 ' -C AT A AAGT AGT ATATGC ATTCC AGTGTT-3 ' (SEQ ID NO : 38)
  • the 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time.
  • Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction.
  • a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers.
  • the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe.
  • the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner.
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative inte ⁇ retation of the data.
  • the 5 ' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700TM Sequence Detection.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer.
  • the system amplifies samples in a 96-well format on a thermocycler.
  • laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD.
  • the system includes software for running the instrument and for analyzing the data.
  • Ct 5' Nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence.
  • the ⁇ Ct values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results.
  • Table 6 describes the stage, T stage and N stage of various primary tumors which were used to screen the PRO1800, PR0539, PR03434 and PR01927 compounds of the invention. Table 6 Primary Lung and Colon Tumor Profiles
  • DNA was prepared from cultured cell lines, primary tumors, normal human blood. The isolation was performed using purification kit, buffer set and protease and all from Quiagen, according to the manufacturer's instructions and the description below.
  • Cell culture lysis :
  • Cells were washed and trypsinized at a concentration of 7.5 x 10 8 per tip and pelleted by centrifuging at 1000 ⁇ m for 5 minutes at 4°C, followed by washing again with 1/2 volume of PBS recentrifugation. The pellets were washed a third time, the suspended cells collected and washed 2x with PBS. The cells were then suspended into 10 ml PBS. Buffer Cl was equilibrated at 4°C. Qiagen protease #19155 was diluted into 6.25 ml cold ddH 2 0 to a final concentration of 20 mg/ml and equilibrated at 4°C.
  • 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse A stock (100 mg/ml) to a final concentration of 200 ⁇ g/ml. Buffer C l ( 10 ml, 4°C) and ddH20 (40 ml, 4°C) were then added to the 10 ml of cell suspension, mixed by inverting and incubated on ice for 10 minutes. The cell nuclei were pelleted by cent ⁇ fuging m a Beckman swinging bucket rotor at 2500 ⁇ m at 4°C for 15 minutes The supernatant was discarded and the nuclei were suspended with a vortex mto 2 ml Buffer Cl (at 4°C) and 6 ml ddH,0, followed by a second 4°C centrifugation at 2500 ⁇ m for 15 minutes The nuclei were then resuspended mto the residual buffer using 200 ⁇ l per tip G2 buffer (10 ml) was added to the suspended nucle
  • Solid human tumor sample preparation and lysis Tumor samples were weighed and placed mto 50 ml conical tubes and held on ice Processing was limited to no more than 250 mg tissue per preparation (1 tip/preparation)
  • the protease solution was freshly prepared by diluting mto 6.25 ml cold ddH-,0 to a final concentration of 20 mg/ml and stored at 4°C G2 buffer (20 ml) was prepared by diluting DNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock)
  • the tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds using the large tip of the polytron in a laminar- flow TC hood in order to avoid inhalation of aerosols, and held at room temperature Between samples, the polytron was cleaned by spinning at 2 x 30 seconds each m 2L ddH-,0, followed by G2 buffer (50 ml) If tissue was still present on the generator tip, the apparatus was disassembled and cleaned
  • Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) with 10 ml QBT buffer QF elution buffer was equilibrated at 50°C The samples were vortexed for 30 seconds, then loaded onto equilibrated tips and drained by gravity The tips were washed with 2 x 15 ml QC buffer The DNA was eluted into 30 ml silamzed, autoclaved 30 ml Corex tubes with 15 ml QF buffer (50"C) Isopropanol (10 5 ml) was added to each sample, the tubes covered with parafin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centrifugation in the SS-34 rotor at 15,000 ⁇ m for 10 minutes at 4°C The pellet location was marked, the supernatant discarded, and 10 ml 70% ethanol (4°C) was added Samples were pelleted again by centrifugation on the SS 34 rotor at 10,000 ⁇ m for 10 minutes
  • DNA levels in each tube were quantified by standard A 260 , A, 80 spectrophotometry on a 1 20 dilution (5 ⁇ l DNA + 95 ⁇ l ddH,0) using the 0 1 ml quartz cuvetts in the Beckman DU640 spectrophotometer A 260 /A 280 ratios were in the range of 1 8-1 9
  • Each DNA samples was then diluted further to approximately 200 ng/ml in TE (pH 8 5) If the original material was highly concentrated (about 700 ng/ ⁇ l), the material was placed at 50°C for several hours until resuspended
  • Fluoromet ⁇ c DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up for about 15 minutes
  • the Hoechst dye working solution (#H33258, 10 ⁇ l, prepared within 12 hours of use) was diluted into 100 ml 1 x TNE buffer A 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zeroed pGEM 3Zf(+) (2 ⁇ l, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units An additional 2 ⁇ l of pGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 +/ 10 units Each sample was then read at least m triplicate When 3 samples were found to be within 10% of each other, their average was taken and this value was used as the quantification value
  • the fluoromet ⁇ cly determined concentration was then used to dilute each sample to 10 ng/ ⁇ l in ddH 2 0 This was done simultaneously on all template samples for a single TaqMan plate assay, and with enough material to run 500-1000 assays
  • the samples were tested in triplicate with TaqmanTM primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls
  • the diluted samples were used provided that the CT value of normal human DNA subtracted from test DNA was +/- 1 Ct
  • the diluted, lot-qualified genomic DNA was stored m l 0 ml ahquots at -80°C Ahquots which were subsequently to be used in the gene amplification assay were stored at 4°C Each 1 ml aliquot is enough for 8-9 plates or 64 tests Gene amplification assay
  • the PRO1800, PR0539, PR03434 and PR01927 compounds of the invention were screened in the following primary tumors and the resulting ⁇
  • This assay shows that certain polypeptides of the invention act to induce an increase in the number of pancreatic ⁇ -cell precursor cells and, therefore, are useful for treating various insulin deficient states in mammals, including diabetes mellitus.
  • the assay is performed as follows. The assay uses a primary culture of mouse fetal pancreatic cells and the primary readout is an alteration in the expression of markers that represent either ⁇ -cell precursors or mature ⁇ -cells. Marker expression is measured by real time quantitative PCR (RTQ- PCR); wherein the marker being evaluated is a transcription factor called Pdxl .
  • RTQ- PCR real time quantitative PCR
  • the pancreata are dissected from E14 embryos (CD1 mice). The pancreata are then digested with collagenase/dispase in F12/DMEM at 37°C for 40 to 60 minutes (collagenase/dispase, 1.37 mg/ml, Boehringer Mannheim, #1097113). The digestion is then neutralized with an equal volume of 5 % BSA and the cells are washed once with RPMI1640. At day 1 , the cells are seeded into 12-well tissue culture plates (pre-coated with laminin, 20 ⁇ g/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata from 1-2 embryos are distributed per well.
  • the culture medium for this primary cuture is 14F/1640.
  • the media is removed and the attached cells washed with RPMI/ 1640. Two mis of minimal media are added in addition to the protein to be tested.
  • the media is removed and RNA prepared from the cells and marker expression analyzed by real time quantitative RT-PCR. A protein is considered to be active in the assay if it increases the expression of the relevant ⁇ -cell marker as compared to untreated controls.
  • 14F/1640 is RPMI 1640 (Gibco) plus the following: group A 1: 1000 group B 1 : 1000 recombinant human insulin 10 ⁇ g/ml
  • Bovine pituitary extract 60 ⁇ g/ml
  • Gentamycin 100 ng/ml Group A (in 10ml PBS) Transferrin, lOOmg (Sigma T2252)
  • transferrin 10 ⁇ g/ml
  • insulin 1 ⁇ g/ml
  • gentamycin 100 ng/ml
  • aprotinin 50 ⁇ g/ml
  • BPE 15 ⁇ g/ml
  • This assay shows that certain polypeptides of the invention act to induce differentiation of pancreatic ⁇ -cell precursor cells into mature pancreatic ⁇ cells and, therefore, are useful for treating various insulin deficient states in mammals, including diabetes melhtus
  • the assay is performed as follows The assay uses a primary culture of mouse fetal pancreatic cells and the primary readout is an alteration m the expression of markers that represent either ⁇ -cell precursors or mature ⁇ -cells Marker expression is measured by real time quantitative PCR (RTQ-PCR), wherein the marker being evaluated is insulin
  • the pancreata are dissected from E14 embryos (CD1 mice) The pancreata are then digested with collagenase/dispase in F12/DMEM at 37°C for 40 to 60 minutes (collagenase/dispase, 1 37 mg/ml, Boehringer Mannheim, #1097113) The digestion is then neutralized with an equal volume of 5 % BSA and the cells are washed once with RPMI1640 At day 1 , the cells are seeded into 12 well tissue culture plates (pre-coated with laminin, 20 ⁇ g/ml in PBS, Boehringer Mannheim, #124317) Cells from pancreata from 1 2 embryos are distributed per well The culture medium for this primary cuture is 14F/1640 At day 2, the media is removed and the attached cells washed with RPMI/ 1640 Two mis of minimal media are added in addition to the protein to be tested At day 4, the media is removed and RNA prepared from the cells and marker expression analyzed by real time quantitative RT-PCR A
  • Epidermal Growth Factor lOO ⁇ g (BRL 100004) Trnodothyronme,10 ⁇ l of 5x10 6 M (Sigma T5516) Ethanolamine, lOO ⁇ l of 10 ' M (Sigma E0135) Phosphoethalamine, lOO ⁇ l of 10 ' M (Sigma P0503) Selenium, 4 ⁇ l of 10 ' M (Aesar #12574)
  • Hydrocortisone 2 ⁇ l of 5X10 3 M (Sigma #H0135)
  • Progesterone lOO ⁇ l of 1 10 3 M (Sigma #P6149)
  • This assay shows that certain polypeptides of the invention act to induce proliferation of mammalian kidney mesangial cells and, therefore, are useful for treating kidney disorders associated with decreased mesangial cell function such as Berger disease or other nephropathies associated with Schonlem-Henoch pu ⁇ ura, celiac disease, dermatitis he ⁇ etiformis or Crohn disease
  • the assay is performed as follows On day one, mouse kidney mesangial cells are plated on a 96 well plate in growth media (3 1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium, 95 % fetal bovine serum, 5 % supplemented with 14 mM HEPES) and grown overnight On day 2, PRO polypeptides are diluted at 2 concentrat ⁇ ons(l % and 0 1 %) m serum-free medium and added to the cells Control samples are serum free medium alone On day 4, 20 ⁇ l of the Cell Titer 96 Aqueous one solution reagent (Progema)
  • EXAMPLE 20 Fibroblast (BHK-21) Proliferation This assay shows that certain pol> peptides of the invention act to induce proliferation of mammalian fibroblast cells in culture and, therefore, function as useful growth factors in mammalian systems
  • the assay is performed as follows BHK-21 fibroblast cells plated in standard growth medium at 2500 cells/well m a total volume of 100 ⁇ l
  • the PRO polypeptide, ⁇ -FGF (positive control) or nothing (negative control) are then added to the wells in the presence of 1 ⁇ g/ml of hepa ⁇ n for a total final volume of 200 ⁇ l
  • the cells are then incubated at 37°C for 6 to 7 days After incubation, the media is removed, the cells are washed with PBS and then an acid phosphatase substrate reaction mixture (100 ⁇ l/well) is added The cells are then incubated at 37°C for 2 hours 10 ⁇ l per well of IN NaOH is then added to stop the acid phosphatas
  • This assay shows that certain pol ⁇ peptides of the invention act to induce redifferentiation of chondrocytes, therefore, are expected to be useful for the treatment of various bone and/or cartilage disorders such as, for example, sports injuries and arthritis
  • the assay is performed as follows Porcine chondrocytes are isolated by overnight collagenase digestion of articulary cartilage of metaca ⁇ ophalangeal joints of 4 6 month old female pigs The isolated cells are then seeded at 25,000 cells/cm 1 in Ham F-12 containing 10% FBS and 4 ⁇ g/ml gentamycin The culture media is changed every third day and the cells are then seeded in 96 well plates at 5,000 cells/well m lOO ⁇ l of the same media without serum and 100 ⁇ l of the test PRO polypeptide, 5 nM staurosporm (positive control) or medium alone (negative control) is added to give a final volume of 200 ⁇ l/well After 5 days of incubation at 37°C , a picture of each
  • EXAMPLE 22 Use of PRO as a hybridization probe The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe
  • DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions
  • Hybridization of radiolabeled PRO-de ⁇ ved probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH
  • DNAs having a desired sequence identity with the DNA encodmg full-length native sequence PRO can then be identified using standard techniques known in the art
  • This example illustrates preparation of an unglycosylated form of PRO by recombinant expression in E cob
  • the DNA sequence encoding PRO is initially amplified using selected PCR primers
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector
  • a variety of expression vectors may be employed
  • An example of a suitable vector is pBR322 (derived from E cob, see Bolivar et al , Gene, 2_95 (1977)) which contains genes for ampicillin and tetracycline resistance
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then ligated into the vector
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a t ⁇ promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene
  • the hgation mixture is then used to transform a selected E cob strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight m liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, du ⁇ ng which the expression promoter is turned on
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation
  • the cell pellet obtained by the centrifugation can be solubihzed using various agents known in the art, and the solubihzed PRO protem can then be purified using a metal chelating column under conditions that allow tight binding of the protein

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Abstract

L'invention a trait à de nouveaux polypeptides et à des molécules d'acides nucléiques codant pour ces polypeptides. L'invention concerne également des vecteurs et des cellules hôtes contenant ces séquences d'acides nucléiques, des molécules polypeptidiques chimères contenant les polypeptides de l'invention fusionnés à des séquences polypeptidiques hétérologues, des anticorps qui se lient aux polypeptides de la présente invention et des procédés de production des polypeptides de l'invention.
EP99962992A 1998-12-16 1999-12-01 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ceux-ci Withdrawn EP1141285A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DK02016081T DK1300417T3 (da) 1999-01-12 1999-12-01 Secerneret og transmembrant polypeptid og nucleinsyre kodende derfor
EP02016080A EP1283215B1 (fr) 1999-02-10 1999-12-01 Polypeptide sécrété ainsi que les acides nucléiques le codant
EP02016081A EP1300417B1 (fr) 1999-01-12 1999-12-01 Polypeptide sécrété et transmembranaire ainsi que les acide nucléique codant pour celle-ci
EP02016082A EP1251173B1 (fr) 1998-12-16 1999-12-01 Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
DK02016082T DK1251173T3 (da) 1998-12-16 1999-12-01 Secernerede og transmembrane polypeptider og nucleinsyrer kodende for disse
EP02016083A EP1277833B1 (fr) 1999-02-12 1999-12-01 Gène amplifié dans des tumeurs et matériaux et méthodes associées
DK02016083T DK1277833T3 (da) 1999-02-12 1999-12-01 Gener, der opformeres i tumorer, samt relaterede materialer og metoder

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US11285198P 1998-12-16 1998-12-16
US11314598P 1998-12-16 1998-12-16
US113145P 1998-12-16
US112851P 1998-12-16
US11351198P 1998-12-22 1998-12-22
US113511P 1998-12-22
US11555899P 1999-01-12 1999-01-12
US11573399P 1999-01-12 1999-01-12
US11556599P 1999-01-12 1999-01-12
US115733P 1999-01-12
US115558P 1999-01-12
US115565P 1999-01-12
US11934199P 1999-02-09 1999-02-09
US119341P 1999-02-09
US11953799P 1999-02-10 1999-02-10
US119537P 1999-02-10
US11996599P 1999-02-12 1999-02-12
US119965P 1999-02-12
PCT/US1999/012252 WO1999063088A2 (fr) 1998-06-02 1999-06-02 Proteines membranaires et acides nucleiques codant ces proteines
WOPCT/US99/12252 1999-06-02
PCT/US1999/028634 WO2000036102A2 (fr) 1998-12-16 1999-12-01 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ceux-ci

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EP02016081A Division EP1300417B1 (fr) 1999-01-12 1999-12-01 Polypeptide sécrété et transmembranaire ainsi que les acide nucléique codant pour celle-ci
EP02016080A Division EP1283215B1 (fr) 1999-02-10 1999-12-01 Polypeptide sécrété ainsi que les acides nucléiques le codant
EP02016082A Division EP1251173B1 (fr) 1998-12-16 1999-12-01 Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP02016083A Division EP1277833B1 (fr) 1999-02-12 1999-12-01 Gène amplifié dans des tumeurs et matériaux et méthodes associées

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