WO2000037640A2 - Compositions and methods for the treatment of tumor - Google Patents

Compositions and methods for the treatment of tumor Download PDF

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Publication number
WO2000037640A2
WO2000037640A2 PCT/US1999/030095 US9930095W WO0037640A2 WO 2000037640 A2 WO2000037640 A2 WO 2000037640A2 US 9930095 W US9930095 W US 9930095W WO 0037640 A2 WO0037640 A2 WO 0037640A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
prol
pro509
pro201
Prior art date
Application number
PCT/US1999/030095
Other languages
French (fr)
Other versions
WO2000037640A9 (en
WO2000037640A3 (en
Inventor
David Botstein
Audrey Goddard
Austin L. Gurney
Kenneth Hillan
David A. Lawrence
Margaret Ann Roy
William I. Wood
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/US1999/005028 external-priority patent/WO1999046281A2/en
Priority claimed from PCT/US1999/012252 external-priority patent/WO1999063088A2/en
Priority claimed from PCT/US1999/020111 external-priority patent/WO2000012708A2/en
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/en
Priority claimed from PCT/US1999/028313 external-priority patent/WO2000032221A2/en
Priority claimed from PCT/US1999/028409 external-priority patent/WO2000032778A2/en
Priority claimed from PCT/US1999/028301 external-priority patent/WO2000032776A2/en
Priority claimed from PCT/US1999/028565 external-priority patent/WO2000037638A2/en
Priority to KR1020017007877A priority Critical patent/KR20010102960A/en
Priority to AU21928/00A priority patent/AU2192800A/en
Priority to IL14321299A priority patent/IL143212A0/en
Priority to JP2000589694A priority patent/JP2003524390A/en
Priority to EP99966374A priority patent/EP1141289A2/en
Priority to CA002353775A priority patent/CA2353775A1/en
Priority to MXPA01006345A priority patent/MXPA01006345A/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to CA002361840A priority patent/CA2361840A1/en
Priority to AU28837/00A priority patent/AU2883700A/en
Priority to JP2000603377A priority patent/JP2004513602A/en
Priority to PCT/US2000/004341 priority patent/WO2000053756A2/en
Priority to KR1020017011399A priority patent/KR20030002292A/en
Priority to EP00907314A priority patent/EP1263948A2/en
Priority to PCT/US2000/004342 priority patent/WO2000078961A1/en
Priority to AU28839/00A priority patent/AU2883900A/en
Priority to PCT/US2000/004414 priority patent/WO2001004311A1/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/en
Priority to CA002362427A priority patent/CA2362427A1/en
Priority to EP00913764A priority patent/EP1220905A2/en
Priority to JP2000603379A priority patent/JP2004516227A/en
Priority to KR1020017011406A priority patent/KR20010103046A/en
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to AU37743/00A priority patent/AU3774300A/en
Priority to PCT/US2000/008439 priority patent/WO2000073454A1/en
Priority to JP2001500766A priority patent/JP2004522402A/en
Priority to AT00916675T priority patent/ATE478145T1/en
Priority to EP00916675A priority patent/EP1210418B1/en
Priority to CA002383254A priority patent/CA2383254A1/en
Priority to PCT/US2000/013705 priority patent/WO2000073445A2/en
Priority to CA002376116A priority patent/CA2376116A1/en
Priority to ES00939307T priority patent/ES2307515T3/en
Priority to JP2001500757A priority patent/JP4297317B2/en
Priority to DK00939307T priority patent/DK1212417T3/en
Priority to AU54412/00A priority patent/AU5441200A/en
Priority to AT00939307T priority patent/ATE393825T1/en
Priority to PT00939307T priority patent/PT1212417E/en
Priority to EP00939307A priority patent/EP1212417B1/en
Priority to EP07004855A priority patent/EP1820860A3/en
Priority to CA002373915A priority patent/CA2373915A1/en
Priority to AT00941164T priority patent/ATE357518T1/en
Priority to DK00941164T priority patent/DK1185648T3/en
Priority to EP07005021A priority patent/EP1873244A3/en
Priority to PCT/US2000/014941 priority patent/WO2000073348A2/en
Priority to ES00941164T priority patent/ES2287020T3/en
Priority to EP07005023A priority patent/EP1867719A3/en
Priority to EP00941164A priority patent/EP1185648B1/en
Priority to EP07005022A priority patent/EP1870464A3/en
Priority to JP2001500672A priority patent/JP2003524406A/en
Publication of WO2000037640A2 publication Critical patent/WO2000037640A2/en
Priority to AT00959474T priority patent/ATE419348T1/en
Priority to CA002384055A priority patent/CA2384055A1/en
Priority to JP2001520865A priority patent/JP3988821B2/en
Priority to ES00959474T priority patent/ES2317847T3/en
Priority to DE60041266T priority patent/DE60041266D1/en
Priority to EP00959474A priority patent/EP1208201B9/en
Priority to PCT/US2000/023522 priority patent/WO2001016319A2/en
Priority to AU70793/00A priority patent/AU7079300A/en
Publication of WO2000037640A9 publication Critical patent/WO2000037640A9/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
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Priority to EP06000589A priority patent/EP1661997A1/en
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Priority to EP00983846A priority patent/EP1250426A2/en
Priority to CA002490853A priority patent/CA2490853A1/en
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Priority to AU20554/01A priority patent/AU2055401A/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to CA002494705A priority patent/CA2494705A1/en
Publication of WO2000037640A3 publication Critical patent/WO2000037640A3/en
Priority to US09/866,028 priority patent/US6642360B2/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,759 priority patent/US20030077654A1/en
Priority to US09/902,853 priority patent/US20020192659A1/en
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Priority to US09/902,634 priority patent/US20030082540A1/en
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Priority to US09/903,925 priority patent/US20030096233A1/en
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Priority to US09/905,056 priority patent/US20030054441A1/en
Priority to US09/905,125 priority patent/US6664376B2/en
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Priority to US09/904,859 priority patent/US20030036060A1/en
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Priority to US09/904,920 priority patent/US6806352B2/en
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Priority to US09/944,896 priority patent/US7189566B2/en
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Priority to US10/011,833 priority patent/US6951920B2/en
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Priority to JP2006225770A priority patent/JP4072181B2/en
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Priority to JP2006225771A priority patent/JP4074645B2/en
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Priority to US11/796,725 priority patent/US20090197301A1/en
Priority to JP2007172879A priority patent/JP5512073B2/en
Priority to JP2007326609A priority patent/JP2008148701A/en
Priority to JP2007326424A priority patent/JP2008167749A/en
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Priority to JP2008145176A priority patent/JP2009019032A/en
Priority to JP2011138276A priority patent/JP2011236224A/en

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/46—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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to compositions and methods for the diagnosis and treatment of tumor.
  • Malignant tumors are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin.. 43:7 [ 1993]).
  • Cancer is characterized by an increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis).
  • a cell proliferates under conditions in which normal cells would not grow.
  • Cancer manifests itself in a wide variety of forms, characterized by different d ⁇ grees of invasiveness and aggressiveness. Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which are a common feature of all cancers.
  • tumor suppression genes which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth.
  • tumor suppression genes which would normally function to prevent malignant cell growth
  • oncogenes certain dominant genes, such as oncogenes, that act to promote malignant growth.
  • Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter, Cell. 64: 1129 [1991 ] and Bishop,
  • a well known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification. This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al., Adv. Cancer Res.. 47:235-281 [1986]). It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made.
  • Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer.
  • the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor (p 185 HER2 ; HER2) related to the epidermal growth factor receptor EGFR)
  • erbB2 also known as her2, or c-erbB-2
  • p 185 HER2 a transmembrane glycoprotein receptor
  • HER2 transmembrane glycoprotein receptor
  • a recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or HerceptinTM) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol., 14:737-744 [1996]).
  • anti-HER2 humanized anti-ErbB2
  • the present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans.
  • the present invention is based on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment.
  • the present invention concerns an isolated antibody which binds to a polypeptide designated herein as a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • the isolated antibody specifically binds to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide.
  • the antibody induces the death of a cell which expresses a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • the cell that expresses the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type.
  • the antibody is a monoclonal antibody, which preferably has non- human complementarity determining region (CDR) residues and human framework region (FR) residues.
  • the antibody may be labeled and may be immobilized on a solid support.
  • the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody which binds, preferably specifically, to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • the invention concerns a composition of matter which comprises an antibody which binds, preferably specifically, to a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in admixture with a pharmaceutically acceptable carrier.
  • the composition of matter comprises a therapeutically effective amount of the antibody.
  • the composition comprises 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 isolated nucleic acid molecules which encode anti- PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies, and vectors and recombinant host cells comprising such nucleic acid molecules.
  • the invention concerns a method for producing an anti-PRO201 , anti- PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PR01 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture.
  • the invention further concerns antagonists of a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide that inhibit one or more of the biological and/or immunological functions or activities of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PRO715,PRO1017, PROl 112, PRO509, PRO853 or PR0882 polypeptide or the complement thereof.
  • Theisolated nucleic acid molecule is preferably DNA, and hybridization preferably occurs under stringent hybridization and wash conditions.
  • Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the transcription and/or translation of the respective amplified genes, or as antisense primers in amplification reactions.
  • sequences can be used as part of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes.
  • the invention provides a method for determining the presence of a PRO201 ,
  • PR0292 PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509,
  • PR0344, PR0343.PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide wherein the method comprises exposing the sample to an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PRO 1 112, anti- PRO509, anti-PR0853 or anti-PR0882 antibody and determining binding of the antibody to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in the sample.
  • the invention provides a method for determining the presence of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PROlOl 7, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and determining binding of the antibody to the cell.
  • the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained.
  • the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti- PR0882 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of acomplex between theanti-PRO201, anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR
  • 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.
  • test sample is usually obtained from an individual suspected to have neoplastic cell growth or proliferation (e.g. cancerous cells).
  • the present invention concerns a cancer diagnostic kit comprising an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PROlOl 7, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 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 presence of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in a sample suspected of containing the same.
  • the invention concerns a method for inhibiting the growth of tumor cells comprising exposing tumor cells which express a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to an effective amount of an agent which inhibits a biological and/or immunological activity and/or the expression of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, wherein growth of the tumor cells is thereby inhibited.
  • the agent preferably is an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule.
  • the agent e.g., the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti- PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 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 container; wherein the composition is effective for inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882 polypeptide as compared to a normal cell of the same tissue type.
  • the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • the active agent is an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti- PR0853 or anti-PR0882 antibody or an antisense oligonucleotide.
  • the invention also provides a method for identifying a compound that inhibits an activity of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, comprising contacting a candidate compound with a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether a biological and or immunological activity of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide is inhibited.
  • either the candidate compound or the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is immobilized on a solid support.
  • the non-immobilized component carries a detectable label.
  • this method comprises the steps of (a) contacting cells and a candidate compound to be screened in the presence of the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
  • the invention provides a method for identifying a compound that inhibits the expression of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is inhibited.
  • this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and (b) determining the inhibition of expression of said polypeptide.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about
  • 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 99% sequence identity to (a) a DNA molecule encoding a PRO201, PR0292, PR03
  • 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 about 99%
  • 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 98% sequence identity and yet more more 80% sequence identity,
  • Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • soluble extracellular domains of the herein described PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 1 12, anti-PRO509, anti-PR0853
  • 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 in 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 in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleo
  • novel fragments of aPRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence fragment(s) are novel.
  • PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequences are contemplated herein.
  • PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide fragments encoded by these nucleotide molecule fragments preferably those PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357,
  • PR0357 anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
  • the invention provides isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention concerns an isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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% 5 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
  • sequence identity yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically
  • the invention concerns an isolated PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 99% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.
  • the invention concerns an isolated PRO201, PR0292, PR0327, PR01265, PR0344,
  • PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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
  • the invention provides an isolated PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and recovering the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide from the cell culture.
  • Another aspect of the invention provides an isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide and recovering the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide from the cell culture.
  • the invention concerns antagonists of a native PRO201 , PR0292, PR0327,
  • the antagonist is an anti-PRO201, anti-PR0292, anti- PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody or a small molecule.
  • the invention concerns a method of identifying antagonists to a PRO201 ,
  • PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PR01112, PRO509, PR0853 or PR0882 polypeptide which comprise contacting the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO201, PR0292, PR0327, PRO 1265, PRO344, PRO343, PRO347, PRO357, PRO715,PRO1017, PROl 1 12, PRO509.PRO853 or PR0882 polypeptide.
  • the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is a native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • the invention concerns a composition of matter comprising a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, or an antagonist of a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882polypeptide as herein described, or an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, or an antagonist thereof as hereinbefore described, or an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti- PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, an antagonist thereof or an anti
  • 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, yeast, or Baculovirus-infected insect cells.
  • a process for producing any of the herein described polypeptides is further provided and comprises culturing 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.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO: 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PRO201 , wherein the nucleotide sequence (SEQ ID NO: 1 ) is a clone designated herein as DNA30676-1223. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 2 shows the amino acid sequence (SEQ ID NO:2) of a native sequence PRO201 polypeptide as derived from the coding sequence of SEQ ID NO: 1 shown in Figure 1.
  • Figure 3 shows the nucleotide sequence (SEQ ID NO:5) of a cDNA containing a nucleotide sequence encoding native sequence PR0292, wherein the nucleotide sequence (SEQ ID NO:5) is a clone designated herein as DNA35617. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 4 shows the amino acid sequence (SEQ ID NO:6) of a native sequence PR0292 polypeptide as derived from the coding sequence of SEQ ID NO:5 shown in Figure 3.
  • Figure 5 shows the nucleotide sequence (SEQ ID NO:7) of a cDNA containing a nucleotide sequence encoding native sequence PR0327, wherein the nucleotide sequence (SEQ ID NO:7) is a clone designated herein as DNA381 13-1230. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 6 shows the amino acid sequence (SEQ ID NO:8) of a native sequence PR0327 polypeptide as derived from the coding sequence of SEQ ID NO:7 shown in Figure 5.
  • Figure 7 shows the nucleotide sequence (SEQ ID NO: 12) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1265, wherein the nucleotide sequence (SEQ ID NO: 12) is a clone designated herein as DNA60764-1533. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 8 shows the amino acid sequence (SEQ ID NO: 13) of a native sequence PRO 1265 polypeptide as derived from the coding sequence of SEQ ID NO: 12 shown in Figure 7.
  • Figure 9 shows the nucleotide sequence (SEQ ID NO: 14) of a cDNA containing a nucleotide sequence encoding native sequence PR0344, wherein the nucleotide sequence (SEQ ID NO: 14) is a clone designated herein as DNA40592-1242. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 10 shows the amino acid sequence (SEQ ID NO: 15) of a native sequence PR0344 polypeptide as derived from the coding sequence of SEQ ID NO: 14 shown in Figure 9.
  • Figure 1 1 shows the nucleotide sequence (SEQ ID NO:22) of a cDNA containing a nucleotide sequence encoding native sequence PR0343, wherein the nucleotide sequence (SEQ ID NO:22) is a clone designated herein as DNA43318-1217. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 12 shows the amino acid sequence (SEQ ID NO:23) of a native sequence PR0343 polypeptide as derived from the coding sequence of SEQ ID NO:22 shown in Figure 1 1.
  • Figure 13 shows the nucleotide sequence (SEQ ID NO:27) of a cDNA containing a nucleotide sequence encoding native sequence PR0347, wherein the nucleotide sequence (SEQ ID NO:27) is a clone designated herein as DNA44176-1244. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 14 shows the amino acid sequence (SEQ ID NO:28) of a native sequence PR0347 polypeptide as derived from the coding sequence of SEQ ID NO:27 shown in Figure 13.
  • Figure 15 shows the nucleotide sequence (SEQ ID NO:32) of a cDNA containing a nucleotide sequence encoding native sequence PR0357, wherein the nucleotide sequence (SEQ ID NO:32) is a clone designated herein as DNA44804- 1248. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 16 shows the amino acid sequence (SEQ ID NO:33) of a native sequence PR0357 polypeptide as derived from the coding sequence of SEQ ID NO:32 shown in Figure 15.
  • Figure 17 shows the nucleotide sequence (SEQ ID NO:39) of a cDNA containing a nucleotide sequence encoding native sequence PR0715, wherein the nucleotide sequence (SEQ ID NO:39) is a clone designated herein as DNA52722-1229. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 18 shows the amino acid sequence (SEQ ID NO:40) of a native sequence PR0715 polypeptide as derived from the coding sequence of SEQ ID NO:39 shown in Figure 17.
  • Figure 19 shows the nucleotide sequence (SEQ ID NO:41 ) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1017, wherein the nucleotide sequence (SEQ ID NO:41 ) is a clone designated herein as DNA561 12-1379. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 20 shows the amino acid sequence (SEQ ID NO:42) of a native sequence PRO1017 polypeptide as derived from the coding sequence of SEQ ID NO:41 shown in Figure 19.
  • Figure 21 shows the nucleotide sequence (SEQ ID NO:43) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 112, wherein the nucleotide sequence (SEQ ID NO:43) is a clone designated herein as DNA57702-1476. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 22 shows the amino acid sequence (SEQ ID NO:44) of a native sequence PROl 112 polypeptide as derived from the coding sequence of SEQ ID NO:43 shown in Figure 21.
  • Figure 23 shows the nucleotide sequence (SEQ ID NO:45) of a cDNA containing a nucleotide sequence encoding native sequence PRO509, wherein the nucleotide sequence (SEQ ID NO:45) is a clone designated herein as DNA50148. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 24 shows the amino acid sequence (SEQ ID NO:46) of a native sequence PRO509 polypeptide as derived from the coding sequence of SEQ ID NO:45 shown in Figure 23.
  • Figure 25 shows the nucleotide sequence (SEQ ID NO:47) of a cDNA containing a nucleotide sequence encoding native sequence PR0853, wherein the nucleotide sequence (SEQ ID NO:47) is a clone designated herein as DNA48227-1350. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 26 shows the amino acid sequence (SEQ ID NO:48) of a native sequence PR0853 polypeptide as derived from the coding sequence of SEQ ID NO:47 shown in Figure 25.
  • Figure 27 shows the nucleotide sequence (SEQ ID NO:52) of a cDNA containing a nucleotide sequence encoding native sequence PR0882, wherein the nucleotide sequence (SEQ ID NO:52) is a clone designated herein as DNA58125. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 28 shows the amino acid sequence (SEQ ID NO:53) of a native sequence PR0882 polypeptide as derived from the coding sequence of SEQ ID NO:52 shown in Figure 27.
  • Figure 29 is a map of chromosome 19 showing the mapping regions of DNA30676- 1223, DNA381 13-1230 and DNA60764-1533.
  • Figure 30 is a map of chromosome 11 showing the mapping region of DNA35617.
  • Figure 31 is a map of chromosome 16 showing the mapping region of DNA43318- 1217and DNA58125.
  • Figure 32 is a map of chromosome 7 showing the mapping region of DNA561 12-1379.
  • Figure 33A is map of chromosome 17 showing the mapping region of DNA52722-1229.
  • Figure 33B is a map of chromosome 17 showing the mapping region of DNA48227-1350.
  • Figure 34 is a map of chromosome 16 showing the mapping region of DNA44804-1248.
  • amplification and “gene duplication” are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line.
  • the duplicated region (a stretch of amplified DNA) is often referred to as "amplicon.”
  • amplicon a stretch of amplified DNA
  • mRNA messenger RNA
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • the "pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc.
  • 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, horses, cats, cattle, pigs, sheep, etc. Preferably, 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) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • 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 album
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., I 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (" Ara- C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carmin
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter 1 , entitled “Cell cycle regulation, oncogens, and antineoplastic drugs” by Murakami etal., (WB Saunders: Philadelphia, 1995), especially p. 13.
  • Doxorubicin is an anthracycline antibiotic.
  • the full chemical name of doxorubicin is (8S-cis)-10-[(3- amino-2,3,6-trideoxy- ⁇ -L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,l l-trihydroxy-8-(hydroxyacetyl)-l- methoxy-5, 12-naphthacenedione.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet- growth factor;
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions. j_4:375-382, 615th Meeting, Harbor (1986), and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug Delivery, Borchardt et al., (ed.), pp. 147-267, Humana Press (1985).
  • the prodrugs of this invention include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • An "effective amount" of a polypeptide disclosed herein or an antagonist thereof, in reference to inhibition of neoplastic cell rowth, tumor growth or cancer cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells.
  • the term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells.
  • An "effective amount" of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide antagonist for purposes of inhibiting neoplastic cell growth, tumor growth or cancer cell growth, may be determined empirically and in a routine manner.
  • a “therapeutical ly effective amount”, in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects : ( 1 ) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder.
  • a "therapeutical ly effective amount" of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner.
  • a "growth inhibitory amount" of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo.
  • a "growth inhibitory amount" of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
  • a "cytotoxic amount” of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 antagonist is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo.
  • a "cytotoxic amount” of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
  • PR0357”, “PR0715”, “PRO1017”, “PROl 1 12", “PRO509”, “PR0853” or “PR0882” polypeptide or protein when used herein encompass native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides and PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide variants (which are further defined herein).
  • PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods.
  • a “native sequence PRO201 ", "native sequence PR0292”, “native sequence PR0327”, “native sequence PR01265", “native sequence PR0344”, “native sequence PR0343”, “native sequence PR0347”, “native sequence PR0357”, “native sequence PR0715”, “native sequence PRO 1017”, “native sequence PROl 112”, “native sequence PRO509”, “native sequence PR0853” or “native sequence PR0882” comprises a polypeptide having the same amino acid sequence as the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide as derived from nature.
  • Such native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means.
  • PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally- occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 and PR0882 polypeptides.
  • naturally-occurring truncated or secreted forms e.g., an extracellular domain sequence
  • naturally- occurring variant forms e.g., alternatively spliced forms
  • the native sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide is a mature or full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide as shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:
  • extracellular domain or “ECD” of a polypeptide disclosed herein refers to a form of the polypeptide
  • transmembrane domain 20 which is essentially free of the transmembrane and cytoplasmic domains.
  • a polypeptide ECD will have less than about 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than about 0.5% of such domains.
  • any transmembrane domain(s) identified for the 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
  • the extracellular domain of a polypeptide of the present invention comprises amino acids 1 to X of the mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side of the extracellular domain/transmembrane domain boundary.
  • the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng.. K): l-6 (1997) and von Heinje et al, Nucl. Acids. Res., J4:4683-4690 (1986)). Moreover, it is also
  • Such PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide variants include, for instance, PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence.
  • a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino 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% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence
  • PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 orPR0882 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.
  • Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALI
  • Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO” represents the amino acid sequence of a hypothetical PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide of interest, "Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared, "PRO- DNA” represents a hypothetical PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 112-, PRO509-, PR0853- or PR0882-encoding
  • filel and file2 are two dna or two protein sequences
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /tmp to hold info about traceback
  • static nm matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[lP LINE] ; /* output line */ static char starfP ] 3]; /* set by stars() *//
  • *ps[i] toupper(*ps[i]); po[i] + + ; ps[i] + + ;
  • *py++ toupper(*px); if (index("ATGCU",*(py-l))) natgc++; ⁇ ⁇
  • Page2ofnwsubr.c ...readjmps if (j ⁇ 0 && dxfdmax]. offset && fj) ⁇
  • Percent (%) amino acid sequence identity with respect to the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 and PR0882 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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, ALIGN-2 or Megalign (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 obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % 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:
  • % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program 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:
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest.
  • amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
  • PRO201 variant polypeptide "PRO0292 variant polypeptide”, “PR0327 variant polypeptide”, “PRO 1265 variant polypeptide”, “PR0344 variant polypeptide”, “PR0343 variant polypeptide”, “PR0347 variant polypeptide”, “PR0357 variant polypeptide”, “PR0715 variant polypeptide”, “PRO 1017 variant polypeptide”, “PROl 112 variant polypeptide”, “PRO509 variant polypeptide", "PR0853 variant polypeptide” and “PR0882 variant polypeptide” or "PRO201 variant nucleic acid sequence", “PR0292 variant nucleic acid sequence”, “PR0327 variant nucleic acid sequence”, “PRO 1265 variant nucleic acid sequence”, “PR0344 variant nucleic acid sequence”, “PR0343 variant nucleic acid sequence”, “PR0347 variant nucleic acid sequence”, “PR0357 variant nucleic acid sequence”, “PR0715 variant nucleic acid sequence”, “PRO 1017 variant nucleic acid sequence”, “PRO 1 112 variant nucleic acid sequence", “PRO509 variant nucle
  • a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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
  • PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 the PRO201 , PR0292, PR0327, PRO 1265,
  • PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853and PR0882 polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent nucleic 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, ALIGN-2 or Megalign (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, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % 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 above using the ALIGN-2 sequence comparison computer program. However, % 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)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov.
  • % 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:
  • 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 of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest.
  • nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
  • PRO201 PRO0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357,
  • PRO715,PRO1017,PROl 112,PRO509,PRO853 and PR0882 variant polynucleotides are nucleic acid molecules that encode an active PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715,
  • PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 variant polypeptides may be those that are encoded by a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 variant polynucleotide.
  • amino acid residues in the sequences compared that are not only identical, but also those that have similar properties are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest.
  • the % value of positives of a given amino acid sequence A to, with, or against a given amino 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. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. 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-proteinaceous 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • the isolated nucleic acid is free of association with all components with which it is naturally associated.
  • An isolated PRO201 -, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-encoding nucleic acid molecule or an anti-PRO201 -, anti-PR0292-, anti-PR0327-, anti-PRO 1265-, anti- PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti-PROl 017-, anti-PROl 112-, anti- PRO509-, anti-PR0853- or anti-PR0882-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature.
  • Isolated nucleic acid molecules therefore are distinguished from the PRO201-, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding nucleic acid molecule or the anti-PRO201 -, anti-PR0292-, anti-PR0327-, anti-PROl 265-, anti-PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti-PROl 017-, anti- PROl 112-, anti-PRO509-, anti-PR0853- or anti-PR0882-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide or an anti- PRO201, anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody includes PRO201-, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-nucleic acid molecules and anti-PRO201-, anti-PR0292-, anti- PR0327-,
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the 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- PRO201, anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 monoclonal antibodies (including antagonist, and neutralizing antibodies),anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti- PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody compositions with polyepitopic specificity, single chain anti-PRO201 , anti- PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347
  • the term "monoclonal antibody” as used herein 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 % S
  • Modely stringent conditions may 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 than those described above.
  • moderately stringent conditions is overnight incubation at 37"C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium 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 35°C-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • “Active” or “activity” for the purposes herein refers to form(s) of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides which retain a biological and/or an immunological activity/property of a native or naturally-occurring PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, wherein "biological” activity refers to a function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 112, PRO509, PR0853 or PR08
  • Bio activity in the context of an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins or otherwise interfere with the transcription or translation of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • a preferred biological activity is growth inhibition of a target tumor cell.
  • Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell.
  • biological activity in the context of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide means the ability of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017,
  • PRO 1 112, PRO509, PR0853 or PR0882 polypeptide to induce neoplastic cell growth or uncontrolled cell growth.
  • immunological cross-reactivity means immunological cross-reactivity with at least one epitope of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • “Immunological cross-reactivity” as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having this activity with polyclonal antisera raised against the known active PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide.
  • Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 8-times, most preferably at least about 10-times higher) than the binding affinity of that molecule to any other known native polypeptide.
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide disclosed herein or the transcription or translation thereof.
  • Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments, peptides, small organic molecules, anti-sense nucleic acids, etc.
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • antibody is used in the broadest sense and specifically covers, without limitation intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • “Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions.
  • CDRs complementarity-determining regions
  • FR framework regions.
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91 -3242, Vol. I, pages 647-669 ( 1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD.
  • CDR complementarity determining region
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata etal. Protein Eng. , 8(10): 1057-1062 [ 1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its 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 in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L di er. 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 affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) 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 cysteine residue(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 (K) 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.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein 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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [1975], or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature. 352:624-628 [1991 ] and Marks et al.. J. Mol. Biol., 222:581 -597 (1991 ), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851 -6855 [1984]).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences
  • 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 are human immunoglobulins (recipient antibody) in which residues from a 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.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • 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 sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • "Single-chain Fv” or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in 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.
  • a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • the term "diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L ).
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • 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.
  • Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-21 1 , Cu-67, Bi-212, and Pd-109.
  • the label may also be a non-detectable entity such as a toxin.
  • 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
  • the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.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 domain 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.
  • immunoglobulin such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882.
  • cDNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 polypeptides has been identified and isolated, as disclosed in 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 protein, and will not be changed.
  • the proteins encoded by the herein disclosed nucleic acid sequences as well as all further native homologues and variants included in the foregoing definition of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 will be referred to as "PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882", regardless of their origin or mode of preparation.
  • cDNA clones have been deposited with the ATCC.
  • the actual nucleotide sequence of the clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art.
  • the predicted amino acid sequences can be determined from the nucleotide sequences using routine skill.
  • PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptides and encoding nucleic acid described herein Applicants have identified what are believed to be the reading frames best identifiable with the sequence information available at the time.
  • PRO201 In addition to the full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO347,PRO357,PRO715, PRO1017,PROl 1 12, PRO509, PRO853 and PR0882 polypeptides described herein, it is contemplated that PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 variants can be prepared.
  • PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 and PR0882 variants can be prepared by introducing appropriate nucleotide changes into the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 DNA, and/or by synthesis of the desired PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • amino acid changes may alter post-translational processes of the PRO201, PR0292, PR0327,
  • PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 or in various domains of the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 or PR0882 that results in a change in the amino acid sequence of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 as compared with the native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017,
  • PROl 1 12, PRO509, PR0853 or PR0882 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized.
  • An alternative approach involves generating PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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).
  • PCR polymerase chain reaction
  • Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
  • PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide fragments share at least one biological and/or immunological activity with the native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • Substantial modifications in function or immunological identity of the 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
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • 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.. J0:6487 (1987)]
  • cassette mutagenesis [Wells et al, Gene, 34:315 ( 1985)]
  • restriction selection mutagenesis [Wells etal, Philos. Trans. R. Soc. London SerA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine 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 amino 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 isoteric amino acid can be used.
  • PRO201 Modifications of PRO201 , PRQ292, PRQ327.
  • One type of covalent modification includes reacting targeted amino acid residues of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PROl 017, anti-PRO 1 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies, and vice- versa.
  • crosslinking agents include, e.g., 1 , 1 -bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • Another type of covalent modification of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PRO347, PRO357, PRO715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Addition of glycosylation sites to thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 (for O-linked glycosylation sites).
  • PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981 ).
  • Removal of carbohydrate moieties present on the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 1 18: 131 (1981).
  • 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. Enzvmol., 138:350 (1987).
  • PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 comprises linking the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to one of a variety of nonproteinaceous 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 or 4,179,337.
  • PEG polyethylene glycol
  • ThePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PRO 1 112, PRO509, PR0853 or PR0882 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of the PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882.
  • epitope-tagged forms of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can be detected using an antibody against the tag polypeptide.
  • provision of the epitope tag enables the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • 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 etal., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology.
  • tag polypeptides include the Flag-peptide [Hopp etal, BioTechnology, 6: 1204- 1210 ( 1988)] ; the KT3 epitope peptide [Martin et al, Science, 255: 192-194 (1992)]; an ⁇ -tubulin epitope peptide [Skinner et al, J. Biol.
  • the chimeric molecule may comprise a fusion of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 with an immunoglobulin or a particular region of an immunoglobulin.
  • an immunoglobulin or a particular region of an immunoglobulin.
  • a bivalent form of the chimeric molecule also referred to as an "immunoadhesin”
  • such a fusion could be to the Fc region of an IgG molecule.
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide in 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 IgGl molecule.
  • the description below relates primarily to production of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 by culturing cells transformed or transfected with a vector containing PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343,
  • PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 sequence, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al, 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.
  • Various portions of the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 orPR0882 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • DNA encoding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 may be obtained from a cDNA library prepared from tissue believed to possess the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 mRNA and to express it at a detectable level.
  • human PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples.
  • PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the PRO201 , PR0292, PR0327, PR01265,
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook etal, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
  • 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.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 production and cultured in 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. In general, 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.
  • DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
  • polycations e.g., polybrene, polyornithine.
  • 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 eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli Kl 2 strain MM294 (ATCC 31 ,446); E. coli X 1776 (ATCC 31 ,537); E. coli strain W31 10 (ATCC 27,325) and E. coli strain K5 772 (ATCC 53,635).
  • suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.
  • Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations.
  • the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W31 10 strain 1 A2, which has the complete genotype tonA ; E. coli W31 10 strain 9E4, which has the complete genotype tonA ptr3; E.
  • coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompTka ⁇ ' ;
  • E. coli W31 10 strain 37D6 which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kan r ;
  • E. coli W31 10 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No.4,946,783 issued 7 August 1990.
  • in 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 PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 112-, PRO509-, PR0853- or PR0882-encoding vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S.
  • Patent No. 4,943,529; Fleer etal, Bio/Technology.9: 968-975 (1991 )) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacteriol.. 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Vanden Berg et al., Bio/Technology. 8:135 (1990)), K . thermotolerans, and K.
  • K. lactis MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacteriol.. 737 [1983]
  • K. fragilis ATCC 12,424)
  • K. bulgaricus ATCC 16,045)
  • Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 1 12:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [19831; Yelton etal. Proc. Natl. Acad. Sci. USA.81 :1470-1474 [19841) andA.
  • 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 PRO201 , PR0292, PR0327, PROl 265, PR0344,
  • PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • the selection of the appropriate host cell is deemed to be within the skill in the art.
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • Various vectors are publicly available.
  • the vector may, for example, be in 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. In general, DNA is inserted into an appropriate restriction endonuclease site(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 ligation techniques which are known to the skilled artisan.
  • PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • 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 PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-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, penicillinase, lpp, or heat-stable enterotoxin 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 in U.S. Patent No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • 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 in 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 toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-.PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-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 etal, Proc. Natl. Acad. Sci. USA, 77:4216 (1980).
  • a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 [Stinchcomb et al, Nature, 282:39 (1979); Kingsman et al. Gene, 7:141 (1979); Tschemper ef ⁇ /.. Gene, 10: 157 (1980)1.
  • the trp ⁇ 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 PRO201 -, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-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..8 :4057 ( 1980); EP 36,776] , and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882.
  • S.D. Shine-Dalgarno
  • 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- ⁇ hosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 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, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 2,21 1 ,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 actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,21
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 coding sequence, but is preferably located at a site 5' from the promoter.
  • 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 cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 orPR0882.
  • Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in recombinant vertebrate cell culture are described in Gething etal, Nature.293:620-625 (1981); Mantei et al., Nature, 281:40-46 ( 1979); EP 1 17,060; and EP 1 17,058.
  • 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 staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.
  • the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against an exogenous sequence fused to PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA and encoding a specific antibody epitope.
  • PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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., Triton- X 100) or by enzymatic cleavage.
  • a suitable detergent solution e.g., Triton- X 100
  • Cells employed in expression of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • PRO201 may be desired to purify PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882.
  • the present invention is based on the identification and characterization of genes that are amplified in certain cancer cells.
  • the genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements.
  • One is the preservation and propagation of DNA as the genetic information in its original form, to guarantee stable inheritance through multiple generations.
  • cells or organisms must be able to adapt to lasting environmental changes.
  • the adaptive mechanisms can include qualitative or quantitative modifications of the genetic material.
  • Qualitative modifications include DNA mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein.
  • Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i.e., a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene.
  • MTX cytotoxic drug methotrexate
  • DHFR dihydrofolate reductase
  • Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation. Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell.
  • One of the most common genetic changes observed in human malignancies are mutations of the p53 protein. p53 controls the transition of cells from the stationary (Gl) to the replicative (S) phase and prevents this transition in the presence of DNA damage.
  • Gl stationary
  • S replicative
  • one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA damage, i.e., genetic changes.
  • Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and gross, structural alterations, such as translocations.
  • the amplification of DNA sequences may indicate a specific functional requirement as illustrated in the
  • the bcl-2 protein was found to be amplified in certain types of non-Hodgkin ' s lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells.
  • Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells less susceptible to limiting amounts of available growth factor.
  • Examples include the amplification of the androgen receptor in recurrent prostate cancer during androgen deprivation therapy and the amplification of the growth factor receptor homologue ERB2 in breast cancer.
  • genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation. This is illustrated by the amplification of the bcl-1 and ras genes in various epithelial and lymphoid neoplasms.
  • CGH comparative genomic hybridization
  • Tumor and normal DNA are hybridized simultaneously onto metaphases of normal cells and the entire genome can be screened by image analysis for DNA sequences that are present in the tumor at an increased frequency.
  • image analysis for DNA sequences that are present in the tumor at an increased frequency.
  • this type of analysis has revealed a large number of recurring amplicons (a stretch of amplified DNA) in a variety of human neoplasms.
  • CGH is more sensitive than classical cytogenetic analysis in identifying amplified stretches of DNA, it does not allow a rapid identification and isolation of coding sequences within the amplicon by standard molecular genetic techniques.
  • PCR-based assays The most sensitive methods to detect gene amplification are polymerase chain reaction (PCR)-based assays. These assays utilize very small amount of tumor DNA as starting material, are extremely sensitive, provide DNA that is amenable to further analysis, such as sequencing and are suitable for high-volume throughput analysis.
  • the above-mentioned assays are not mutually exclusive, but are frequently used in combination to identify amplifications in neoplasms. While cytogenetic analysis and CGH represent screening methods to survey the entire genome for amplified regions, PCR-based assays are most suitable for the final identification of coding sequences, i.e., genes in amplified regions.
  • such genes have been identified by quantitative PCR (S. Gelmini et al., Clin. Chem.. 43:752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. Quantitative PCR was performed using a TaqMan instrument (ABI). Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs.
  • Human lung carcinoma cell lines include A549 (SRCC768), Calu- 1 (SRCC769), Calu-6 (SRCC770), H 157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832),and H810 (SRCC833), all available from ATCC.
  • SRCC724 (adenocarcinoma, abbreviated as "AdenoCa")(LTl), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTla), SRCC726 (adenocarcinoma)(LT2), SRCC727 (adenocarcinoma)(LT3), SRCC728 (adenocarcinoma)(LT4), SRCC729 (squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cell carcinoma)(LT7), SRCC731 (adenocarcinoma)(LT9), SRCC732 (squamous cell carcinoma)(LT10), SRCC733 (squamous cell
  • human lung tumors designated SRCC1125 [HF-000631 ], SRCC1 127 [HF-000641 ], SRCC1 129 [HF-000643], SRCC1 133 [HF-000840], SRCC1 135 [HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297], SRCC1235 [HF-001299], and SRCC1236 [HF-001300].
  • Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr.
  • ATCC cell lines SW480 adenocarcinoma, SRCC776)
  • SW620 lymph node metastasis of colon adenocarcinoma, SRCC777
  • Colo320 carcinoma, SRCC778
  • HT29 adenocarcinoma, SRCC779
  • HM7 a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr.
  • Primary colon tumors include colon adenocarcinomas designated CT2 (SRCC742), CT3 (SRCC743) ,CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1 (SRCC751 ), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT1 1 (SRCC757), CT18 (SRCC758), CT19 (adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21 (adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinoma, SRCC91 1
  • SRCC1051 [HF-000499]
  • SRCC1052 [HF-000539]
  • SRCC1053 [HF-000575]
  • SRCC1054 [HF-000698]
  • SRCC1 142 [HF-000762]
  • SRCC1 144 [HF-000789]
  • SRCC1 146 [HF-000795]
  • SRCC1 148[HF-00081 1] human colon tumor centers
  • Human breast carcinoma cell lines include, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468(SRCC762), MB 175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545]. Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC1101, and human breast-met-lung-NS tumor designated SRCC893 [LT 32].
  • Human kidney tumor centers include SRCC989 [HF-000611] and SRCC1014 [HF-000613].
  • Human testis tumor center includes SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF-
  • Human parathyroid tumor includes SRCC1002 [HF-000831 ] and SRCC1003 [HF-000832].
  • results of the gene amplification assays herein can be verified by further studies, such as, by determining mRNA expression in various human tissues.
  • gene amplification and/or gene expression in various tissues may be measured 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.
  • Gene expression in various tissues may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • immunological methods such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.
  • the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 DNA and encoding a specific antibody epitope.
  • General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided hereinbelow.
  • the gene can be mapped to a particular chromosome, e.g., by radiation-hybrid analysis. The amplification level is then determined at the location identified, and at the neighboring genomic region. Selective or preferential amplification at the genomic region to which the gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival. Chromosome mapping includes both framework and epicenter mapping. For further details see, e.g., Stewart et al., Genome Research, 7:422-433 (1997).
  • the results of the gene amplification study can be further verified by antibody binding studies, in which the ability of anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 11 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies to inhibit the expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides on tumor (cancer) cells is tested.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
  • Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).
  • ком ⁇ онентs rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody.
  • the amount of target protein (encoded by a gene amplified in a tumor cell) in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • Cell-based assays and animal models for tumors can be used to verify the findings of the gene amplification assay, and further understand the relationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth.
  • the role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein. Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above.
  • cells of a cell type known to be involved in a particular tumor are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed.
  • Suitable cells include, for example, stable tumor cells lines such as, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorogenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC). Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cancer.
  • stable tumor cells lines such as, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth
  • Animal models of tumors and cancers include both non- recombinant and recombinant (transgenic) animals.
  • Non-recombinant animal models include, for example, rodent, e.g., murine models.
  • Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue.
  • standard techniques e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue.
  • mice Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice.
  • the autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, AS W, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL.
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any of the above-listed tumor cell lines, and, for example, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); r ⁇ s-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers.
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer. 48:689-696 [1983]).
  • Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures.
  • the subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
  • Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions.
  • tumor tissue fragments of suitable size are introduced into the s.c. space.
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously.
  • Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue. Boven and Winograd (1991), supra.
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or rcew-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al.. PNAS USA, 83:9129-9133 (1986).
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals.
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al., Cancer Research, 54:4726-4728 ( 1994) and Too et al., Cancer Research, 55:681 -684 (1995). This model is based on the so-called "METAMOUSE” sold by AntiCancer, Inc., (San Diego, California).
  • Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.
  • Meth A, CMS4, CMS5, CMS21 , and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med., 146:720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol., 138:4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOxlO 6 to lOxlO 7 cells/ml.
  • the animals are then infected subcutaneously with 10 to 100 ⁇ l of the cell suspension, allowing one to three weeks for a tumor to appear.
  • the Lewis lung (3LL) carcinoma of mice which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL).
  • SCCL small cell carcinoma of the lung
  • This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al, Br. J. Cancer, 4Lsuppl. 4:309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see, Zacharski, Haemostasis. 16:300-320 [1986]).
  • One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment.
  • the size of implanted tumors has been measured with a slide caliper in two or three dimensions.
  • the measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula.
  • the measurement of tumor size is very inaccurate.
  • the therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay.
  • Another important variable in the description of tumor growth is the tumor volume doubling time.
  • Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int.
  • necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis.
  • Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals.
  • Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys.
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., Proc. Natl.
  • transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals").
  • the transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al, Proc. Natl. Acad. Sci. USA. 89:6232- 636 (1992).
  • the expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals are further examined for signs of tumor or cancer development.
  • "knock out" animals can be constructed which have a defective or altered gene encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PR01112, PRO509, PR0853 or PR0882 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal.
  • cDNA encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques.
  • a portion of the genomic DNA encoding a particular PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see, e.g., Thomas and Capecchi, Cell, 5 _:503 (1987) for a description of homologous recombination vectors].
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see, e.g., Li et al, Cell, 69:915 (1992)].
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 1 13-152].
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined 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 recombined DNA.
  • Knockout animals can be characterized for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • SCC feline oral squamous cell carcinoma
  • Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor.
  • These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor.
  • each cat Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves.
  • CT computed tomography
  • Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck.
  • CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
  • fibrosarcoma adenocarcinoma
  • lymphoma adenocarcinoma
  • chrondroma adenocarcinoma of dogs, cats
  • baboons a preferred model as its appearance and behavior are very similar to those in humans.
  • the use of this model is limited by the rare occurrence of this type of tumor in animals.
  • Screening assays for drug candidates are designed to identify compounds that bind or complex with the 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.
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, ( ⁇ oly)peptide-immunoglobulin fusions, 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.
  • 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 are common in that they call for contacting the drug candidate with a 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 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 by coating the solid surface with a solution of the polypeptide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the 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 detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PR01017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide encoded by a gene identified herein
  • its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, cross- linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co- workers [Fields and Song, Nature.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain.
  • 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 in which the target protein is fused to the DNA- binding domain of GAL4.
  • GALl -/ ⁇ cZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction of a PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-,
  • PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encoding gene identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, 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 reaction(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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide indicates that the compound is an antagonist to the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • antagonists may be detected by combining the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and a potential antagonist with membrane-bound PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide can be labeled, such as by radioactivity, such that the number of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting.
  • RNA is prepared from a cell responsive to the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
  • Transfected cells that are grown on glass slides are exposed to labeled PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357.PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • Another potential PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide 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..
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO201 , PR0292, PR0327, PRO 1265 , PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide (antisense - Okano, Neurochem, 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988).
  • ohgonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • antisense DNA oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in 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 in length, about 55 bases in length, about 60 bases in 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.
  • 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, thereby blocking the normal biological activity of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non- peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecules capableof 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 (published
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides.
  • the base composition of these ohgonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Hoogsteen base-pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • compositions and Methods for the Treatment of Tumors include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc., that inhibit the expression and/or activity of the target gene product.
  • antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • 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 (published
  • Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides.
  • the base composition of these ohgonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Hoogsteen base pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments which may inhibit the production or the gene product of the amplified genes identified herein and/or reduce the activity of the gene products.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide or a fusion protein thereof.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715 , anti-PRO 1017, anti-PRO 11 12, anti-PRO509, anti-PR0853 or anti-PR0882 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 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide, including fragments, or a fusion protein of such protein or a fragment 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. Usually, 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.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • 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 (ATCC), Manassas, Virginia.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
  • 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). Such techniques and assays are known in the art.
  • 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. Alternatively, 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
  • 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, supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • a 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.
  • Theanti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies 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
  • donor antibody non-human species
  • 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); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • 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, Science, 239: 1534- 1536 ( 1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "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 Boerner 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(1 ):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. This approach is described, for example, in U.S. Patent Nos.
  • ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
  • the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01 145) to an active anti-cancer drug.
  • a prodrug e.g., a peptidyl chemotherapeutic agent, see WO 81/01 1405
  • an active anti-cancer drug See, for example, WO 88/07378 and U. S. Patent No. 4,975,278.
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, glycosidase, glucose oxidase, human lysosyme, human glucuronidase, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti -cancer drug 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amin
  • antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328:457-458 (1987)).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
  • the enzymes of this invention can be covalently bound to the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti- PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking agents discussed above.
  • fusion proteins comprising at least the antigen binding region of the antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g. , Neuberger et al, Nature, 3 2:604-608 (1984)).
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • 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]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • 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.
  • 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 using chemical linkage. Brennan et al, Science, 229:81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 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 polypeptide herein.
  • an anti-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 polypeptide.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular polypeptide.
  • These antibodies possess a polypeptide-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 polypeptide and further binds tissue factor (TF).
  • 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]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, 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 mefhyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • cysteine residue(s) may be introduced in 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, J. Exp. Med., 176: 1 191 -1 195 ( 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 etal, Cancer Research.53:2560- 2565 (1993).
  • an antibody can be engineered which 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 small molecule toxin), or a radioactive isotope (i.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 small molecule toxin), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • Small molecule toxins include, for example, calicheamicins, maytansinoids, palytoxin and CCl 065.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 13 V 31 In, »°Y and lll ⁇ Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987).
  • Carbon- 14-labeled 1 -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/1 1026.
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting 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) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a "receptor” such as streptavidin
  • 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. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (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 etal, J. Biol. Chem., 257:286-288 (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See, Gabizon etal, J. National Cancer Inst., 81( 19): 1484 (1989).
  • Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
  • the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred.
  • lipofections 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 which specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 [1993]).
  • Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. [1980]), in the form of lyophilized 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 and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and w-cresol); 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 glycine, glutamine, asparagine, histidine,
  • 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 a cytotoxic agent, cytokine or growth inhibitory agent.
  • cytotoxic agent cytokine or growth inhibitory agent.
  • Such molecules are suitably present in 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, hydroxymethylcellulose or gelatin-microcapsules and poly-
  • microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, macroemulsions.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • 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. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein.
  • Exemplary conditions or disorders to be treated with such antibodies and other compounds include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
  • benign or malignant tumors e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung
  • the anti-tumor agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
  • Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e.g., antibodies of the instant invention.
  • the patient to be treated with such anti-cancer agents may also receive radiation therapy.
  • a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD ( 1992).
  • the chemotherapeutic agent may precede, or follow administration of the anti-tumor agent, e.g., antibody, or may be given simultaneously therewith.
  • the antibody may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
  • antibodies against other tumor associated antigens such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF).
  • VEGF vascular endothelial factor
  • two or more antibodies binding the same or two or more different antigens disclosed herein may be co- administered to the patient.
  • the antibodies herein are co-administered with a growth inhibitory agent.
  • the growth inhibitory agent may be administered first, followed by an antibody of the present invention.
  • simultaneous administration or administration of the antibody of the present invention first is also contemplated.
  • Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein.
  • the appropriate dosage of an anti-tumor agent e.g., an antibody herein will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician.
  • the agent is suitably administered to the patient at one time or over a series of treatments.
  • ⁇ g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is usually an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e.g., an antibody.
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline,
  • Ringer's solution and dextrose solution may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • tumors While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells find additional use in the diagnosis and prognosis of tumors.
  • antibodies directed against the protein products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics.
  • antibodies, including antibody fragments can be used to qualitatively or quantitatively detect the expression of proteins encoded by the amplified genes ("marker gene products").
  • the antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. These techniques are particularly suitable, if the amplified gene encodes a cell surface protein, e.g., a growth factor. Such binding assays are performed essentially as described in section 5 above.
  • In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy.
  • a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample.
  • This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
  • the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press N.Y., 1989; Ausubel etal, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989; Innis etal, PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., N.Y., 1990; Harlow etal, Antibodies: A Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor. 1988; Gait, Oligonucleotide Synthesis. IRL Press, Oxford, 1984; R.I. Freshney, Animal Cell Culture, 1987; Coligan et al.. Current Protocols in Immunology, 1991.
  • EXAMPLE 1 Extracellular Domain Homology 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. LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzvmology.
  • consensus DNA sequences were assembled relative to the other identified EST sequences using phrap.
  • consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
  • 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 in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
  • 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 into 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.
  • 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
  • polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc., (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private (LIFESEQ ® , Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases.
  • the signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration.
  • the nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons.
  • the second is not examined. If neither meets the requirement, the candidate sequence is not scored.
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
  • EXAMPLE 3 Isolation of cDNA Clones Encoding Human PRO201
  • An expressed sequence tag (EST) DNA database ( LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified (1328938, also designated DNA28710) which was in a fetal pancreas library and which shared significant identity with the adaptor protein She.
  • EST expressed sequence tag
  • a full length cDNA corresponding to the isolated EST was cloned from a human fetal kidney library using an in vivo cloning technique in pRK5.
  • the cDNA libraries used to isolate the cDNA clones encoding human PRO201 were constructed by standard methods using commercially available reagents such as those from
  • 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 into 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.
  • 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 )
  • Probes based on the Incyte EST no. 1328938 were used to screen a cDNA library derived from the human fetal kidney library:
  • Analysis of the full-length PRO201 sequence shown in Figure 2 evidences the presence of a variety of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above.
  • Analysis of the full-length PRO201 polypeptide shown in Figure 2 evidences the presence of the following: a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 142 to about amino acid 146; N-myristoylation sites from about amino acid 41 to about amino acid 47, from about amino acid 107 to about amino acid 111, from about amino acid 164 to about amino acid 170, from about amino acid 203 to about amino acid 209, from about amino acid 243 to about amino acid 249, from about amino acid 343 to about amino acid 349, from about amino acid 460 to about amino acid 466, from about amino acid 546 to about amino acid 552, and from about amino acid 551 to about amino acid 557; an amidation site from about amino acid 97 to about amino acid
  • PRO201 shows amino acid sequence identity to both Sck and She proteins.
  • PR0292 is identical with the human death asscoiated protein DAP-7, also called cathepsin D.
  • the amino acid sequence of this 412 amino acid protein is present in the public Dayhoff database under Accession Nos. CATD_HUMAN and P_R74207, and is shown in Figure 4 (SEQ ID NO:6).
  • the nucleotide sequence of the DNA encoding PR0292 is shown in Figure 3 (SEQ ID NO:5).
  • SEQ ID NO:6 Analysis of the full-length PR0292 sequence shown in Figure 4 (SEQ ID NO:6) evidences the presence of a variety of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 20; N-glycosylation sites from about amino acid 134 to about amino acid 138, and from about amino acid 263 to about amino acid 267; a tyrosine kinase phosphorylation site from about amino acid 72 to about amino acid 81 ; N-myristoylation sites from about amino acid 145 to about amino acid 151 , from about amino acid 248 to about amino acid 254, and from about amino acid 282 to about amino acid 288; and a leucine zipper pattern from about amino acid 335 to about amino acid 357.
  • DAP-7 has also been disclosed in WO 95/10630 published on April 20, 1995, and in Faust et al., Proc. Natl. Acad. Sci. USA, 82:4910-4914 (1985).
  • DNA38110 This consensus sequence is designated herein as DNA38110. Based on the DNA38110 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 PR0327. A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer:
  • hybridization probe 5'-CAAGTGCGCTGCAACCCCTTTGGCATCTATGGCTCCAAGAAAGCCGGGAT-3' (SEQ ID NO: 11 )
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue (LIB26).
  • DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA38113-1230 [ Figure 5, SEQ ID NO:7]; and the derived protein sequence for PR0327.
  • a signal peptide from about amino acid 1 to about amino acid 30; N-glycosylation sites from about amino acid 92 to about amino acid 96, from about amino acid 104 to about amino acid 108, from about amino acid 140 to about amino acid 144, from about amino acid 168 to about amino acid 172, from about amino acid 292 to about amino acid 296, and from about amino acid 382 to about amino acid 386; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 413 to about amino acid 417; casein kinase II phosphorylation sites from about amino acid 44 to about amino acid 48, from about amino acid 183 to about amino acid 187, and from about amino acid 205 to about amino acid 209; N-myristoylation sites from about amino acid 30 to about amino acid 36, from about amino acid 37 to about amino acid 43, from about amino acid 73 to about amino acid 79,
  • Clone DNA381 13-1230 has been deposited with the ATCC on September 10, 1997 and is assigned ATCC deposit no. 209530.
  • the full-length PR0327 protein shown in Figure 6 has an estimated molecular weight of about 46,302 daltons and a pi of about 9.42.
  • PR0327 may be a novel prolactin binding protein.
  • EXAMPLE 6 Isolation of cDNAs Encoding Human PROl 265 DNA60764-1533 was identified by applying the proprietary signal sequence finding algorithm described in Example 3 above. Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the LIFESEQ ® database, designated Incyte EST cluster no. 86995. 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 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 as DNA55717. In light of the sequence homology between the DNA55717 sequence and Incyte EST no. 20965, Incyte EST no. 20965 was purchased and the cDNA insert was obtained and sequenced. The sequence of this cDNA insert is shown in Figure 7 (SEQ ID NO:12) and is herein designated as DNA60764-1533.
  • DNA34398 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 (34398.fl ):
  • hybridization probe 5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3' (SEQ ID NO:21 )
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.
  • DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA40592-1242 [ Figure 9, SEQ ID NO: 14]; and the derived protein sequence for PR0344.
  • DNA40592-1242 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229, and an apparent stop codon at nucleotide positions 956-958.
  • the predicted polypeptide precursor is 243 amino acids long.
  • Analysis of the full-length PR0344 sequence shown in Figure 10 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • EXAMPLE 8 Isolation of cDNA Clones Encoding Human PRQ343
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein identified as DNA30895. Based on the DNA30895 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 PR0343.
  • forward PCR primer 5'-CGTCTCGAGCGCTCCATACAGTTCCCTTGCCCCA-3' (SEQ ID NO:24) reverse PCR primer:
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue (LIB26).
  • DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA43318-1217 [ Figure 11 , SEQ ID NO:22]; and the derived protein sequence for PR0343.
  • DNA43318-1217 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 53-55, and an apparent stop codon at nucleotide positions 1004-1006.
  • the predicted polypeptide precursor is 317 amino acids long.
  • Analysis of the full-length PR0343 sequence shown in Figure 12 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 32; an N-glycosylation site from about amino acid 70 to about amino acid 74; a glycosaminoglycan attachment site from about amino acid 178 to about amino acid 182; N-myristoylation sites from about amino acid 5 to about amino acid 11 , from about amino acid 12 to about amino acid 18, from about amino acid 13 to about amino acid 19, from about amino acid 16 to about amino acid 22, from about amino acid 52 to about amino acid 58, from about amino acid 71 to about amino acid 77, from about amino acid 77 to about amino acid 83, from about amino acid 1 12 to about amino acid 1 18, from about amino acid 273 to about amino acid 279, and from about amino acid 310 to about amino acid 316; a prokaryotic membrane hpoprotein lipid attachment site from about amino acid 4 to about amino acid 15; and a serine proteases, trypsin family, his
  • 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 PR0347.
  • forward PCR primer 5'-AGGAACTTCTGGATCGGGCTCACC-3' (SEQ ID NO:29) reverse PCR primer:
  • a synthetic oligonucleotide hybridization probe was constructed from the DNA39499 consensus sequence: 5'-GCCAAGGACTCCTTCCGCTGGGCCACAGGGGAGCACCAGGCCTTC-3' (SEQID NO:31 )
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228). DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA44176-1244 [ Figure 13, SEQ ID NO:27]; and the derived protein sequence for PR0347.
  • DNA44176-1244 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 123-125, and an apparent stop codon at nucleotide positions 1488-1490.
  • the predicted polypeptide precursor is 455 amino acids long.
  • Analysis of the full-length PR0347 sequence shown in Figure 14 (SEQ ID NO:28) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 26; N-glycosylation sites from about amino acid 144 to about amino acid 148, and from about amino acid 243 to about amino acid 247; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 45 to about amino acid 49; N-myristoylation sites from about amino acid 22 to about amino acid 28, from about amino acid 99 to about amino acid 105, from about amino acid 131 to about amino acid 137, from about amino acid 201 to about amino acid 207, from about amino acid 213 to about amino acid 219, from about amino acid 287 to about amino acid 293, from about amino acid 288 to about amino acid 294, from about amino acid 331 to about amino acid 337, and from about amino acid 398 to about amino acid 404; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 204 to about amino acid 2
  • reverse PCR primer 1 5'-CGGTTCTGGGGACGTTAGGGCTCG-3' (SEQ ID NO:35) forward PCR primer 2:
  • hybridization probe 1 Two synthetic oligonucleotide hybridization probes were constructed from the DNA37162 consensus sequence: hybridization probe 1 :
  • RNA for construction of the cDNA libraries was isolated from human fetal liver tissue.
  • DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA44804-1248 [Figure 15, SEQ ID NO:32]; and the derived protein sequence for PR0357.
  • the entire coding sequence of DNA44804-1248 is included in Figure 15 (SEQ ID NO:32).
  • DNA44804-1248 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139, and an apparent stop codon at nucleotide positions 1931-1933.
  • the predicted polypeptide precursor is 598 amino acids long.
  • Analysis of the full-length PR0357 sequence shown in Figure 16 (SEQ ID NO:33) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 23; a transmembrane domain from about amino acid 501 to about amino acid 520; N- glycosylation sites from about amino acid 198 to about amino acid 202, from about amino acid 425 to about amino acid 429, and from about amino acid 453 to about amino acid 457; a tyrosine kinase phosphorylation site from about amino acid 262 to about amino acid 270; N-myristoylation sites from about amino acid 23 to about amino acid 29, from about amino acid 27 to about amino acid 33, from about amino acid 1 12 to about amino acid 1 18, from about amino acid 273 to about amino acid 279, from about amino acid 519 to about amino acid 525, and from about amino acid 565 to about amino acid 571 ; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 14 to about amino acid 25; an EGF-like domain cysteine
  • Clone DNA44804- 1248 has been deposited with the ATCC on December 10, 1997 and is assigned ATCC deposit no. 209527.
  • the full-length PR0357 protein shown in Figure 16 has an estimated molecular weight of about 63,030 daltons and a pi of about 7.24.
  • DNA sequencing of the isolated clone isolated as described above gave the full-length DNA sequence for DNA52722-1229 [ Figure 17, SEQ ID NO:39]; and the derived protein sequence for PR0715.
  • DNA52722-1229 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1 14-1 16, and an apparent stop codon at nucleotide positions 864-866.
  • the predicted polypeptide precursor is 250 amino acids long.
  • Analysis of the full-length PR0715 sequence shown in Figure 18 (SEQ ID NO:40) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 40; an N-glycosylation site from about amino acid 124 to about amino acid 128; a tyrosine kinase phosphorylation site from about amino acid 156 to about amino acid 164; N-myristoylation sites from about amino acid 36 to about amino acid 42, from about amino acid 40 to about amino acid 46, from about amino acid 179 to about amino acid 185, and from about amino acid 242 to about amino acid 248; and a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 34 to about amino acid 45.
  • PR0715 is a novel tumor necrosis factor protein.
  • EXAMPLE 12 Isolation of cDNA Clones Encoding Human PROl 017 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein designated " ⁇ consen01>", sometimes called DNA53235. Based on the assemblies presented herein and the consensus sequences, EST AA243086 (Merck clone 664402) was further examined and sequenced.
  • DNA561 12-1379 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 128-130, and an apparent stop codon at nucleotide positions 1370-1372.
  • the predicted polypeptide precursor is 414 amino acids long.
  • Analysis of the full-length PRO 1017 sequence shown in Figure 20 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 31 ; N-glycosylation sites from about amino acid 134 to about amino acid 138, from about amino acid 209 to about amino acid 213, from about amino acid 280 to about amino acid 284, and from about amino acid 370 to about amino acid 374; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 85 to about amino acid 89, and from about amino acid 236 to about amino acid 240; and N- myristoylation sites from about amino acid 77 to about amino acid 83, from about amino acid 164 to about amino acid 170, and from about amino acid 295 to about amino acid 301.
  • DNA57702-1476 was identified by applying the proprietary signal sequence finding algorithm described in Example 3 above.
  • Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from public (e.g., GenBank) and/or private LIFESEQ ® Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases.
  • the clustering and assembling of the public and private ESTs into one or several consensus sequences to create a candidate sequence was performed using repeated cycles of the computer program phrap. (Phil Green,
  • 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. The consensus sequence obtained therefrom is herein designated as DNA56108.
  • Analysis of the full-length PROl 1 12 sequence shown in Figure 22 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • Analysis of the full-length PROl 112 sequence shown in Figure 22 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 13; transmembrane domains from about amino acid 58 to about amino acid 76, from about amino acid 99 to about amino acid 1 13, from about amino acid 141 to about amino acid 159, and from about amino acid 203 to about amino acid 222; and N-myristoylation sites from about amino acid 37 to about amino acid 43, from about amino acid 42 to about amino acid 48, and from about amino acid 229 to about amino acid 235.
  • cDNA Clones Encoding Human PRO509 To isolate a cDNA for PRO509 (also called HVEM), a bacteriophage library of human retinal cDNA (commercially available from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on an EST sequence (GenBank locus AA021617), which showed some degree of homology to members of the TNFR family. Five positive clones (containing cDNA inserts of 1.8-1.9 kb) were identified in the cDNA library, and the positive clones were confirmed to be specific by PCR using the above hybridization probe as a PCR primer. Single phage plaques containing each of the five positive clones were isolated by limiting dilution and the DNA was purified using a Wizard Lambda Prep DNA purification kit (commercially available from Promega).
  • the cDNA inserts from three of the five bacteriophage clones were excised from the vector arms by digestion with EcoRI, gel-purified, and subcloned into pRK5 and sequenced on both strands.
  • the three clones contained an identical open reading frame (with the exception of an intron found in one of the clones).
  • the entire sequence of DNA50148 (HVEM) is shown in Figure 23 (SEQ ID NO:45).
  • the cDNA contained one open reading frame with a translational initiation site assigned to the ATG codon at nucleotide positions 82-84.
  • the open reading frame ends at the termination codon TGA at nucleotide positions 931 -933.
  • the predicted amino acid sequence of the full-length PRO509 contains 283 amino acids as shown in Figure 24 (SEQ ID NO:46).
  • the full-length PRO509 protein shown in Figure 24 has an estimated molecular weight of about 30,420 daltons and a pl of about 7.34.
  • Analysis of the full-length PRO509 sequence shown in Figure 24 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 36; a transmembrane domain from about amino acid 203 to about amino acid 222; N-glycosylation sites from about amino acid 1 10 to about amino acid 1 14, and from about amino acid 173 to about amino acid 177; and N-myristoylation sites from about amino acid 81 to about amino acid 87, from about amino acid 89 to about amino acid 95, from about amino acid 104 to about amino acid 1 10, from about amino acid 120 to about amino acid 126, from about amino acid 153 to about amino acid 159, from about amino acid 193 to about amino acid 199, from about amino acid 195 to about amino acid 201 , and from about amino acid 220 to about amino acid 226.
  • the sequence differs from the PRO509 (HVEM) sequence reported in Montgomery etal, supra, in at least two amino acids: codon 108 encodes a serine and codon 140 encodes an alanine.
  • An alignment using the ALIGN computer program) of a 58 amino acid long cytoplasmic region of PRO509 (HVEM) with other known members of the human TNF receptor family showed some similarity, in particular to CD40 (12 identities) and LT-beta receptor (11 identities).
  • Example 1 The analysis identified the single Incyte clone no. 2645134.
  • the Incyte 2645134 sequence was then extended using repeated cycles of BLAST and phrap to extend the sequence as far as possible using the sources of EST sequences discussed above. This extended assembled consensus sequence is herein designated
  • DNA43050 Based on the DNA43050 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 PR0853.
  • forward PCR primer (43050.fl): 5'-CTTCATGGCCTTGGACTTGGCCAG-3' (SEQ ID NO:49) reverse PCR primer (43Q50.rl ):
  • a synthetic oligonucleotide hybridization probe was constructed from the DNA43050 consensus sequence: 5'-CTTTCTGAGCTCTGAGCCACGGTTGGACATCCTCATCCACAATGC-3' (SEQID NO:51)
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228). DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA48227-1350 [ Figure 25, SEQ ID NO:47]; and the derived protein sequence for PR0853.
  • DNA48227-1350 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 128-130, and an apparent stop codon at nucleotide positions 1259-1261.
  • the predicted polypeptide precursor is 377 amino acids long.
  • Analysis of the full-length PR0853 sequence shown in Figure 26 (SEQ ID NO:48) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above.
  • a signal peptide from about amino acid 1 to about amino acid 16; a glycosaminoglycan attachment site from about amino acid 46 to about amino acid 50; N-myristoylation sites from about amino acid 9 to about amino acid 15, from about amino acid 29 to about amino acid 35, from about amino acid 32 to about amino acid 38, from about amino acid 43 to about amino acid 49, from about amino acid 124 to about amino acid 130, and from about amino acid 312 to about amino acid 318; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 118 to about amino acid 129; and short-chain alcohol dehydrogenase family sites from about amino acid 37 to about amino acid 50, and from about amino acid 1 14 to about amino acid 125.
  • Clone DNA48227-1350 has been deposited with the ATCC on April 28, 1998 and is assigned ATCC deposit no. 209812.
  • the full-length PR0853 protein shown in Figure 26 has an estimated molecular weight of about 40,849 daltons and a pl of about 7.98.
  • PR0882 (DNA58125) is identical with cardiotrophin-1.
  • the amino acid sequence of this 201 amino acid protein is present in the public Dayhoff database under Accession Nos. P_R83967, P_W29238 and CTF1_HUMAN, among others.
  • the nucleotide sequence of DNA58125 encoding the PR0882 is shown in Figure 27 (SEQ ID NO:52).
  • EXAMPLE 17 Gene Amplification This example shows that the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding 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.
  • Therapeutic agents may take the form of antagonists of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptides, for example, murine-human chimeric, humanized or human antibodies against a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
  • the starting material for the screen was genomic DNA isolated from a variety of cancers.
  • the DNA is quantitated precisely, e.g., fluorometrically.
  • DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown).
  • the 5' nuclease assay for example, TaqManTM
  • real-time quantitative PCR for example, ABI Prizm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied Biosystems Division, Foster City, CA)
  • ABI Prizm 7700 Sequence Detection SystemTM Perkin Elmer, Applied Biosystems Division, Foster City, CA
  • the results were used to determine whether the DNA encoding PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 is over-represented in 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 4. An explanation of the abbreviations used for the designation of the primary tumors listed in Table 4 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore.
  • 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 PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PR01017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encoding gene.
  • Regions of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
  • primers and probes forward, reverse and probe
  • PRO201 The sequences for the primers and probes (forward, reverse and probe) used for the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO347,PRO357, PRO715,PRO1017, PROl 1 12, PRO509, PRO853 or PR0882 gene amplification analysis were as follows:
  • PRO201 (DNA30676-1223): 30676.tm.f
  • PRQ292 (DNA35617): 35617.tm.f
  • PRQ327 (DNA381 13-1230): 381 13.tm.f 5'-CTCAAGAAGCACG CGTACTGC-3' (SEQ ID NO: 60)
  • PRQ344 (DNA40592-1242): 40592.tm.fl 5'-TGGCAAGGAATGGGAACAGT-3' (SEQ ID NO: 66)
  • PRQ715 (DNA52722-1229): 52722.tm.fl 5'-TGGCTCCCAGCTTGGAAGA-3' (SEQ ID NO: 78)
  • PRO1017 (DNA561 12-1379): 56112.tm.fl
  • PRO! 1 12 (DNA57702-1476): 57702.tm.fl
  • PRO509 (DNA50148): 50148.tm.fl
  • PR0853 (DNA48227-1350) 48227.tm.fl 5'-GGCACTTCATGGTCCTTGAAA-3' (SEQ ID NO: 90)
  • 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 interpretation 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 4 describes the stage, T stage and N stage of various primary tumors which were used to screen the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 compounds of the invention.
  • Table 4 Primary Lung and Colon Tumor Profiles

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Abstract

The invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The invention is based upon the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product as compared to normal cells of the same tissue type and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment. The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR
Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of tumor.
Background of the Invention Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin.. 43:7 [ 1993]).
Cancer is characterized by an increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different dςgrees of invasiveness and aggressiveness. Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter, Cell. 64: 1129 [1991 ] and Bishop,
Cel], 64:235-248 [1991]).
A well known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification. This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al., Adv. Cancer Res.. 47:235-281 [1986]). It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made.
Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor (p 185HER2; HER2) related to the epidermal growth factor receptor EGFR), is overexpressed in about 25% to 30% of human breast cancer (Slamon etal.. Science. 235:177-182 [1987]; Slamon et al., Science, 244:707-712 [1989]).
It has been reported that gene amplification of a proto-oncogene is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al., Genes Chromosomes Cancer, ]_: 181 - 193 [ 1990] ; Alitalo et al., supra). Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., [ 1987] and [ 1989] , supra; Ravdin and Chamness, Gene, 159: 19-27 [ 1995] ; and Hynes and Stern, Biochim. Biophys. Acta, 1 198: 165-184 [1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga etal., Oncology, 1 1 (3 Suppl l):43-48 [1997]). However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-positive patients responding clinically to treatment with taxanes were greater than three times those of HER2-negati ve patients (Ibid). A recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin™) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol., 14:737-744 [1996]). In light of the above, there is obvious interest in identifying novel methods and compositions which are useful for diagnosing and treating tumors which are associated with gene amplification.
Summary of the Invention A. Embodiments The present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The present invention is based on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment.
In one embodiment, the present invention concerns an isolated antibody which binds to a polypeptide designated herein as a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. In one aspect, the isolated antibody specifically binds to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide. In another aspect, the antibody induces the death of a cell which expresses a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. Often, the cell that expresses the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type. In yet another aspect, the antibody is a monoclonal antibody, which preferably has non- human complementarity determining region (CDR) residues and human framework region (FR) residues. The antibody may be labeled and may be immobilized on a solid support. In yet another aspect, the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody which binds, preferably specifically, to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
In another embodiment, the invention concerns a composition of matter which comprises an antibody which binds, preferably specifically, to a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition of matter comprises a therapeutically effective amount of the antibody. In another aspect, the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent. Preferably, the composition is sterile. In a further embodiment, the invention concerns isolated nucleic acid molecules which encode anti- PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies, and vectors and recombinant host cells comprising such nucleic acid molecules.
In a still further embodiment, the invention concerns a method for producing an anti-PRO201 , anti- PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PR01 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture.
The invention further concerns antagonists of a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide that inhibit one or more of the biological and/or immunological functions or activities of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
In a further embodiment, the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PRO715,PRO1017, PROl 112, PRO509, PRO853 or PR0882 polypeptide or the complement thereof. Theisolated nucleic acid molecule is preferably DNA, and hybridization preferably occurs under stringent hybridization and wash conditions. Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the transcription and/or translation of the respective amplified genes, or as antisense primers in amplification reactions. Furthermore, such sequences can be used as part of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes.
In another embodiment, the invention provides a method for determining the presence of a PRO201 ,
PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509,
PR0853 or PR0882 polypeptide in a sample suspected of containing a PRO201 , PR0292, PR0327, PROl 265,
PR0344, PR0343.PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, wherein the method comprises exposing the sample to an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PRO 1 112, anti- PRO509, anti-PR0853 or anti-PR0882 antibody and determining binding of the antibody to a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in the sample. In another embodiment, the invention provides a method for determining the presence of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PROlOl 7, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and determining binding of the antibody to the cell.
In yet another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti- PR0882 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of acomplex between theanti-PRO201, anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal. 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 to have neoplastic cell growth or proliferation (e.g. cancerous cells).
In another embodiment, the present invention concerns a cancer diagnostic kit comprising an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PROlOl 7, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and a carrier (e.g., a buffer) in suitable packaging. The kit preferably contains instructions for using the antibody to detect the presence of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in a sample suspected of containing the same. In yet another embodiment, the invention concerns a method for inhibiting the growth of tumor cells comprising exposing tumor cells which express a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to an effective amount of an agent which inhibits a biological and/or immunological activity and/or the expression of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, wherein growth of the tumor cells is thereby inhibited. The agent preferably is an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule. In a specific aspect, the agent, e.g., the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti- PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, induces cell death. In a further aspect, the tumor cells are further exposed to radiation treatment and/or a cytotoxic or chemotherapeutic agent. In a further embodiment, 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 inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882 polypeptide as compared to a normal cell of the same tissue type. In particular aspects, the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. In preferred aspects, the active agent is an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti- PR0853 or anti-PR0882 antibody or an antisense oligonucleotide.
The invention also provides a method for identifying a compound that inhibits an activity of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, comprising contacting a candidate compound with a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether a biological and or immunological activity of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide is inhibited. In a specific aspect, either the candidate compound or the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is immobilized on a solid support. In another aspect, the non-immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened in the presence of the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist. In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is inhibited. In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and (b) determining the inhibition of expression of said polypeptide.
B. Additional Embodiments
In other embodiments of the present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. In one aspect, 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 about 99% sequence identity to (a) a DNA molecule encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In other aspects, 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 about 99% sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide cDNA as disclosed herein, the coding sequence of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In a further aspect, 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 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a). Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody or as antisense oligonucleotide probes. Such 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 in 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 in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length and yet more preferably at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of aPRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357,
PR0715, PRO 1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide fragments that comprise a binding site for an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-
PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
In another embodiment, the invention provides isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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% 5 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%
10 sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically
15 defined fragment of the full-length amino acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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%
20 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%
25 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 99% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.
30 In a further aspect, the invention concerns an isolated PRO201, PR0292, PR0327, PR01265, PR0344,
PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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
35 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 about 99% positives when compared with the amino acid sequence of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and recovering the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide from the cell culture.
Another aspect of the invention provides an isolated PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide and recovering the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide from the cell culture. In yet another embodiment, the invention concerns antagonists of a native PRO201 , PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide as defined herein. In a particular embodiment, the antagonist is an anti-PRO201, anti-PR0292, anti- PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody or a small molecule. In a further embodiment, the invention concerns a method of identifying antagonists to a PRO201 ,
PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PR01112, PRO509, PR0853 or PR0882 polypeptide which comprise contacting the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO201, PR0292, PR0327, PRO 1265, PRO344, PRO343, PRO347, PRO357, PRO715,PRO1017, PROl 1 12, PRO509.PRO853 or PR0882 polypeptide. Preferably, the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is a native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. In a still further embodiment, the invention concerns a composition of matter comprising a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, or an antagonist of a PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882polypeptide as herein described, or an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti- PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, or an antagonist thereof as hereinbefore described, or an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti- PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, an antagonist thereof or an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli, yeast, or Baculovirus-infected insect cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
In other embodiments, 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.
In another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody. In yet other embodiments, 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.
Brief Description of the Figures Figure 1 shows the nucleotide sequence (SEQ ID NO: 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PRO201 , wherein the nucleotide sequence (SEQ ID NO: 1 ) is a clone designated herein as DNA30676-1223. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 2 shows the amino acid sequence (SEQ ID NO:2) of a native sequence PRO201 polypeptide as derived from the coding sequence of SEQ ID NO: 1 shown in Figure 1.
Figure 3 shows the nucleotide sequence (SEQ ID NO:5) of a cDNA containing a nucleotide sequence encoding native sequence PR0292, wherein the nucleotide sequence (SEQ ID NO:5) is a clone designated herein as DNA35617. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 4 shows the amino acid sequence (SEQ ID NO:6) of a native sequence PR0292 polypeptide as derived from the coding sequence of SEQ ID NO:5 shown in Figure 3.
Figure 5 shows the nucleotide sequence (SEQ ID NO:7) of a cDNA containing a nucleotide sequence encoding native sequence PR0327, wherein the nucleotide sequence (SEQ ID NO:7) is a clone designated herein as DNA381 13-1230. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 6 shows the amino acid sequence (SEQ ID NO:8) of a native sequence PR0327 polypeptide as derived from the coding sequence of SEQ ID NO:7 shown in Figure 5.
Figure 7 shows the nucleotide sequence (SEQ ID NO: 12) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1265, wherein the nucleotide sequence (SEQ ID NO: 12) is a clone designated herein as DNA60764-1533. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 8 shows the amino acid sequence (SEQ ID NO: 13) of a native sequence PRO 1265 polypeptide as derived from the coding sequence of SEQ ID NO: 12 shown in Figure 7.
Figure 9 shows the nucleotide sequence (SEQ ID NO: 14) of a cDNA containing a nucleotide sequence encoding native sequence PR0344, wherein the nucleotide sequence (SEQ ID NO: 14) is a clone designated herein as DNA40592-1242. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 10 shows the amino acid sequence (SEQ ID NO: 15) of a native sequence PR0344 polypeptide as derived from the coding sequence of SEQ ID NO: 14 shown in Figure 9. Figure 1 1 shows the nucleotide sequence (SEQ ID NO:22) of a cDNA containing a nucleotide sequence encoding native sequence PR0343, wherein the nucleotide sequence (SEQ ID NO:22) is a clone designated herein as DNA43318-1217. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 12 shows the amino acid sequence (SEQ ID NO:23) of a native sequence PR0343 polypeptide as derived from the coding sequence of SEQ ID NO:22 shown in Figure 1 1.
Figure 13 shows the nucleotide sequence (SEQ ID NO:27) of a cDNA containing a nucleotide sequence encoding native sequence PR0347, wherein the nucleotide sequence (SEQ ID NO:27) is a clone designated herein as DNA44176-1244. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 14 shows the amino acid sequence (SEQ ID NO:28) of a native sequence PR0347 polypeptide as derived from the coding sequence of SEQ ID NO:27 shown in Figure 13.
Figure 15 shows the nucleotide sequence (SEQ ID NO:32) of a cDNA containing a nucleotide sequence encoding native sequence PR0357, wherein the nucleotide sequence (SEQ ID NO:32) is a clone designated herein as DNA44804- 1248. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 16 shows the amino acid sequence (SEQ ID NO:33) of a native sequence PR0357 polypeptide as derived from the coding sequence of SEQ ID NO:32 shown in Figure 15.
Figure 17 shows the nucleotide sequence (SEQ ID NO:39) of a cDNA containing a nucleotide sequence encoding native sequence PR0715, wherein the nucleotide sequence (SEQ ID NO:39) is a clone designated herein as DNA52722-1229. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 18 shows the amino acid sequence (SEQ ID NO:40) of a native sequence PR0715 polypeptide as derived from the coding sequence of SEQ ID NO:39 shown in Figure 17. Figure 19 shows the nucleotide sequence (SEQ ID NO:41 ) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1017, wherein the nucleotide sequence (SEQ ID NO:41 ) is a clone designated herein as DNA561 12-1379. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 20 shows the amino acid sequence (SEQ ID NO:42) of a native sequence PRO1017 polypeptide as derived from the coding sequence of SEQ ID NO:41 shown in Figure 19.
Figure 21 shows the nucleotide sequence (SEQ ID NO:43) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 112, wherein the nucleotide sequence (SEQ ID NO:43) is a clone designated herein as DNA57702-1476. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 22 shows the amino acid sequence (SEQ ID NO:44) of a native sequence PROl 112 polypeptide as derived from the coding sequence of SEQ ID NO:43 shown in Figure 21.
Figure 23 shows the nucleotide sequence (SEQ ID NO:45) of a cDNA containing a nucleotide sequence encoding native sequence PRO509, wherein the nucleotide sequence (SEQ ID NO:45) is a clone designated herein as DNA50148. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 24 shows the amino acid sequence (SEQ ID NO:46) of a native sequence PRO509 polypeptide as derived from the coding sequence of SEQ ID NO:45 shown in Figure 23.
Figure 25 shows the nucleotide sequence (SEQ ID NO:47) of a cDNA containing a nucleotide sequence encoding native sequence PR0853, wherein the nucleotide sequence (SEQ ID NO:47) is a clone designated herein as DNA48227-1350. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 26 shows the amino acid sequence (SEQ ID NO:48) of a native sequence PR0853 polypeptide as derived from the coding sequence of SEQ ID NO:47 shown in Figure 25.
Figure 27 shows the nucleotide sequence (SEQ ID NO:52) of a cDNA containing a nucleotide sequence encoding native sequence PR0882, wherein the nucleotide sequence (SEQ ID NO:52) is a clone designated herein as DNA58125. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 28 shows the amino acid sequence (SEQ ID NO:53) of a native sequence PR0882 polypeptide as derived from the coding sequence of SEQ ID NO:52 shown in Figure 27.
Figure 29 is a map of chromosome 19 showing the mapping regions of DNA30676- 1223, DNA381 13-1230 and DNA60764-1533. Figure 30 is a map of chromosome 11 showing the mapping region of DNA35617. Figure 31 is a map of chromosome 16 showing the mapping region of DNA43318- 1217and DNA58125. Figure 32 is a map of chromosome 7 showing the mapping region of DNA561 12-1379. Figure 33A is map of chromosome 17 showing the mapping region of DNA52722-1229. Figure 33B is a map of chromosome 17 showing the mapping region of DNA48227-1350.
Figure 34 is a map of chromosome 16 showing the mapping region of DNA44804-1248.
Detailed Description of the Invention I. Definitions The phrases "gene amplification" and "gene duplication" are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon." Usually, the amount of the messenger RNA (mRNA) produced, i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed. "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc.
"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, horses, cats, cattle, pigs, sheep, etc. Preferably, 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. Examples of 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) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I131, 1125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (" Ara- C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter 1 , entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami etal., (WB Saunders: Philadelphia, 1995), especially p. 13.
"Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3- amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,l l-trihydroxy-8-(hydroxyacetyl)-l- methoxy-5, 12-naphthacenedione.
The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet- growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -a, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G- CSF); interleukins (ILs) such as IL-1, IL- la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions. j_4:375-382, 615th Meeting, Belfast (1986), and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery, Borchardt et al., (ed.), pp. 147-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrugs form for use in this invention include, but are not limited to, those chemotherapeutic agents described above. An "effective amount" of a polypeptide disclosed herein or an antagonist thereof, in reference to inhibition of neoplastic cell rowth, tumor growth or cancer cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells. The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells. An "effective amount" of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide antagonist for purposes of inhibiting neoplastic cell growth, tumor growth or cancer cell growth, may be determined empirically and in a routine manner.
A "therapeutical ly effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects : ( 1 ) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder. A "therapeutical ly effective amount" of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner.
A "growth inhibitory amount" of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "growth inhibitory amount" of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
A "cytotoxic amount" of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 antagonist is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount" of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. The terms "PRO201 ", "PR0292", "PR0327", "PR01265", "PR0344", "PR0343", "PR0347",
"PR0357", "PR0715", "PRO1017", "PROl 1 12", "PRO509", "PR0853" or "PR0882" polypeptide or protein when used herein encompass native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides and PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide variants (which are further defined herein). The PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods.
A "native sequence PRO201 ", "native sequence PR0292", "native sequence PR0327", "native sequence PR01265", "native sequence PR0344", "native sequence PR0343", "native sequence PR0347", "native sequence PR0357", "native sequence PR0715", "native sequence PRO 1017", "native sequence PROl 112", "native sequence PRO509", "native sequence PR0853" or "native sequence PR0882" comprises a polypeptide having the same amino acid sequence as the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide as derived from nature. Such native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native sequence" PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally- occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 and PR0882 polypeptides. In one embodiment of the invention, the native sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide is a mature or full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide as shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 5 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), respectively. Also, while the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 and PR0882 polypeptides disclosed in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24
10 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), respectively, are shown to begin with the methionine residue designated therein as amino acid position 1 , it is conceivable and possible that another methionine residue located either upstream or downstream from amino acid position 1 in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure
15 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), respectively, may be employed as the starting amino acid residue for the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
The "extracellular domain" or "ECD" of a polypeptide disclosed herein refers to a form of the polypeptide
20 which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a polypeptide ECD will have less than about 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than about 0.5% of such domains. It will be understood that any transmembrane domain(s) identified for the 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
25 about 5 amino acids at either end of the domain as initially identified and as shown in the appended figures. As such, in one embodiment of the present invention, the extracellular domain of a polypeptide of the present invention comprises amino acids 1 to X of the mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side of the extracellular domain/transmembrane domain boundary.
The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are
30 shown in the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng.. K): l-6 (1997) and von Heinje et al, Nucl. Acids. Res., J4:4683-4690 (1986)). Moreover, it is also
35 recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
"PRO201 polypeptide variant", "PR0292 polypeptide variant", "PR0327 polypeptide variant", "PRO 1265 polypeptide variant", "PR0344 polypeptide variant", "PR0343 polypeptide variant", "PR0347 polypeptide variant", "PR0357 polypeptide variant", "PR0715 polypeptide variant", "PROl 017 polypeptide variant", "PROl 1 12 polypeptide variant", "PRO509 polypeptide variant", "PR0853 polypeptide variant" or "PR0882 polypeptide variant" means an active PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide sequence as disclosed herein, a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide sequence as disclosed herein. Such PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide variants include, for instance, PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino 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% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide sequence as disclosed herein, a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide sequence as disclosed herein. Ordinarily, PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 orPR0882 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more. As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
In addition, Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO- DNA" represents a hypothetical PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 112-, PRO509-, PR0853- or PR0882-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.
Table 1
/*
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define _M -8 /* value of a match with a stop */ int day[26][26] = {
/* A" B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A*/ 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/*B*/ 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C*/ ■2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D*/ 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3},
/* p */ -4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G*/ 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0},
/*H*/ -1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,JΛ, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
1*1*1 -1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
I* J*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/*K*/ -1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0}, l*L*l -2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-l, 0, 2,-2, 0,-1,-2}, l*U*l -1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1},
/*N*/ 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
1*0*1 _M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1, -1, -3, -1, -1, ,-5, .-1, , 0, -2, , 0, ,-L ,-3, ,-2. ,-1, , M. ,6, ,0,0, 1,0,0,-1,-6,0,-5,0}, /*Q*/ 0, 1, -5, 2, 2, -5, -1, 3, -2, 0, 1, -2, -1, 1, M, 0, 4, 1,-1,-1,0,-2,-5,0,-4, 3}, /*R*/ -2, 0, -4, -1, -1,-4, .-3, ,2, .-2, , 0, ,3, ,-3, , o, 0, M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /*S*/ 1, o, 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1,- -1,0,2, 1,0,-1,-2,0,-3,0}, /* f */ 1, 0, -2, o, 0, -3, 0, -1, 0, 0, 0, -1, -1, 0,' M, 0,- -1,-1, 1,3,0,0,-5,0,-3,0},
/*U*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,' M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* v */ 0, -2, -2, -2, -2, ,-1, ,-L ,-2, ,4, , o, ,-2.. ,2, .2, -2, ~ , A, ,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2},
/* w*/ -6, -5, -8, -7, ,-7.. , 0, -7. ,-3. ,-5. , o, ,-3. ,-2 ,-4 ,-4. , M ,-6. ,-5,2,-2,-5,0,-6,17,0,0,-6}, /*x*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, o, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y */ -3, -3, 0, -4, -4, , 7, -5, 0, -1, 0, -4, ,-L ,-2, -2, " M. ,-5, ,-4,-4,-3,-3,0,-2,0,0,10,-4},
/*z*/ 0, 1, -5, 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Page 1 of day. h /* */
#include < stdio.h > #include <ctyρe.h>
#defιne MAXJMP 16 /* max jumps in a diag */
#defme MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp */ define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#defme DINSO 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) * 7 unsigned short x[MAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2Λ16 -1 * */ struct diag { int score; /* score at last jmp */ long offset; /* offset of prev block */ short ijmp; /* current jmp index */ struct jmp jp; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; /* output file name */ char *namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best diag: nw() */ int dmaxO; /* final diag */ int dna; /* set if dna: main() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds path for seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char *getseq(), *g_calloc();
Page 1 of nw.h /* Needleman-Wunsch alignment program
*
* usage progs filel file2
* where filel and file2 are two dna or two protein sequences
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ',', ' > ' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file ' align out" *
* The program may create a tmp file in /tmp to hold info about traceback
* Original version developed under BSD 43 on a vax 8650 */
^include ' nw h" #include "day h' static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }. static _pbval[26] = {
1,2|(1<<('D'-'A'))|(K<('N'-'A')), 4, 8, 16,32,64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1 < <21, 1< <22,
1<<23, 1<<24, 1<<25|(1<<(,E,-'A'))|(1<<('Q'-'A')) }, maιn(ac, aavv)) main int ac, char *av[], prog = = av[0], if (ac ' = 3){ fpπntf(stderr, ' usage %s filel file2\n", prog), fpπntf(stderr, "where filel and file2 are two dna or two protein sequences \n"), fpπntf(stderr,"The sequences can be in upper- or lower case\n"), fpπntf(stderr, " Any lines beginning with ',' or ' < ' are ιgnored\n"), fpπntf(stderr, "Output is in the file \ 'align outY \n' ), exιt(l),
} namex[0] = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0], &len0), seqxfl] = getseq(namex[l], &lenl), xbm = (dna)9 dbval _pbval, endgaps = 0, /* 1 to penalize endgaps */ ofile = "align out", /* output file */ nw(), /* fill in the matrix, get the possible jmps */ readjmpsO, /* get the actual jmps */ print(), /* print stats, alignment */ cleanup(O), /* unlink any tmp files */
Page 1 of nw.c /* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*/ nw() nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping rowO, rowl */ int mis; /* score for each type */ int insO, insl; /* insertion penalties */ register id; /* diagonal index */ register ϋ; /* jmp index */ register *col0, *coll; /* score for curr, last row */ register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO + lenl + 1, sizeof(struct diag)); ndely = (int *)g_calloc("to get ndely", lenl + 1 , sizeof(int)); dely = (int *)g_calloc("to get dely", lenl + 1 , sizeof(int)); coIO = (int *)g_calloc("to get colO", lenl + 1, sizeof(int)); coll = (int *)g_calloc("to get coll ", lenl + 1, sizeof(int)); insO = (dna)? DINS0 : PINS0; insl = (dna)? DINS 1 : PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1; yy < = lenl ; yy + +) { col0[yy] = dely[yy] = col0[yy-l] - insl; ndely[yy] = yy;
} col0[0] = 0; /* Waterman Bull Math Biol 84 */
} else for (yy = 1; yy < = lenl; yy+ +) dely[yy] = -insO;
/* fill in match matrix */ for (px = seqx[0], xx = 1 ; xx < = lenO; px+ + , xx+ +) { /* initialize first entry in col */ if (endgaps) { if (xx = = 1) coll [0] = delx = -(insO + insl); else coll [0] = delx = col0[0] - insl ; ndelx = xx;
} else { coll[0] = 0; delx = -insO; ndelx = 0;
Page 2 of nw.c ...nw seqx[l], yy = 1; yy < = lenl ; py + + , yy+ +) { mis = col0[yy-l]; if (dna) mis + = (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS; else mis + = _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely[yy] < MAXGAP) { if (colOfyy] - insO > = dely[yy]) { delyfyy] = colOfyy] - (insO + insl); ndely[yy] = 1 ; } else { dely[yy] -= insl; ndely [yy] + + ;
} } else { if (colO[yy] - (insO+insl) > = delyfyy]) { dely[yy] = colOfyy] - (insO+insl); ndely [yy] = 1; } else ndely[yy] + + ; }
/* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { delx = coll[yy-l] - (insO + insl); ndelx = 1; } else { delx -= insl; ndelx+ + ; }
} else { if (coIl[yy-l] - (insO+insl) > = delx) { delx = coIl[yy-l] - (insO+insl); ndelx = 1;
} else ndelx+ + ;
}
/* pick the maximum score; we're favoring * mis over any del and delx over dely
*/
Page 3 of nw.c ...n id = xx - yy + lenl - 1 ; if (mis > = delx && mis > = delyfyy]) coll fyy] = mis; else if (delx > = delyfyy]) { collfyy] = delx; ij = dxfid]. ijmp; if (dxfid] .jp.nfO] && (!dna 1 1 (ndelx > = MAXJMP && xx > dx[id].jp.x[ij] +MX) 1 1 mis > dx[id].score+DINSO)) { dx[id].ijmp+ + ; if (+ + ij > = MAXJMP) { writejmps(id); ij = dxfid]. ijmp = 0; dxfid]. offset = offset; offset + = sizeof (struct jmp) + sizeof(offset); } } dxfid] jp.nfij] = ndelx; dxfid] .jp.xfij] = xx; dxfid]. score = delx;
} else { coll fyy] = delyfyy]; ij = dxfid]. ijmp; if (dxfid] .jp.nfO] && (!dna | | (ndelyfyy] > = MAXJMP
&& xx > dx[id].jp.x[ij] +MX) | j mis > dx[id].score+DINSO)) { dxfid]. ijmp+ + ; if (+ +ij > = MAXJMP) { writejmps(id); ij = dxfid]. ijmp = 0; dxfid]. offset = offset; offset + = sizeof (struct jmp) + sizeof (offset);
} dx[id].jp.n[ij] = -ndelyfyy]; dxfid] .jp.xfij] = xx; dxfid]. score = delyfyy];
} if (xx = = lenO && yy < lenl) { /* last col
*/ if (endgaps) collfyy] -= ins0+insl*(lenl-yy); if (col 1 [yy] > smax) { smax = coll fyy]; dmax = id; }
} } if (endgaps && xx < lenO) coll [yy-l] -= ins0+insl *(len0-xx); if (collfyy- 1] > smax) { smax = coll[yy-l]; dmax = id;
} tmp = colO; colO = coll ; coll = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0)
(void) free((char *)coll)
Page 4 of nw.c * print() -- only routine visible outside this module
*
* static:
* getmat() — trace back best path, count matches: print()
* pr alignO — print alignment of described in array p[]: print()
* dumpblock() — dump a block of lines with numbers, stars: pr_align()
* numsO - put out a number line: dumpblock()
* putline() — put out a line (name, [num], seq, [num]): dumpblockO
* stars() - -put a line of stars: dumpblockO
* stripnameO — strip any path and prefix from a seqname */
^include "nw.h"
#define SPC 3
^define P LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx; /* output file */ printo print
{ int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) = = 0) { fprintf(stderr, " %s: can't write %s\n", prog, ofile); cleanup(l);
} fprintf(fx, " < first sequence: %s (length = %d)\n", namexfO], lenO); fprintf(fx, " < second sequence: %s (length = %d)\n", namexfl], lenl); olen = 60; lx = lenO; ly = lenl; firstgap = lastgap = 0; if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1 ; ly -= pp[0].spc;
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1); lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1; lx -= lastgap;
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly -= lastgap;
} getmat(lx, ly, firstgap, lastgap); pr alignO;
Page 1 of nwprint.c * trace back the best path, count matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /* "core" (minus endgaps) */ int firstgap, lastgap; /* leading trailing overlap */ int ran, iO, il , sizO, sizl; char outx[32]; double pet; register nO, nl; register char *p0, *pl;
/* get total matches, score */ iO = il = sizO = sizl = 0; pO = seqxfO] + pp[l].spc; pi = seqxfl] + pp[0].spc; nO = pp[l].spc + 1; nl = pp[0].spc + 1; nm = 0; while ( *p0 && *pl ) { if (sizO) { pl + + ; nl + + ; sizO— ;
} else if (sizl) { p0+ +; n0+ + ; sizl—;
} else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ; if (nO+ + = = pp[0].x[iO]) sizO = pp[0].n[iO + +]; if (nl + + = = pp[l].x[il]) sizl = pp[l].n[il + +]; p0+ + ; pl + + ;
/* pet homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)? lenO : lenl; else lx = (lx < ly)? lx : ly; pet = 100.*(double)nm/(double)lx; fprintf(fx, "\n"); fprintf(fx, " < %d match%s in an overlap of %ά: %.2f percent similarity\n" nm, (nm = = 1)? "" : "es", lx, pet);
Page2 ofnwprint.c fprintf(fx, " < gaρs in first sequence: %d", gapx); ..getmat if (gapx) {
(void) sprintf(outx, " (%d %s%s)", ngapx, (dna)? "base": "residue", (ngapx = = 1 I )V?? "" "":: ""s"); fprintf(fx," %s", outx); fprintf(fx, ", gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base": "residue", (ngapy = = 1)? "": "s"); fprintf(fx, " %s" , outx);
} if (dna) fprintf(fx,
"\n< score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n" smax, DMAT, DMIS, DINSO, DINS1); else fprintf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n", smax, PINSO, PINS1); if (endgaps) fprintf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d s%s\n", firstgap, (dna)? "base" : "residue", (firstgap = = 1)? " " : "s", lastgap, (dna)? "base" : "residue", (lastgap = = 1)? "" : "s"); else fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[lP LINE] ; /* output line */ static char starfP ] 3]; /* set by stars() */
/*
* print alignment of described in struct path ppf]
*/ static pr alignO pr align
{ int nn; /* char count */ int more; register i; for (i = 0, lmax = 0; i < 2; i+ +) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn; ncfi] = 1; nifi] = 1 ; sizfi] = ijfi] = 0; psfi] = seqxfi]; pofi] = outfi];
Page 3 of nwprint.c for (nn = nm = 0, more = 1 ; more; ) { ...pr align for (i = more = 0; i < 2; i + +) { /* * do we have more of this sequence?
*/ if(!*ps[i]) continue; more+ + ; if (pp[i].spc) { /* leading space */ *po[i] + + = ' '; pp[i].spc-;
} else if (sizfi]) { /* in a gap */
*po[i] + + = '-'; sizfil— ;
} else { /* we're putting a seq element
*/ *po[i] = *ps[i]; if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]); po[i] + + ; ps[i] + + ;
/*
* are we at next gap for this seq?
*/ if(ni[i] ==pp[i].x[ij[i]]){ /*
* we need to merge all gaps
* at this location */ sizfi] = pp[i].n[ij[i] + +]; while (nifi] == pp[i].x[ij[i]]) sizfi] +=pp[i].n[ij[i] + +]
} ni[i] + +;
}
} if (+ +nn = = olen 11 !more && nn) { dumpblockO; for(i = 0; i < 2; i++) pofi] = outfi]; nn = 0; }
/*
* dump a block of lines, including numbers, stars: pr alignO
*/ static dumpblockO dumpblock
{ register i; for(i = 0; i < 2; i + +) *po[i]- = '\0';
Page 4 of nwprint.c .dumpblock
(void) putc('\n', fx); for(i = 0; i < 2; i++){ if (*out[i] && (*out[i] ! = *(po[i]) != ")){ if(i ==0) nums(i); if (i = = 0 && *out[l]) stars(); putline(i); if (i = = 0 && *out[l]) fprintf(fx, star); if(i == 1) nums(i);
/*
* put out a number line: dumpblockO */ static nu s(ix) nums int lx ; /* index in outf] holding seq line */ char nlinefP LINE]; register U; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax + P_SPC; i++,pn++)
*pn = ' '; for (i = ncfix], py = outfix]; *py; py++, pn++) { if (*py = = = " ll *py == '-')
*pn = ' '; else { if (i%10 == 0 || (i == 1 && ncfix] ! = D){ j = (i < 0)? -i : i; for (px = pn;j;j /= 10, px-)
*px = j%10 + '0'; if (i < 0)
*px = '-';
} else
*pn = i + + ;
}
}
*pn = '\0'; ncfix] = i; for (pn = nline; *pn; pn+ +) (void) putc(*pn, fx); (void)putc('\n\ fx);
}
/*
* put out a line (name, [num], seq, [num]): dumpblockO */ static putline(ix) putline
Page 5 of nwprint.c .putline int register char px; for (px = namexfix], i = 0; *px && *px ':'; ρx++, i++)
(void) putc(*px, fx); for (; i < lmax+P SPC; i++)
(void) putc(' ', fx);
/* these count from 1 :
* nif] is current element (from 1)
* ncf] is number at start of current line */ for (px = outfix]; *px; px++)
(void) putc(*px&0x7F, fx); (void) putc('\n', fx);
/*
* put a line of stars (seqs always in outfO], outfl]): dumpblockO
*/ static stars() stars
{ int register char *p0, *pl, ex, *px; if (!*out[0] 11 (*out[0] == ' '&& *(po[0]) == ' ') || !*outfl] I I (*out[l] == " && *(po[l]) == ' ')) return; px = star; for (i = lmax+P SPC; i; i~)
*px++ = ' '; for(p0 = out[0],pl = outfl]; *p0 && *pl; p0+ + , pl + +) { if (isalpha(*p0) && isalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ;
} else if (!dna &&_day[*pO-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px+ + = ex;
*px++ = '\n'; *px = '\0';
Page 6 of nwprint.c /*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) Stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px = = V) py = px + 1; if (py)
(void) strcpy(pn, py); return(strlen(pn)) ;
Page7 ofnwprint.c /*
* cleanupO — cleanup any tmp file
* getseq() — read in seq, set dna, len, maxlen
* g_calloc() - calloc() with error checkin
* readjmpsO — get the good jmps, from tmp file if necessary
* writejmpsO — write a filled array of jmps to a tmp file: nw() */
^include "nw.h" ^include < sys/file.h > char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */
FILE *0; int cleanupO; /* cleanup tmp file */ long lseek();
/* * remove any tmp file if we blow
*/ cleanupO) cleanup int i; { if ( j)
(void) unlink(jname); exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; ', ' < ', or ' > '
* seq in upper or lower case
*/ char * getseq(file, len) getseq char *file; /* file name */ int *len; /* seq len */
{ char linefl024], *pseq: register char *px, *py; int natgc, tlen;
FILE *fp; if ((fp = fopen(file,"r")) = = 0) { fprintf(stderr, " %s: can't read %s\n", prog, file); exit(l);
} tlen = natgc = 0; while (fgets(line, 1024, fp)) { if (*line = = ' ;' | | *line = = ' < ' | | *line = = ' > ') continue; for (px = line; *px ! = '\n'; px+ +) if (isuρper(*px) 1 1 islower(*px)) tlen+ + ;
} if ((pseq = maIloc((unsigned)(tlen+6))) = = 0) { fprintf(stderr," %s: malloc() failed to get %d bytes for %s\n", prog, tlen+6, file); exit(l);
} pseqfO] = pseqfl] = pseq[2] = pseq[3] = '\0' ;
Page 1 of nwsubr.c ...getseq py = pseq + 4; *len = tlen; rewind(φ); while (fgets(line, 1024, fp)) { if (*line == ';' 11 *line == '<' | | *line == '>') continue; for (px = line; *px != '\n'; px++) { if (isupper(*px))
*py + + = *pχ; else if (islower(*px))
*py++ = toupper(*px); if (index("ATGCU",*(py-l))) natgc++; } }
*py++ = '\0'; *py = '\0'; (void) fclose(φ); dna = natgc > (tlen/3); return(pseq+4);
} char * g_calloc(msg, nx, sz) g_CallθC char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */
{ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { φrintf(stderr, "%s: g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz); exit(l); } } return(px);
}
/* * get final jmps from dxf] or tmp file, set ppf], reset dmax: main()
*/ readjmpsO readjmps
{ int fd = -1; int siz, iO, il; register i, j, xx; if(fj){
(void) fclose(fj); if ((fd = open(jname, 0_RDONLY, 0)) < 0) { φrintf(stderr, "%s: can't open() %s\n", prog, jname); cleanup(l); } } for (i = iO = il = 0, dmaxO = dmax, xx = lenO; ; i + +) { while (1) { for (j = dxfdmax].ijmp; j > = 0 && dx[dmax].jp.xfj] > = xx; j--)
Page2ofnwsubr.c ...readjmps if (j < 0 && dxfdmax]. offset && fj) {
(void) lseek(fd, dxfdmax]. offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax]. offset, sizeof(dx[dmax] . offset)); dxfdmax]. ijmp = MAXJMP- 1 ;
} else break;
} if (i > = JMPS) { φrintf(stderr, " %s: too many gaps in alignment\n", prog); cleanupO); } tf α > = o) { siz = dxfdmax] .jp.nfj]; xx = dxfdmaxj.jp. xfj]; dmax + = siz; if (siz < 0) { /* gap in second seq */ pp[l].n[il] = -siz; xx + = siz;
/* id = xx - yy + lenl - 1 */ pp[l].x[il] = xx - dmax + lenl - 1; gapy+ + ; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)? -siz : MAXGAP; il + + ;
} else if (siz > 0) { /* gap in first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx+ + ; ngapx + = siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP 1 1 endgaps)? siz : MAXGAP; i0+ + ; } } else break; }
/* reverse the order of jmps */ for (j = 0, iO-; j < iO; j + + , i0~) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].xD]; pp[0].x[j] = pp[0].x[i0]; pp[0] .x[i0] = i;
} for (j = 0, il-; j < il; j + + , il-) { i = pp[l].n[j]; ppfl].n[j] = pp[l].π[il]; pp[l] .n[il] = i; i = pp[l].x[j]; pp[l].x[j] = PPfl].x[il]; pp[l].x[ϋ] = i;
} if (fd > = 0)
(void) close(fd); if ( j) {
(void) unlink(jname); fj = 0; offset = 0; }
} Page 3 of nwsubr.c /*
* write a filled jmp struct offset of the prev one (if any): nw() */ writejmps(ix) ritejmpS int ix;
{ char *mktemp(); if (iff) { if (mktemp(jname) < 0) { φrintf(stderr, " %s: can't mktempO %s\n", prog, jname); cleanupO);
} if ((fj = fopen(jname, "w")) = = 0) { φrintf(stderr, " %s: can't write %s\n", prog, jname); exit(l); } }
(void) fwrite((char *)&dx[ix].jp, sizeof (struct jmp), 1, fj); (void) fwrite((char *)&dx[ix]. offset, sizeof(dx[ix]. offset), 1, fj);
Page4 ofnwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 15 = 33.3 %
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3%
"Percent (%) amino acid sequence identity" with respect to the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 and PR0882 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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, ALIGN-2 or Megalign (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 obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program 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. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask - yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e- value = 0.01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al, Methods in Enzvmology, 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span - 1 , overlap fraction = 0.125, word threshold (T) = 11 , and scoring matrix = BLOSUM62. For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest. For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
"PRO201 variant polypeptide", "PR0292 variant polypeptide", "PR0327 variant polypeptide", "PRO 1265 variant polypeptide", "PR0344 variant polypeptide", "PR0343 variant polypeptide", "PR0347 variant polypeptide", "PR0357 variant polypeptide", "PR0715 variant polypeptide", "PRO 1017 variant polypeptide", "PROl 112 variant polypeptide", "PRO509 variant polypeptide", "PR0853 variant polypeptide" and "PR0882 variant polypeptide" or "PRO201 variant nucleic acid sequence", "PR0292 variant nucleic acid sequence", "PR0327 variant nucleic acid sequence", "PRO 1265 variant nucleic acid sequence", "PR0344 variant nucleic acid sequence", "PR0343 variant nucleic acid sequence", "PR0347 variant nucleic acid sequence", "PR0357 variant nucleic acid sequence", "PR0715 variant nucleic acid sequence", "PRO 1017 variant nucleic acid sequence", "PRO 1 112 variant nucleic acid sequence", "PRO509 variant nucleic acid sequence", "PR0853 variant nucleic acid sequence" and "PR0882 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 and PR0882 polypeptide sequence as disclosed herein, a full-length native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 and PR0882 polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 and PR0882 polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 polypeptide sequence as disclosed herein. Ordinarily, a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding a full-length native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 and PR0882 polypeptide sequence as disclosed herein, a full-length native sequence PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 and PR0882 polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 and PR0882 polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 and PR0882 polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence. Ordinarily, PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715,
PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 the PRO201 , PR0292, PR0327, PRO 1265,
PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853and PR0882 polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic 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, ALIGN-2 or Megalign (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, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, 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, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA".
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % 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)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, 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, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al, Methods in Enzymology. 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 1 1, and scoring matrix = BLOSUM62. For purposes herein, 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 of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
In other embodiments, PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357,
PRO715,PRO1017,PROl 112,PRO509,PRO853 and PR0882 variant polynucleotides are nucleic acid molecules that encode an active PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715,
PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl l 12, PRO509, PR0853 or PR0882 polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO:15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), respectively. PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 variant polypeptides may be those that are encoded by a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties. Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A.
"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. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. 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-proteinaceous solutes. In preferred embodiments, 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or an "isolated" nucleic acid encoding an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti- PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody, 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 PRO201-, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PROl 017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding nucleic acid or the anti-PRO201-, anti-PR0292-, anti-PR0327-, anti-PROl 265-, anti-PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti-PROl 017-, anti-PROl 112-, anti- PRO509-,anti- PR0853- or anti-PR0882-encoding nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PRO201 -, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-encoding nucleic acid molecule or an anti-PRO201 -, anti-PR0292-, anti-PR0327-, anti-PRO 1265-, anti- PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti-PROl 017-, anti-PROl 112-, anti- PRO509-, anti-PR0853- or anti-PR0882-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the PRO201-, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding nucleic acid molecule or the anti-PRO201 -, anti-PR0292-, anti-PR0327-, anti-PROl 265-, anti-PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti-PROl 017-, anti- PROl 112-, anti-PRO509-, anti-PR0853- or anti-PR0882-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide or an anti- PRO201, anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody includes PRO201-, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-nucleic acid molecules and anti-PRO201-, anti-PR0292-, anti- PR0327-, anti-PROl 265-, anti-PR0344-, anti-PR0343-, anti-PR0347-, anti-PR0357-, anti-PR0715-, anti- PRO1017-, anti-PROl 112-, anti-PRO509-, anti-PR0853- or anti-PR0882-encoding nucleic acid molecules contained in cells that ordinarily express PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptides or express anti-PRO201 , anti- PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, 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. For example, 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. Generally, "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.
The term "antibody" is used in the broadest sense and specifically covers, for example, single anti- PRO201, anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 monoclonal antibodies (including antagonist, and neutralizing antibodies),anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti- PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody compositions with polyepitopic specificity, single chain anti-PRO201 , anti- PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies, and fragments of anti- PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies (see below). The term "monoclonal antibody" as used herein 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 μg/ml), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" may 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 than 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 trisodium 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 35°C-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
"Active" or "activity" for the purposes herein refers to form(s) of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides which retain a biological and/or an immunological activity/property of a native or naturally-occurring PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, wherein "biological" activity refers to a function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a a native or naturally-occurring PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide.
"Biological activity" in the context of an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins or otherwise interfere with the transcription or translation of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. A preferred biological activity is growth inhibition of a target tumor cell. Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell.
The term "biological activity" in the context of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide means the ability of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017,
PRO 1 112, PRO509, PR0853 or PR0882 polypeptide to induce neoplastic cell growth or uncontrolled cell growth.
The phrase "immunological activity" means immunological cross-reactivity with at least one epitope of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having this activity with polyclonal antisera raised against the known active PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide. Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds. The immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 8-times, most preferably at least about 10-times higher) than the binding affinity of that molecule to any other known native polypeptide.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide disclosed herein or the transcription or translation thereof. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments, peptides, small organic molecules, anti-sense nucleic acids, etc. Included are methods for identifying antagonists of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
A "small molecule" is defined herein to have a molecular weight below about 500 Daltons. "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and specifically covers, without limitation intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91 -3242, Vol. I, pages 647-669 ( 1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD. [ 1991 ]) and/or those residues from a "hypervariable loop" (i.e., residues 26-32 (LI ), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain ; Clothia and Lesk, J. Mol. Biol.. 196:901-917 [1987]). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata etal. Protein Eng. , 8(10): 1057-1062 [ 1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its 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 in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL di er. 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 affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) 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 cysteine residue(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 (K) and lambda (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, 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. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, e, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term "monoclonal antibody" as used herein 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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [1975], or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature. 352:624-628 [1991 ] and Marks et al.. J. Mol. Biol., 222:581 -597 (1991 ), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851 -6855 [1984]).
"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. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a 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. In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, 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 sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature. 321 :522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988]; and Presta, OUT. Op. Struct. Biol., 2:593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
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. In preferred embodiments, 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.
The word "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. Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-21 1 , Cu-67, Bi-212, and Pd-109. The label may also be a non-detectable entity such as a toxin.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of 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. In certain embodiments, depending on the context, 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. As used herein, 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 amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.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 domain 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.
IL Compositions and Methods of the Invention
A. Full-length PRO201. PRQ292. PRQ327. PROl 265. PRQ344. PRQ343. PRQ347. PRQ357. PRQ715. PRO1017. PROl 112. PRO509. PRQ853 and PRQ882 polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882. In particular, cDNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 polypeptides has been identified and isolated, as disclosed in further detail in the Examples below. It is noted that 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 protein, and will not be changed. However, for sake of simplicity, in the present specification the proteins encoded by the herein disclosed nucleic acid sequences as well as all further native homologues and variants included in the foregoing definition of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 will be referred to as "PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882", regardless of their origin or mode of preparation.
As disclosed in the Examples below, cDNA clones have been deposited with the ATCC. The actual nucleotide sequence of the clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequences can be determined from the nucleotide sequences using routine skill. For the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptides and encoding nucleic acid described herein, Applicants have identified what are believed to be the reading frames best identifiable with the sequence information available at the time.
B. PRO201. PRQ292. PRQ327. PRQ1265. PRQ344. PRQ343. PRQ347. PRQ357, PRQ715. PRQ1017. PROl 112. PRO509. PRQ853 and PRQ882 Variants
In addition to the full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO347,PRO357,PRO715, PRO1017,PROl 1 12, PRO509, PRO853 and PR0882 polypeptides described herein, it is contemplated that PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 variants can be prepared. PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 and PR0882 variants can be prepared by introducing appropriate nucleotide changes into the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 DNA, and/or by synthesis of the desired PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO201, PR0292, PR0327,
PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343,
PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 or in various domains of the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 or PR0882 that results in a change in the amino acid sequence of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 as compared with the native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882. Optionally 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882. 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 and PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017,
PROl 1 12, PRO509, PR0853 or PR0882 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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). Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide fragments share at least one biological and/or immunological activity with the native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened. Table 3
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; lys; arg gin
Asp (D) glu glu
Cys (C) ser ser
Gin (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg
He (I) leu; val; met; ala; phe; norleucine leu
Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gin; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity of the 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:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. 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.. J0:6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34:315 ( 1985)], restriction selection mutagenesis [Wells etal, Philos. Trans. R. Soc. London SerA. 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PRO715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine 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 amino 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 isoteric amino acid can be used.
C. Modifications of PRO201 , PRQ292, PRQ327. PROl 265, PRQ344, PRQ343, PRQ347, PRQ357, PRQ715. PROIOI 7. PROl 1 12. PRO509. PRQ853 and PRQ882
Covalent modifications of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 and PR0882 are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882. Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PROl 017, anti-PRO 1 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies, and vice- versa. Commonly used crosslinking agents include, e.g., 1 , 1 -bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PRO347, PRO357, PRO715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 (for O-linked glycosylation sites). The PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981 ).
Removal of carbohydrate moieties present on the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 1 18: 131 (1981). 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. Enzvmol., 138:350 (1987). Another type of covalent modification of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343,
PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 comprises linking the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to one of a variety of nonproteinaceous 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 or 4,179,337.
ThePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PRO 1 112, PRO509, PR0853 or PR0882 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882. The presence of such epitope-tagged forms of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 orPR0882 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 etal., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology. 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp etal, BioTechnology, 6: 1204- 1210 ( 1988)] ; the KT3 epitope peptide [Martin et al, Science, 255: 192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al, J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc. Natl. Acad. Sci. USA. 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also, US Patent No. 5,428,130 issued June 27, 1995.
D. Preparation of PRO201. PRQ292. PRQ327. PROl 265. PRQ344. PRQ343. PRQ347. PRQ357. PRQ715. PROIOI 7. PROl 1 12. PRO509. PRQ853 and PRQ882 Polypeptides
The description below relates primarily to production of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 by culturing cells transformed or transfected with a vector containing PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343,
PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882. For instance, the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al, 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 orPR0882 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882.
a. Isolation of DNA Encoding a PRO201 , PRQ292. PRQ327. PROl 265, PRQ344, PRQ343, PRQ347. PRQ357. PRQ715. PROl 017. PROl 1 12. PRO509, PRQ853 or PRQ882 Polypeptide
DNA encoding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 may be obtained from a cDNA library prepared from tissue believed to possess the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 mRNA and to express it at a detectable level. Accordingly, human PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the PRO201 , PR0292, PR0327, PR01265,
PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, or ohgonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook etal, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PRO201 , PR0292, PR0327, PRO 1265 , PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 is to use PCR methodology [Sambrook et al, supra; Dieffenbach et al, PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. 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 32P-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.
b. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 production and cultured in 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. In general, 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. Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaP04, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al, Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456- 457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen etal, J. Bact. 130:946 (1977) and Hsiao etal, Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see, Keown etal, Methods in Enzvmology, 185:527-537 ( 1990) and Mansour et al, Nature. 336:348-352 (1988).
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 eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli Kl 2 strain MM294 (ATCC 31 ,446); E. coli X 1776 (ATCC 31 ,537); E. coli strain W31 10 (ATCC 27,325) and E. coli strain K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W31 10 strain 1 A2, which has the complete genotype tonA ; E. coli W31 10 strain 9E4, which has the complete genotype tonA ptr3; E. coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompTkaή'; E. coli W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kanr; E. coli W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No.4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 112-, PRO509-, PR0853- or PR0882-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer etal, Bio/Technology.9: 968-975 (1991 )) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacteriol.. 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Vanden Berg et al., Bio/Technology. 8:135 (1990)), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (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, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 1 12:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [19831; Yelton etal. Proc. Natl. Acad. Sci. USA.81 :1470-1474 [19841) andA. niser (Kelly and Hvnes. EMBO J.. 4:475-479 [1985]). 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 PRO201 , PR0292, PR0327, PROl 265, PR0344,
PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of 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 Chasin, Proc. Natl. Acad. Sci. USA. 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51 ). The selection of the appropriate host cell is deemed to be within the skill in the art.
c. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in 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. In general, DNA is inserted into an appropriate restriction endonuclease site(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 ligation techniques which are known to the skilled artisan.
The PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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. In general, the signal sequence may be a component of the vector, or it may be a part of the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-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, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Patent No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
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 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-.PR0357-, PR0715-, PRO1017-, PROl 112-, PRO509-, PR0853- or PR0882-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 etal, Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trpλ gene present in the yeast plasmid YRp7 [Stinchcomb et al, Nature, 282:39 (1979); Kingsman et al. Gene, 7:141 (1979); Tschemper ef α/.. Gene, 10: 157 (1980)1. The trpλ 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 PRO201 -, PR0292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-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 β-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..8 :4057 ( 1980); EP 36,776] , and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882.
Examples of 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-ρhosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other 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, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 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 2,21 1 ,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 actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 coding sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) 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 cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 orPR0882.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in recombinant vertebrate cell culture are described in Gething etal, Nature.293:620-625 (1981); Mantei et al., Nature, 281:40-46 ( 1979); EP 1 17,060; and EP 1 17,058.
d. Detecting Gene Amplification/Expression
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. Alternatively, 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, alternatively, 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 staining 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against an exogenous sequence fused to PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA and encoding a specific antibody epitope.
e. Purification of Polypeptide
Forms of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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., Triton- X 100) or by enzymatic cleavage. Cells employed in expression of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
It may be desired to purify PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882. 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 produced.
E. Amplification of Genes Encoding the PRO201 , PRQ292, PRQ327, PROl 265, PRQ344, PRQ343, PRQ347. PRQ357. PRQ715. PRO 1017. PROl 1 12. PRO509. PRQ853 or PRQ882 Polypeptides in Tumor Tissues and Cell Lines
The present invention is based on the identification and characterization of genes that are amplified in certain cancer cells.
The genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements. One is the preservation and propagation of DNA as the genetic information in its original form, to guarantee stable inheritance through multiple generations. On the other hand, cells or organisms must be able to adapt to lasting environmental changes. The adaptive mechanisms can include qualitative or quantitative modifications of the genetic material. Qualitative modifications include DNA mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein. Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i.e., a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene.
The phenomenon of gene amplification and its underlying mechanisms have been investigated in vitro in several prokaryotic and eukaryotic culture systems. The best-characterized example of gene amplification involves the culture of eukaryotic cells in medium containing variable concentrations of the cytotoxic drug methotrexate (MTX). MTX is a folic acid analogue and interferes with DNA synthesis by blocking the enzyme dihydrofolate reductase (DHFR). During the initial exposure to low concentrations of MTX most cells (>99.9%) will die. A small number of cells survive, and are capable of growing in increasing concentrations of MTX by producing large amounts of DHFR-RNA and protein. The basis of this overproduction is the amplification of the single DHFR gene. The additional copies of the gene are found as extrachromosomal copies in the form of small, supernumerary chromosomes (double minutes) or as integrated chromosomal copies.
Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation. Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell. One of the most common genetic changes observed in human malignancies are mutations of the p53 protein. p53 controls the transition of cells from the stationary (Gl) to the replicative (S) phase and prevents this transition in the presence of DNA damage. In other words, one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA damage, i.e., genetic changes. Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and gross, structural alterations, such as translocations. The amplification of DNA sequences may indicate a specific functional requirement as illustrated in the
DHFR experimental system. Therefore, the amplification of certain oncogenes in malignancies points toward a causative role of these genes in the process of malignant transformation and maintenance of the transformed phenotype. This hypothesis has gained support in recent studies. For example, the bcl-2 protein was found to be amplified in certain types of non-Hodgkin ' s lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells. Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells less susceptible to limiting amounts of available growth factor. Examples include the amplification of the androgen receptor in recurrent prostate cancer during androgen deprivation therapy and the amplification of the growth factor receptor homologue ERB2 in breast cancer. Lastly, genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation. This is illustrated by the amplification of the bcl-1 and ras genes in various epithelial and lymphoid neoplasms.
These earlier studies illustrate the feasibility of identifying amplified DNA sequences in neoplasms, because this approach can identify genes important for malignant transformation. The case of ERB2 also demonstrates the feasibility from a therapeutic standpoint, since transforming proteins may represent novel and specific targets for tumor therapy.
Several different techniques can be used to demonstrate amplified genomic sequences. Classical cytogenetic analysis of chromosome spreads prepared from cancer cells is adequate to identify gross structural alterations, such as translocations, deletions and inversions. Amplified genomic regions can only be visualized, if they involve large regions with high copy numbers or are present as extrachromosomal material. While cytogenetics was the first technique to demonstrate the consistent association of specific chromosomal changes with particular neoplasms, it is inadequate for the identification and isolation of manageable DNA sequences. The more recently developed technique of comparative genomic hybridization (CGH) has illustrated the widespread phenomenon of genomic amplification in neoplasms. Tumor and normal DNA are hybridized simultaneously onto metaphases of normal cells and the entire genome can be screened by image analysis for DNA sequences that are present in the tumor at an increased frequency. (WO 93/18, 186; Gray etal. Radiation Res., 137:275-289 [19941). As a screening method, this type of analysis has revealed a large number of recurring amplicons (a stretch of amplified DNA) in a variety of human neoplasms. Although CGH is more sensitive than classical cytogenetic analysis in identifying amplified stretches of DNA, it does not allow a rapid identification and isolation of coding sequences within the amplicon by standard molecular genetic techniques.
The most sensitive methods to detect gene amplification are polymerase chain reaction (PCR)-based assays. These assays utilize very small amount of tumor DNA as starting material, are exquisitely sensitive, provide DNA that is amenable to further analysis, such as sequencing and are suitable for high-volume throughput analysis. The above-mentioned assays are not mutually exclusive, but are frequently used in combination to identify amplifications in neoplasms. While cytogenetic analysis and CGH represent screening methods to survey the entire genome for amplified regions, PCR-based assays are most suitable for the final identification of coding sequences, i.e., genes in amplified regions.
According to the present invention, such genes have been identified by quantitative PCR (S. Gelmini et al., Clin. Chem.. 43:752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors. Quantitative PCR was performed using a TaqMan instrument (ABI). Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs.
Human lung carcinoma cell lines include A549 (SRCC768), Calu- 1 (SRCC769), Calu-6 (SRCC770), H 157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832),and H810 (SRCC833), all available from ATCC. Primary human lung tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRCC724 (adenocarcinoma, abbreviated as "AdenoCa")(LTl), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTla), SRCC726 (adenocarcinoma)(LT2), SRCC727 (adenocarcinoma)(LT3), SRCC728 (adenocarcinoma)(LT4), SRCC729 (squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cell carcinoma)(LT7), SRCC731 (adenocarcinoma)(LT9), SRCC732 (squamous cell carcinoma)(LT10), SRCC733 (squamous cell carcinoma)(LTl l), SRCC734 (adenocarcinoma)(LT12), SRCC735 (adeno/squamous cell carcinoma)(LT13), SRCC736 (squamous cell carcinoma)(LT15), SRCC737 (squamous cell carcinoma)(LT16), SRCC738 (squamous cell carcinoma)(LTl 7), SRCC739 (squamous cell carcinoma)(LTl 8), SRCC740 (squamous cell carcinoma)(LTl 9), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21 ), SRCC81 1 (adenocarcinoma)(LT22), SRCC825 (adenocarcinoma)(LT8), SRCC886 (adenocarcinoma)(LT25), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31 ), SRCC894 (adenocarcinoma) (LT33). Also included are human lung tumors designated SRCC1125 [HF-000631 ], SRCC1 127 [HF-000641 ], SRCC1 129 [HF-000643], SRCC1 133 [HF-000840], SRCC1 135 [HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297], SRCC1235 [HF-001299], and SRCC1236 [HF-001300].
Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr. Robert Warren, UCSF), CaWiDr (adenocarcinoma, SRCC781 ), HCT1 16 (carcinoma, SRCC782), SKCOl (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), Colo205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC829), HCC2998 (carcinoma, SRCC830), and KM 12 (carcinoma, SRCC831 ). Primary colon tumors include colon adenocarcinomas designated CT2 (SRCC742), CT3 (SRCC743) ,CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1 (SRCC751 ), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT1 1 (SRCC757), CT18 (SRCC758), CT19 (adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21 (adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinoma, SRCC91 1 ), CT25 (adenocarcinoma, SRCC912), CT26 (adenocarcinoma, SRCC913), CT27 (adenocarcinoma, SRCC914),CT28 (adenocarcinoma, SRCC915), CT29 (adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31 (adenocarcinoma, SRCC918), CT32 (adenocarcinoma, SRCC919), CT33 (adenocarcinoma, SRCC920), CT35 (adenocarcinoma, SRCC921 ), and CT36 (adenocarcinoma, SRCC922). Also included are human colon tumor centers designated SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1 142 [HF-000762], SRCC1 144 [HF-000789], SRCC1 146 [HF-000795] and SRCC1 148[HF-00081 1].
Human breast carcinoma cell lines include, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468(SRCC762), MB 175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545]. Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC1101, and human breast-met-lung-NS tumor designated SRCC893 [LT 32].
Human kidney tumor centers include SRCC989 [HF-000611] and SRCC1014 [HF-000613]. Human testis tumor center includes SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF-
000716].
Human parathyroid tumor includes SRCC1002 [HF-000831 ] and SRCC1003 [HF-000832].
F. Tissue Distribution
The results of the gene amplification assays herein can be verified by further studies, such as, by determining mRNA expression in various human tissues.
As noted before, gene amplification and/or gene expression in various tissues may be measured 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. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining 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 PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 DNA and encoding a specific antibody epitope. General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided hereinbelow.
G. Chromosome Mapping
If the amplification of a given gene is functionally relevant, then that gene should be amplified more than neighboring genomic regions which are not important for tumor survival. To test this, the gene can be mapped to a particular chromosome, e.g., by radiation-hybrid analysis. The amplification level is then determined at the location identified, and at the neighboring genomic region. Selective or preferential amplification at the genomic region to which the gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival. Chromosome mapping includes both framework and epicenter mapping. For further details see, e.g., Stewart et al., Genome Research, 7:422-433 (1997).
H. Antibody Binding Studies
The results of the gene amplification study can be further verified by antibody binding studies, in which the ability of anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 11 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies to inhibit the expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides on tumor (cancer) cells is tested. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).
Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein (encoded by a gene amplified in a tumor cell) in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Patent No. 4,376,1 10. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
For immunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
I. Cell-Based Tumor Assays
Cell-based assays and animal models for tumors (e.g., cancers) can be used to verify the findings of the gene amplification assay, and further understand the relationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth. The role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein. Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above. In a different approach, cells of a cell type known to be involved in a particular tumor are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed. Suitable cells include, for example, stable tumor cells lines such as, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorogenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC). Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cancer.
In addition, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al, Mol. Cell. Biol.. 5:642-648 [1985]).
J. Animal Models
A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, including small molecule antagonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non- recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue. (See, e.g., PCT publication No. WO 97/33551, published September 18, 1997).
Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, AS W, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g., The Nude Mouse in Oncology Research. E. Boven and B. Winograd, eds., CRC Press, Inc., 1991. The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any of the above-listed tumor cell lines, and, for example, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); røs-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer. 48:689-696 [1983]).
Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation. Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions. For solid block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue. Boven and Winograd (1991), supra.
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or rcew-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al.. PNAS USA, 83:9129-9133 (1986).
Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals. An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al., Cancer Research, 54:4726-4728 ( 1994) and Too et al., Cancer Research, 55:681 -684 (1995). This model is based on the so-called "METAMOUSE" sold by AntiCancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMS5, CMS21 , and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med., 146:720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol., 138:4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOxlO6 to lOxlO7 cells/ml. The animals are then infected subcutaneously with 10 to 100 μl of the cell suspension, allowing one to three weeks for a tumor to appear. In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al, Br. J. Cancer, 4Lsuppl. 4:309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see, Zacharski, Haemostasis. 16:300-320 [1986]).
One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng eds., Basel, 1989, 301. It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis.
Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals. Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal, Cell, 56:313-321 [1989]); electroporation of embryos (Lo, Mol. Cell Biol.. 3:1803-1814 [ 1983]); sperm-mediated gene transfer (Lavitrano et al., Cell, 57:717-73 [1989]). For review, see, for example, U.S. Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al, Proc. Natl. Acad. Sci. USA. 89:6232- 636 (1992).
The expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals are further examined for signs of tumor or cancer development.
Alternatively, "knock out" animals can be constructed which have a defective or altered gene encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PR01112, PRO509, PR0853 or PR0882 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see, e.g., Thomas and Capecchi, Cell, 5 _:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see, e.g., Li et al, Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 1 13-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined 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 recombined DNA. Knockout animals can be characterized for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
The efficacy of antibodies specifically binding the polypeptides identified herein and other drug candidates, can be tested also in the treatment of spontaneous animal tumors. A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this type of tumor in animals.
K. Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that bind or complex with the 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. Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (ρoly)peptide-immunoglobulin fusions, 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. 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 are common in that they call for contacting the drug candidate with a polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the 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 by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the 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. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. If the candidate compound interacts with but does not bind to a particular PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PR01017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, cross- linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co- workers [Fields and Song, Nature. 340:245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while 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 in which the target protein is fused to the DNA- binding domain of GAL4. and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl -/αcZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction of a PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-,
PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encoding gene identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, 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 reaction(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.
To assay for antagonists, the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide indicates that the compound is an antagonist to the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. Alternatively, antagonists may be detected by combining the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide and a potential antagonist with membrane-bound PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide can be labeled, such as by radioactivity, such that the number of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding 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 ). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. The PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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.
As an alternative approach for receptor identification, labeled PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 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. In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357.PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
Another potential PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 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. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide 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 PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 orPR0882 polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO201 , PR0292, PR0327, PRO 1265 , PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide (antisense - Okano, Neurochem, 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). The ohgonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in 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 in length, about 55 bases in length, about 60 bases in 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.
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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, thereby blocking the normal biological activity of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non- peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules capableof 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 (published
September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these ohgonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551 , supra.
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
L. Compositions and Methods for the Treatment of Tumors The compositions useful in the treatment of tumors associated with the amplification of the genes identified herein include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc., that inhibit the expression and/or activity of the target gene product.
For example, antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
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 (published
September 18, 1997).
Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these ohgonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.
These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
M. Antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments which may inhibit the production or the gene product of the amplified genes identified herein and/or reduce the activity of the gene products.
1. Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in 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 in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 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, bovine thyroglobulin, 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 dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
2. Monoclonal Antibodies The anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715 , anti-PRO 1017, anti-PRO 11 12, anti-PRO509, anti-PR0853 or anti-PR0882 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). In 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. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 11 12, PRO509, PR0853 or PR0882 polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or 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. Usually, 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. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), 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 (ATCC), 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882. Preferably, 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). Such techniques and assays are known in the art. 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).
After the desired hybridoma cells are identified, 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. Alternatively, 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. Once isolated, 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, supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a 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. Alternatively, 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.
3. Human and Humanized Antibodies
Theanti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies 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. In some instances, 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. In general, 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); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, 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, Science, 239: 1534- 1536 ( 1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "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. In practice, 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 Boerner 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(1 ):86-95 (1991)]. Similarly, 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. 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 era/., Nature, 368:856-859 ( 1994); Morrison, Nature. 368 : 812- 13 ( 1994); Fish wild et al. , Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).
4. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01 145) to an active anti-cancer drug. See, for example, WO 88/07378 and U. S. Patent No. 4,975,278.
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form.
Enzymes that are useful in the method of this invention include, but are not limited to, glycosidase, glucose oxidase, human lysosyme, human glucuronidase, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti -cancer drug 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin Vamidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes" can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covalently bound to the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti- PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking agents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of the antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g. , Neuberger et al, Nature, 3 2:604-608 (1984)).
5. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker ef al., EMBO J., 10:3655-3659 (1991). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) 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. For further details of generating bispecific antibodies see, for example, Suresh etal. Methods in Enzymology, 121 :210 (1986).
According to another approach described in WO 96/2701 1 , 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. In this method, 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 using chemical linkage. Brennan et al, Science, 229:81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 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. 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.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol.. 148(5): 1547- 1553 ( 1992). 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 "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J. Immunol, 147:60 (1991 ).
Exemplary bispecific antibodies may bind to two different epitopes on a given polypeptide herein. Alternatively, an anti-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 (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular polypeptide. These antibodies possess a polypeptide-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the polypeptide and further binds tissue factor (TF).
6. Heteroconjugate Antibodies
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]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, 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 mefhyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
7. Effector function engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. For example, cysteine residue(s) may be introduced in 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, J. Exp. Med., 176: 1 191 -1 195 ( 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 etal, Cancer Research.53:2560- 2565 (1993). Alternatively, an antibody can be engineered which 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).
8. Immunoconiugates
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 small molecule toxin), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Small molecule toxins include, for example, calicheamicins, maytansinoids, palytoxin and CCl 065. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 13 V31In, »°Y and lllβRe.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon- 14-labeled 1 -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/1 1026.
In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting 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) which is conjugated to a cytotoxic agent (e.g., a radionucleotide). 9. Immunoliposomes
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. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (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 etal, J. Biol. Chem., 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See, Gabizon etal, J. National Cancer Inst., 81( 19): 1484 (1989).
N. Pharmaceutical Compositions
Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections 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 which 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 which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 [1993]).
Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. [1980]), in the form of lyophilized 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 and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and w-cresol); 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 glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner, using standard techniques well known in the art.
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. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in 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, hydroxymethylcellulose 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 Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
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. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
O. Methods of Treatment It is contemplated that the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein. Exemplary conditions or disorders to be treated with such antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptides, antisense molecules, etc., include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
The anti-tumor agents of the present invention, e.g., antibodies, are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred. Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e.g., antibodies of the instant invention. For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD ( 1992). The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent, e.g., antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
It may be desirable to also administer antibodies against other tumor associated antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co- administered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein. For the prevention or treatment of disease, the appropriate dosage of an anti-tumor agent, e.g., an antibody herein will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments. For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. P. Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is usually an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e.g., an antibody. The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline,
Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Q. Diagnosis and Prognosis of Tumors While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells find additional use in the diagnosis and prognosis of tumors. For example, antibodies directed against the protein products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics. For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect the expression of proteins encoded by the amplified genes ("marker gene products"). The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. These techniques are particularly suitable, if the amplified gene encodes a cell surface protein, e.g., a growth factor. Such binding assays are performed essentially as described in section 5 above.
In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. 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, 10801 University Bl vd. , Manassas, V A 201 10-2209. All original deposits referred to in the present application were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638). Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press N.Y., 1989; Ausubel etal, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989; Innis etal, PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., N.Y., 1990; Harlow etal, Antibodies: A Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor. 1988; Gait, Oligonucleotide Synthesis. IRL Press, Oxford, 1984; R.I. Freshney, Animal Cell Culture, 1987; Coligan et al.. Current Protocols in Immunology, 1991.
EXAMPLE 1 Extracellular Domain Homology 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. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzvmology. 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, Washington).
Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using phrap. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
Based upon the consensus sequences obtained as described above, 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 in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, 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 into 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 Using Signal Algorithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc., (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
EXAMPLE 3 Isolation of cDNA Clones Encoding Human PRO201 An expressed sequence tag (EST) DNA database ( LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified (1328938, also designated DNA28710) which was in a fetal pancreas library and which shared significant identity with the adaptor protein She.
A full length cDNA corresponding to the isolated EST was cloned from a human fetal kidney library using an in vivo cloning technique in pRK5. The cDNA libraries used to isolate the cDNA clones encoding human PRO201 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 into 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.
Probes based on the Incyte EST no. 1328938 were used to screen a cDNA library derived from the human fetal kidney library:
cloning primer:
5'-ACTGAGGCCTGTTGAAAGTGCAGAGCTCAG-3' (SEQ ID NO:3) enrichment primer:
5'-GCTGAAGAAGAGCTTCAG-3' (SEQ ID NO:4) A full length clone [DNA30676-1223]was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 152- 154 and a stop signal at nucleotide positions 1880- 1882 (Figure 1 , SEQ ID NO:l ). The predicted polypeptide precursor is 576 amino acids long, has a calculated molecular weight of approximately 63,094 daltons and an estimated pi of approximately 7.26. Analysis of the full-length PRO201 sequence shown in Figure 2 (SEQ ID NO:2) evidences the presence of a variety of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PRO201 polypeptide shown in Figure 2 evidences the presence of the following: a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 142 to about amino acid 146; N-myristoylation sites from about amino acid 41 to about amino acid 47, from about amino acid 107 to about amino acid 111, from about amino acid 164 to about amino acid 170, from about amino acid 203 to about amino acid 209, from about amino acid 243 to about amino acid 249, from about amino acid 343 to about amino acid 349, from about amino acid 460 to about amino acid 466, from about amino acid 546 to about amino acid 552, and from about amino acid 551 to about amino acid 557; an amidation site from about amino acid 97 to about amino acid 101 ; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 371 to about amino acid 382; and a leucine zipper pattern from about amino acid 184 to about amino acid 206. Clone DNA30676-1223 has been deposited with ATCC on September 23, 1997 and is assigned ATCC deposit no. 209567.
Based on a BLAST and FastA sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID NO:2), PRO201 shows amino acid sequence identity to both Sck and She proteins.
EXAMPLE 4 Isolation of cDNA Clones Encoding Human PRQ292
PR0292 is identical with the human death asscoiated protein DAP-7, also called cathepsin D. The amino acid sequence of this 412 amino acid protein is present in the public Dayhoff database under Accession Nos. CATD_HUMAN and P_R74207, and is shown in Figure 4 (SEQ ID NO:6). The nucleotide sequence of the DNA encoding PR0292 is shown in Figure 3 (SEQ ID NO:5). Analysis of the full-length PR0292 sequence shown in Figure 4 (SEQ ID NO:6) evidences the presence of a variety of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0292 polypeptide shown in Figure 4 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 20; N-glycosylation sites from about amino acid 134 to about amino acid 138, and from about amino acid 263 to about amino acid 267; a tyrosine kinase phosphorylation site from about amino acid 72 to about amino acid 81 ; N-myristoylation sites from about amino acid 145 to about amino acid 151 , from about amino acid 248 to about amino acid 254, and from about amino acid 282 to about amino acid 288; and a leucine zipper pattern from about amino acid 335 to about amino acid 357.
DAP-7 has also been disclosed in WO 95/10630 published on April 20, 1995, and in Faust et al., Proc. Natl. Acad. Sci. USA, 82:4910-4914 (1985).
EXAMPLE 5
Isolation of cDNA Clones Encoding Human PRQ327 An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and various EST sequences were identified which showed certain degrees of homology to human prolactin receptor protein. A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above. This consensus sequence is designated herein as DNA38110. Based on the DNA38110 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 PR0327. A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer:
5'-CCCGCCCGACGTGCACGTGAGCC-3' (SEQ ID NO:9) reverse PCR primer:
5'-TGAGCCAGCCCAGGAACTGCTTG-3' (SEQ ID NO: 10)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA38110 sequence which had the following nucleotide sequence: hybridization probe: 5'-CAAGTGCGCTGCAACCCCTTTGGCATCTATGGCTCCAAGAAAGCCGGGAT-3' (SEQ ID NO: 11 )
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0327 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal lung tissue (LIB26).
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA38113-1230 [Figure 5, SEQ ID NO:7]; and the derived protein sequence for PR0327.
The entire coding sequence of DNA381 13-1230 is included in Figure 5 (SEQ ID NO:7). Clone DNA38113-1230 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 131 -133, and an apparent stop codon at nucleotide positions 1397-1399. The predicted polypeptide precursor is 422 amino acids long. Analysis of the full-length PR0327 sequence shown in Figure 6 (SEQ ID NO:8) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0327 polypeptide shown in Figure 6 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 30; N-glycosylation sites from about amino acid 92 to about amino acid 96, from about amino acid 104 to about amino acid 108, from about amino acid 140 to about amino acid 144, from about amino acid 168 to about amino acid 172, from about amino acid 292 to about amino acid 296, and from about amino acid 382 to about amino acid 386; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 413 to about amino acid 417; casein kinase II phosphorylation sites from about amino acid 44 to about amino acid 48, from about amino acid 183 to about amino acid 187, and from about amino acid 205 to about amino acid 209; N-myristoylation sites from about amino acid 30 to about amino acid 36, from about amino acid 37 to about amino acid 43, from about amino acid 73 to about amino acid 79, from about amino acid 121 to about amino acid 127, from about amino acid 179 to about amino acid 185, from about amino acid 218 to about amino acid 224, from about amino acid 300 to about amino acid 306, from about amino acid 317 to about amino acid 323, from about amino acid 320 to about amino acid 326, from about amino acid 347 to about amino acid 353, from about amino acid 355 to about amino acid 361, and from about amino acid 407 to about amino acid 413; amidation sites from about amino acid 3 to about amino acid 7, from about amino acid 79 to about amino acid 83, and from about amino acid 41 1 to about amino acid 415; and a growth factor and cytokine receptor family signature 2 from about amino acid 325 to about amino acid 332. Clone DNA381 13-1230 has been deposited with the ATCC on September 10, 1997 and is assigned ATCC deposit no. 209530. The full-length PR0327 protein shown in Figure 6 has an estimated molecular weight of about 46,302 daltons and a pi of about 9.42.
An analysis of the full-length PR0327 sequence shown in Figure 6 (SEQ ID NO:8), suggests that it possesses significant homology to the human prolactin receptor binding protein, thereby indicating that PR0327 may be a novel prolactin binding protein.
EXAMPLE 6 Isolation of cDNAs Encoding Human PROl 265 DNA60764-1533 was identified by applying the proprietary signal sequence finding algorithm described in Example 3 above. Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the LIFESEQ® database, designated Incyte EST cluster no. 86995. 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. 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 as DNA55717. In light of the sequence homology between the DNA55717 sequence and Incyte EST no. 20965, Incyte EST no. 20965 was purchased and the cDNA insert was obtained and sequenced. The sequence of this cDNA insert is shown in Figure 7 (SEQ ID NO:12) and is herein designated as DNA60764-1533.
The entire coding sequence of DNA60764-1533 is included in Figure 7 (SEQ ID NO: 12). Clone DNA60764-1533 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 79-81 and ending at the stop codon at nucleotide positions 1780-1782 (Figure 7). The predicted polypeptide precursor is 567 amino acids long (Figure 8; SEQ ID NO: 13). The full-length PROl 265 protein shown in Figure 8 has an estimated molecular weight of about 62,881 daltons and a pi of about 8.97. Analysis of the full- length PROl 265 sequence shown in Figure 8 (SEQ ID NO: 13) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PRO 1265 sequence shown in Figure 8 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 21 ; a transmembrane domain from about amino acid 59 to about amino acid 75; N-glycosylation sites from about amino acid 54 to about amino acid 58, from about amino acid 134 to about amino acid 138, from about amino acid 220 to about amino acid 224, and from about amino acid 559 to about amino acid 563; tyrosine kinase phosphorylation sites from about amino acid 35 to about amino acid 43, and from about amino acid 161 to about amino acid 169; and a D-amino acid oxidase proteins site from about amino acid 61 to about amino acid 81. Clone DNA60764-1533 has been deposited with ATCC on November 10, 1998 and is assigned ATCC deposit no. 203452.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-BLAST2 sequence alignment analysis of the full-length sequence shown in Figure 8 (SEQ ID NO: 13), evidenced significant sequence identity between the PRO 1265 amino acid sequence and Dayhoff sequence no. MMU70429_1. Sequence homology was also found to exist between the full-length sequence shown in Figure 8 (SEQ ID NO: 13) and the following Dayhoff sequences: BC542A , E69899, S76290, MTV014 4, AOFB_HUMAN, ZMJ002204_1, S45812_l , DBRNAPD_1 , and CRTl_SOYBN.
EXAMPLE 7
Isolation of cDNA Clones Encoding Human PRQ344
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above. This assembled consensus sequence is herein identified as DNA34398. Based on the
DNA34398 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
PR0344.
PCR primers (forward and reverse) were synthesized: forward PCR primer (34398.fl ):
5'-TACAGGCCCAGTCAGGACCAGGGG-3" (SEQ ID NO: 16) forward PCR primer (34398.f2):
5'-AGCCAGCCTCGCTCTCGG-3' (SEQ ID NO: 17) forward PCR primer (34398.f3): 5'-GTCTGCGATCAGGTCTGG-3' (SEQ ID NO: 18) reverse PCR primer (34398.r1 ): 5'-GAAAGAGGCAATGGATTCGC-3' (SEQ ID NO: 19) reverse PCR primer (34398.r2): S'-GACTTACACTTGCCAGCACAGCAC-S' (SEQ ID NO:20)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the DNA34398 consensus sequence which had the following nucleotide sequence: hybridization probe (34398.pl): 5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3' (SEQ ID NO:21 )
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0344 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA40592-1242 [Figure 9, SEQ ID NO: 14]; and the derived protein sequence for PR0344.
The entire coding sequence of DNA40592-1242 is included in Figure 9 (SEQ ID NO:14). Clone DNA40592-1242 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229, and an apparent stop codon at nucleotide positions 956-958. The predicted polypeptide precursor is 243 amino acids long. Analysis of the full-length PR0344 sequence shown in Figure 10 (SEQ ID NO: 15) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0344 polypeptide shown in Figure 10 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 15; N-myristoylation sites from about amino acid 1 1 to about amino acid 17, from about amino acid 68 to about amino acid 74, and from about amino acid 216 to about amino acid 222; and a cell attachment sequence from about amino acid 77 to about amino acid 80. Clone DNA40592- 1242 has been deposited with the ATCC on November 21 , 1997 and is assigned ATCC deposit no.209492. The full-length PR0344 protein shown in Figure 10 has an estimated molecular weight of about 25,298 daltons and a pi of about 6.44.
An analysis of the full-length PR0344 sequence shown in Figure 10 (SEQ ID NO: 15), suggests that portions of it possess significant homology to the human and murine complement proteins, thereby indicating that PR0344 may be a novel complement protein.
EXAMPLE 8 Isolation of cDNA Clones Encoding Human PRQ343 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein identified as DNA30895. Based on the DNA30895 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 PR0343.
A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-CGTCTCGAGCGCTCCATACAGTTCCCTTGCCCCA-3' (SEQ ID NO:24) reverse PCR primer:
5'-TGGAGGGGGAGCGGGATGCTTGTCTGGGCGACTCCGGGGGCCCCCTCATGTGCCAGGTGGA-3' (SEQ ID NO:25)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the DNA30895 consensus sequence:
5'-CCCTCAGACCCTGCAGAAGCTGAAGGTTCCTATCATCGACTCGGAAGTCTGCAGCCATCTGTA CTGGCGGGGAGCAGGACAGGGACCCATCACTGAGGACATGCTGTGTGCCGGCTACT-3'(SEQIDNO:26)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0343 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal lung tissue (LIB26).
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA43318-1217 [Figure 11 , SEQ ID NO:22]; and the derived protein sequence for PR0343.
The entire coding sequence of DNA43318-1217 is included in Figure 11 (SEQ ID NO:22). Clone DNA43318-1217 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 53-55, and an apparent stop codon at nucleotide positions 1004-1006. The predicted polypeptide precursor is 317 amino acids long. Analysis of the full-length PR0343 sequence shown in Figure 12 (SEQ ID NO:23) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0343 polypeptide shown in Figure 12 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 32; an N-glycosylation site from about amino acid 70 to about amino acid 74; a glycosaminoglycan attachment site from about amino acid 178 to about amino acid 182; N-myristoylation sites from about amino acid 5 to about amino acid 11 , from about amino acid 12 to about amino acid 18, from about amino acid 13 to about amino acid 19, from about amino acid 16 to about amino acid 22, from about amino acid 52 to about amino acid 58, from about amino acid 71 to about amino acid 77, from about amino acid 77 to about amino acid 83, from about amino acid 1 12 to about amino acid 1 18, from about amino acid 273 to about amino acid 279, and from about amino acid 310 to about amino acid 316; a prokaryotic membrane hpoprotein lipid attachment site from about amino acid 4 to about amino acid 15; and a serine proteases, trypsin family, histidine active site from about amino acid 86 to about amino acid 92. Clone DNA43318-1217 has been deposited with the ATCC on November 21 , 1997 and is assigned ATCC deposit no. 209481. The full-length PR0343 protein shown in Figure 12 has an estimated molecular weight of about 33,732 daltons and a pi of about 7.90. EXAMPLE 9 Isolation of cDNA Clones Encoding Human PRQ347 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein designated "<consen01 >" and as DNA39499. Based on the "<consen01>" and DNA39499 consensus sequences, 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 PR0347.
A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-AGGAACTTCTGGATCGGGCTCACC-3' (SEQ ID NO:29) reverse PCR primer:
5'-GGGTCTGGGCCAGGTGGAAGAGAG-3' (SEQ ID NO:30)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the DNA39499 consensus sequence: 5'-GCCAAGGACTCCTTCCGCTGGGCCACAGGGGAGCACCAGGCCTTC-3' (SEQID NO:31 )
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0347 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228). DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA44176-1244 [Figure 13, SEQ ID NO:27]; and the derived protein sequence for PR0347.
The entire coding sequence of DNA44176-1244 is included in Figure 13 (SEQ ID NO:27). Clone DNA44176-1244 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 123-125, and an apparent stop codon at nucleotide positions 1488-1490. The predicted polypeptide precursor is 455 amino acids long. Analysis of the full-length PR0347 sequence shown in Figure 14 (SEQ ID NO:28) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0347 polypeptide shown in Figure 14 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 26; N-glycosylation sites from about amino acid 144 to about amino acid 148, and from about amino acid 243 to about amino acid 247; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 45 to about amino acid 49; N-myristoylation sites from about amino acid 22 to about amino acid 28, from about amino acid 99 to about amino acid 105, from about amino acid 131 to about amino acid 137, from about amino acid 201 to about amino acid 207, from about amino acid 213 to about amino acid 219, from about amino acid 287 to about amino acid 293, from about amino acid 288 to about amino acid 294, from about amino acid 331 to about amino acid 337, and from about amino acid 398 to about amino acid 404; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 204 to about amino acid 215; EGF-like domain cysteine pattern signatures from about amino acid 249 to about amino acid 261 , and from about amino acid 280 to about amino acid 292; and a C-type lectin domain signature from about amino acid 417 to about amino acid 442. Clone DNA44176- 1244 has been deposited with the ATCC on December 10, 1997 and is assigned ATCC deposit no. 209532. The full-length PR0347 protein shown in Figure 14 has an estimated molecular weight of about 50,478 daltons and a pi of about 8.44. Analysis of the amino acid sequence of the full-length PR0347 polypeptide suggests that portions of it possess significant homology to various cysteine-rich secretory proteins, thereby indicating that PR0347 may be a novel cysteine-rich secretory protein.
EXAMPLE 10 Isolation of cDNA Clones Encoding Human PRQ357 The sequence expression tag "2452972" by Incyte Pharmaceuticals, Palo Alto, CA, was used to begin a database search for ESTs which overlapped with a portion of "2452972". A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein designated as DNA37162. Based on the DNA37162 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 PR0357. PCR primers (forward and reverse) were synthesized: forward PCR primer 1 :
5'-CCCTCCACTGCCCCACCGACTG-3' (SEQ ID NO:34) reverse PCR primer 1 : 5'-CGGTTCTGGGGACGTTAGGGCTCG-3' (SEQ ID NO:35) forward PCR primer 2:
5'-CTGCCCACCGTCCACCTGCCTCAAT-3' (SEQ ID NO:36)
Additionally, two synthetic oligonucleotide hybridization probes were constructed from the DNA37162 consensus sequence: hybridization probe 1 :
5'-AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCACC-3' (SEQID NO:37) hybridization probe 2:
5 -ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTGGG-3' (SEQ ID NO:38)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0357 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal liver tissue.
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA44804-1248 [Figure 15, SEQ ID NO:32]; and the derived protein sequence for PR0357. The entire coding sequence of DNA44804-1248 is included in Figure 15 (SEQ ID NO:32). Clone
DNA44804-1248 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139, and an apparent stop codon at nucleotide positions 1931-1933. The predicted polypeptide precursor is 598 amino acids long. Analysis of the full-length PR0357 sequence shown in Figure 16 (SEQ ID NO:33) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0357 polypeptide shown in Figure 16 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 23; a transmembrane domain from about amino acid 501 to about amino acid 520; N- glycosylation sites from about amino acid 198 to about amino acid 202, from about amino acid 425 to about amino acid 429, and from about amino acid 453 to about amino acid 457; a tyrosine kinase phosphorylation site from about amino acid 262 to about amino acid 270; N-myristoylation sites from about amino acid 23 to about amino acid 29, from about amino acid 27 to about amino acid 33, from about amino acid 1 12 to about amino acid 1 18, from about amino acid 273 to about amino acid 279, from about amino acid 519 to about amino acid 525, and from about amino acid 565 to about amino acid 571 ; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 14 to about amino acid 25; an EGF-like domain cysteine pattern signature from about amino acid 355 to about amino acid 367; and leucine zipper patterns from about amino acid 122 to about amino acid 144, and from about amino acid 194 to about amino acid 216. Clone DNA44804- 1248 has been deposited with the ATCC on December 10, 1997 and is assigned ATCC deposit no. 209527. The full-length PR0357 protein shown in Figure 16 has an estimated molecular weight of about 63,030 daltons and a pi of about 7.24.
Anaylsis of the amino acid sequence of the full-length PR0357 polypeptide suggests that portions of it possess significant homology to ALS, thereby indicating that PR0357 may be a novel leucine rich repeat protein related to ALS.
EXAMPLE 1 1
Isolation of cDNA Clones Encoding Human PRQ715 An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and various EST sequences were identified which showed homology to human TNF-α. This search resulted in the identification of Incyte EST No. 2099855. A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above. This consensus sequence is designated herein as DNA52092. Based upon the alignment of the various EST clones described above, a single clone (725887, Accession no. AA292358) was identified and was sequenced.
DNA sequencing of the isolated clone isolated as described above gave the full-length DNA sequence for DNA52722-1229 [Figure 17, SEQ ID NO:39]; and the derived protein sequence for PR0715.
The entire coding sequence of DNA52722-1229 is included in Figure 17 (SEQ ID NO:39). Clone DNA52722-1229 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1 14-1 16, and an apparent stop codon at nucleotide positions 864-866. The predicted polypeptide precursor is 250 amino acids long. Analysis of the full-length PR0715 sequence shown in Figure 18 (SEQ ID NO:40) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0715 polypeptide shown in Figure 18 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 40; an N-glycosylation site from about amino acid 124 to about amino acid 128; a tyrosine kinase phosphorylation site from about amino acid 156 to about amino acid 164; N-myristoylation sites from about amino acid 36 to about amino acid 42, from about amino acid 40 to about amino acid 46, from about amino acid 179 to about amino acid 185, and from about amino acid 242 to about amino acid 248; and a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 34 to about amino acid 45. Clone DNA52722-1229 has been deposited with the ATCC on January 7, 1998 and is assigned ATCC deposit no. 209883. The full-length PR0715 protein shown in Figure 18 has an estimated molecular weight of about 27,433 daltons and a pi of about 9.85.
An analysis of the full-length PR0715 sequence shown in Figure 18 (SEQ ID NO:40), suggests that it possesses significant homology to members of the tumor necrosis factor family of proteins, thereby indicating that PR0715 is a novel tumor necrosis factor protein.
EXAMPLE 12 Isolation of cDNA Clones Encoding Human PROl 017 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This assembled consensus sequence is herein designated "<consen01>", sometimes called DNA53235. Based on the assemblies presented herein and the consensus sequences, EST AA243086 (Merck clone 664402) was further examined and sequenced.
DNA sequencing of the isolated clone isolated as described above gave the full-length DNA sequence for DNA56112-1379 [Figure 19, SEQ ID NO:41]; and the derived protein sequence for PRO1017. The entire coding sequence of DNA56112-1379 is included in Figure 19 (SEQ ID NO:41 ). Clone
DNA561 12-1379 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 128-130, and an apparent stop codon at nucleotide positions 1370-1372. The predicted polypeptide precursor is 414 amino acids long. Analysis of the full-length PRO 1017 sequence shown in Figure 20 (SEQ ID NO:42) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PROl 017 polypeptide shown in Figure 20 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 31 ; N-glycosylation sites from about amino acid 134 to about amino acid 138, from about amino acid 209 to about amino acid 213, from about amino acid 280 to about amino acid 284, and from about amino acid 370 to about amino acid 374; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 85 to about amino acid 89, and from about amino acid 236 to about amino acid 240; and N- myristoylation sites from about amino acid 77 to about amino acid 83, from about amino acid 164 to about amino acid 170, and from about amino acid 295 to about amino acid 301. Clone DNA561 12- 1379 has been deposited with the ATCC on May 20, 1998 and is assigned ATCC deposit no. 209883. The full-length PRO 1017 protein shown in Figure 20 has an estimated molecular weight of about 48,414 daltons and a pi of about 9.54. Analysis of the amino acid sequence of the full-length PRO 1017 polypeptide suggests that portions of it possess sequence identity with HNK-1 sulfotransferase, thereby indicating that PROl 017 may be a novel sulfotransferase. EXAMPLE 13
Isolation of cDNA Clones Encoding Human PROl 1 12
DNA57702-1476 was identified by applying the proprietary signal sequence finding algorithm described in Example 3 above. Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from public (e.g., GenBank) and/or private LIFESEQ® Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases. The clustering and assembling of the public and private ESTs into one or several consensus sequences to create a candidate sequence was performed using repeated cycles of the computer program phrap. (Phil Green,
University of Washington, Seattle Washington). Candidate sequences with a sufficient score were further examined
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. The consensus sequence obtained therefrom is herein designated as DNA56108.
Based on the discoveries and information provided herein, Merck EST AA223646, clone 650953, from library 318, a human neuroepithelium tissue library, was further examined. DNA sequencing of the clone gave DNA57702-1476 (Figure 21 , SEQ ID NO:43), which includes the full-length DNA sequence for PROl 112.
The entire coding sequence of DNA57702-1476 is included in Figure 21 (SEQ ID NO:43). Clone DNA57702-1476 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 20-22 and ending at the stop codon at nucleotide positions 806-808 (Figure 21 ). The predicted polypeptide precursor is 262 amino acids long (Figure 22; SEQ ID NO:44). The full-length PROl 1 12 protein shown in Figure 22 has an estimated molecular weight of about 29,379 daltons and a pi of about 8.93. Analysis of the full-length PROl 1 12 sequence shown in Figure 22 (SEQ ID NO:44) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PROl 112 sequence shown in Figure 22 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 13; transmembrane domains from about amino acid 58 to about amino acid 76, from about amino acid 99 to about amino acid 1 13, from about amino acid 141 to about amino acid 159, and from about amino acid 203 to about amino acid 222; and N-myristoylation sites from about amino acid 37 to about amino acid 43, from about amino acid 42 to about amino acid 48, and from about amino acid 229 to about amino acid 235. Clone DNA57702- 1476 has been deposited with ATCC on June 9, 1998 and is assigned ATCC deposit no. 209951. An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-BLAST2 sequence alignment analysis of the full-length sequence shown in Figure 22 (SEQ ID NO:44), evidenced significant sequence identity between the PROl 112 amino acid sequence and the following Dayhoff sequences: MTY20B11_13 (a mycobacterium tuberculosis peptide), F64471 , AE000690_6, XLU16364_1, E43259 (H+-transporting ATP synthase) and PIGSLADRXE_1 (MHC class II histocompatibility antigen).
EXAMPLE 14
Isolation of cDNA Clones Encoding Human PRO509 To isolate a cDNA for PRO509 (also called HVEM), a bacteriophage library of human retinal cDNA (commercially available from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on an EST sequence (GenBank locus AA021617), which showed some degree of homology to members of the TNFR family. Five positive clones (containing cDNA inserts of 1.8-1.9 kb) were identified in the cDNA library, and the positive clones were confirmed to be specific by PCR using the above hybridization probe as a PCR primer. Single phage plaques containing each of the five positive clones were isolated by limiting dilution and the DNA was purified using a Wizard Lambda Prep DNA purification kit (commercially available from Promega).
The cDNA inserts from three of the five bacteriophage clones were excised from the vector arms by digestion with EcoRI, gel-purified, and subcloned into pRK5 and sequenced on both strands. The three clones contained an identical open reading frame (with the exception of an intron found in one of the clones). The entire sequence of DNA50148 (HVEM) is shown in Figure 23 (SEQ ID NO:45). The cDNA contained one open reading frame with a translational initiation site assigned to the ATG codon at nucleotide positions 82-84. The open reading frame ends at the termination codon TGA at nucleotide positions 931 -933.
The predicted amino acid sequence of the full-length PRO509 (HVEM) contains 283 amino acids as shown in Figure 24 (SEQ ID NO:46). The full-length PRO509 protein shown in Figure 24 has an estimated molecular weight of about 30,420 daltons and a pl of about 7.34. Analysis of the full-length PRO509 sequence shown in Figure 24 (SEQ ID NO:46) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full- length PRO509 sequence shown in Figure 24 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 36; a transmembrane domain from about amino acid 203 to about amino acid 222; N-glycosylation sites from about amino acid 1 10 to about amino acid 1 14, and from about amino acid 173 to about amino acid 177; and N-myristoylation sites from about amino acid 81 to about amino acid 87, from about amino acid 89 to about amino acid 95, from about amino acid 104 to about amino acid 1 10, from about amino acid 120 to about amino acid 126, from about amino acid 153 to about amino acid 159, from about amino acid 193 to about amino acid 199, from about amino acid 195 to about amino acid 201 , and from about amino acid 220 to about amino acid 226.
The sequence differs from the PRO509 (HVEM) sequence reported in Montgomery etal, supra, in at least two amino acids: codon 108 encodes a serine and codon 140 encodes an alanine. An alignment (using the ALIGN computer program) of a 58 amino acid long cytoplasmic region of PRO509 (HVEM) with other known members of the human TNF receptor family showed some similarity, in particular to CD40 (12 identities) and LT-beta receptor (11 identities).
EXAMPLE 15
Isolation of cDNA Clones Encoding Human PRQ853
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above. The analysis identified the single Incyte clone no. 2645134. The Incyte 2645134 sequence was then extended using repeated cycles of BLAST and phrap to extend the sequence as far as possible using the sources of EST sequences discussed above. This extended assembled consensus sequence is herein designated
"<consen01>" or DNA43050. Based on the DNA43050 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 PR0853.
A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer (43050.fl): 5'-CTTCATGGCCTTGGACTTGGCCAG-3' (SEQ ID NO:49) reverse PCR primer (43Q50.rl ):
5'-ACGCCAGTGGCCTCAAGCTGGTTG-3' (SEQ ID NO:50)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the DNA43050 consensus sequence: 5'-CTTTCTGAGCTCTGAGCCACGGTTGGACATCCTCATCCACAATGC-3' (SEQID NO:51)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0853 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228). DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA48227-1350 [Figure 25, SEQ ID NO:47]; and the derived protein sequence for PR0853.
The entire coding sequence of DNA48227-1350 is included in Figure 25 (SEQ ID NO:47). Clone DNA48227-1350 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 128-130, and an apparent stop codon at nucleotide positions 1259-1261. The predicted polypeptide precursor is 377 amino acids long. Analysis of the full-length PR0853 sequence shown in Figure 26 (SEQ ID NO:48) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0853 polypeptide shown in Figure 26 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 16; a glycosaminoglycan attachment site from about amino acid 46 to about amino acid 50; N-myristoylation sites from about amino acid 9 to about amino acid 15, from about amino acid 29 to about amino acid 35, from about amino acid 32 to about amino acid 38, from about amino acid 43 to about amino acid 49, from about amino acid 124 to about amino acid 130, and from about amino acid 312 to about amino acid 318; a prokaryotic membrane Hpoprotein lipid attachment site from about amino acid 118 to about amino acid 129; and short-chain alcohol dehydrogenase family sites from about amino acid 37 to about amino acid 50, and from about amino acid 1 14 to about amino acid 125. Clone DNA48227-1350 has been deposited with the ATCC on April 28, 1998 and is assigned ATCC deposit no. 209812. The full-length PR0853 protein shown in Figure 26 has an estimated molecular weight of about 40,849 daltons and a pl of about 7.98.
EXAMPLE 16 Isolation of cDNA Clones Encoding Human PRQ882
PR0882 (DNA58125) is identical with cardiotrophin-1. The amino acid sequence of this 201 amino acid protein is present in the public Dayhoff database under Accession Nos. P_R83967, P_W29238 and CTF1_HUMAN, among others. The nucleotide sequence of DNA58125 encoding the PR0882 is shown in Figure 27 (SEQ ID NO:52).
Analysis of the full-length PR0882 sequence shown in Figure 28 (SEQ ID NO:53) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0882 polypeptide shown in Figure 28 evidences the presence of the following: N-myristoylation sites from about amino acid 166 to about amino acid 172, and from about amino acid 174 to about amino acidl 80; and a leucine zipper pattern from about amino acid 37 to about amino acid 59. The full-length PR0882 protein shown in Figure 28 has an estimated molecular weight of about 21 ,227 daltons and a pl of about 9.30. Cardiotrophin-1 has also been disclosed in WO9730146, published on 21 August 1997 and W09529237, published on 2 November 1995.
EXAMPLE 17 Gene Amplification This example shows that the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encoding 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. Therapeutic agents may take the form of antagonists of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptides, for example, murine-human chimeric, humanized or human antibodies against a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
The starting material for the screen was genomic DNA isolated from a variety of cancers. The DNA is quantitated precisely, e.g., fluorometrically. As a negative control, DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown). The 5' nuclease assay (for example, TaqMan™) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer, Applied Biosystems Division, Foster City, CA)), were used to find genes potentially amplified in certain cancers. The results were used to determine whether the DNA encoding PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 is over-represented in 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 4. An explanation of the abbreviations used for the designation of the primary tumors listed in Table 4 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore.
The results of the TaqMan™ are reported in delta (Δ) 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 TaqMan™ fluorescent probe derived from the PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PR01017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encoding gene. Regions of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO347,PRO357, PRO715,PRO1017, PROl 1 12, PRO509, PRO853 or PR0882 gene amplification analysis were as follows:
PRO201 (DNA30676-1223): 30676.tm.f
5'-CGCAGACACCCTTCTTCACA-3' (SEQ ID NO: 54)
30676.tm.r
5'-CGACTCCTTTGGTCTCTTCTGG-3' (SEQ ID NO: 55)
30676.tm.p 5'-CCGGGACCCCCAGGTπτrGC-3' (SEQ ID NO: 56)
PRQ292 (DNA35617): 35617.tm.f
5'-GATCCTGGG CGACGTCTTC-3' (SEQ ID NO: 57)
35617.tm.p 5'-TCGGCCGCTACTACACTGTGTTTGACC-3' (SEQ ID NO: 58) 35617.tm.r 5'-GCCCACCCTGTTGTTGTCA-3' (SEQ ID NO: 59)
PRQ327 (DNA381 13-1230): 381 13.tm.f 5'-CTCAAGAAGCACG CGTACTGC-3' (SEQ ID NO: 60)
38113.tm.p
5'-CCAACCTCAGCTTCCGCCTCTACGA-3' (SEQ ID NO: 61 )
38113.tm.r 5'-CATCCAGGCTCGCCACTG-3' (SEQ ID NO: 62)
PRQ1265 (DNA60764-1533): 60764.tm.fl
5'-TGACCTGGCAAAGGAAGAA-3' (SEQ ID NO: 63)
60764.tm.pl 5 -CAGCCACCCTCCAGTCCAAGG-3' (SEQ ID NO: 64) 60764.tm.rl
5'-GGGTCGTGTTTTGGAGAGA-3' (SEQ ID NO: 65)
PRQ344 (DNA40592-1242): 40592.tm.fl 5'-TGGCAAGGAATGGGAACAGT-3' (SEQ ID NO: 66)
40592.tm.pl
5'-ATGCTGC CAGACCTGAT CGCAGACA-3' (SEQ ID NO: 67) 40592.tm.rl 5'-G GGCAGAAATC CAGCCACT-3' (SEQ ID NO: 68)
PRQ343 (DNA43318-1217):
43318.tm.fl
5'-TCTACATCAGCCTCTCTGCGC-3' (SEQ ID NO: 69)
43318.tm.pl
5'-CGATCTTCTCCACCCAGGAGCGG-3' (SEQ ID NO: 70) 43318.tm.rl
5'-GGAGCTGCACCCCTTGC-3' (SEQ ID NO: 71 )
PRQ347 (DNA44176-1244): 44176.tm.fl
5'-CCCTTCGCCTGCTTTTGA-3, (SEQ ID NO: 72) 44176.tm.pl
5'-GCCATCTAATTGAAGCCCATCTTCCCA-3' (SEQ ID NO: 73)
44176.tm.rl
5'-CTGGCGGTGT CCTCTCCTT-3' (SEQ ID NO: 74)
PRQ357 (DNA44804-1248): 44804.tm.fl
5'-CCTCGGTCTCCTCATCTGTGA-3' (SEQ ID NO: 75)
44804.tm.pl
5'-TGGCCCAGCTGACGAGCCCT-3' (SEQ ID NO: 76)
44804.tm.rl 5'-CTCATAGGCACTCGGTTCTGG-3' (SEQ ID NO: 77)
PRQ715 (DNA52722-1229): 52722.tm.fl 5'-TGGCTCCCAGCTTGGAAGA-3' (SEQ ID NO: 78)
52722.tm.pl
5'-CAGCTCTTGGCTGTCTCCAGTATGTACCCA-3' (SEQ ID NO: 79)
52722.tm.rl 5'-GATGCCTCTGTTCCTGCACAT-3' (SEQ ID NO: 80)
PRO1017 (DNA561 12-1379): 56112.tm.fl
5'-CCTCCTCCGAGACTGAAAGCT-3' (SEQ ID NO: 81 ) 56112.tm.pl
5'-TCGCGTTGCTTTTTCTCGCGTG-3' (SEQ ID NO: 82)
561 12.tm.rl
5'-GCGTGCGTC AGGTTCCA-3' (SEQ ID NO: 83)
PRO! 1 12 (DNA57702-1476): 57702.tm.fl
5'-GTCCCTTCACTGTTTAGAGCATGA-3' (SEQ ID NO: 84)
57702.tm.pl
5'-ACTCTCCCCCTCAACAGCCTCCTGAG-3' (SEQ ID NO: 85)
57702.tm.rl 5 -GTGG TCAGGGCAGA TCCTTT-3' (SEQ ID NO: 86)
PRO509 (DNA50148): 50148.tm.fl
5'-GGAGGAGACAATACCCTCATTCA-3' (SEQ ID NO: 87)
50148.tm.pl 5'-AGCAGCCGTCGCTCCAGGTATCTC-3' (SEQ ID NO: 88)
50148.tm.rl 5'-CCA GGTGGACAGCCTCTTTC-3' (SEQ ID NO: 89)
PR0853 (DNA48227-1350) 48227.tm.fl 5'-GGCACTTCATGGTCCTTGAAA-3' (SEQ ID NO: 90)
48227.tm.pl
5'-CGGATGTGTGTGAGGCCATGCC-3' (SEQ ID NO: 91 )
48227.tm.rl 5'-GAAAGTA ACCACGGAGG TCAAGAT-3' (SEQ ID NO: 92) PRQ882 (DNA58125) 58125.tm.fl
5'-πCCCAGCCTCTCTTTGCTTT-3' (SEQ ID NO: 93)
58125.tm.pl 5'-TGCCCCGπCTCTTAACTCTTGGACCC-3' (SEQ ID NO: 94) 58125.tm.rl 5'-TCAGACGGAGTTACCATGCAGA-3' (SEQ ID NO: 95)
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. During the amplification reaction, 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 interpretation 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. During amplification, 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.
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 ΔCt 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 4 describes the stage, T stage and N stage of various primary tumors which were used to screen the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 compounds of the invention. Table 4 Primary Lung and Colon Tumor Profiles
Primary Tumor Stage Other Stage Dukes Stage T Stage N Stage
Human lung tumor AdenoCa (SRCC724) [LT1 ] IIA Tl Nl Human lung tumor SqCCa (SRCC725) [LT1 a] IIB T3 NO
Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 NO
Human lung tumor AdenoCa (SRCC727) [LT3] IIIA Tl N2
Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 NO
Human lung tumor SqCCa (SRCC729) [LT6] IB T2 NO Human lung tumor Aden/SqCCa (SRCC730) [LT7] IA Tl NO
Human lung tumor AdenoCa (SRCC731 ) [LT9] IB T2 NO
Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 Nl
Human lung tumor SqCCa (SRCC733) [LT11 ] IIA Tl Nl
Human lung tumor AdenoCa (SRCC734) [LT12] IV T2 NO Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2 NO
Human lung tumor SqCCa (SRCC736) [LT15] IB T2 NO
Human lung tumor SqCCa (SRCC737) [LT16] IB T2 NO
Human lung tumor SqCCa (SRCC738) [LT17] IIB T2 Nl
Human lung tumor SqCCa (SRCC739) [LT18] IB T2 NO Human lung tumor SqCCa (SRCC740) [LT19] IB T2 NO
Human lung tumor LCCa (SRCC741 ) [LT21 ] IIB T3 Nl
Human lung AdenoCa (SRCC81 1 ) [LT22] 1 A Tl NO
Human colon AdenoCa (SRCC742) [CT2] Ml D pT4 NO
Human colon AdenoCa (SRCC743) [CT3] B pT3 NO Human colon AdenoCa (SRCC 744) [CT8] B T3 NO
Human colon AdenoCa (SRCC745) [CT10] A pT2 NO
Human colon AdenoCa (SRCC746) [CT12] MO, Rl B T3 NO
Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pNO
Human colon AdenoCa (SRCC748) [CT15] M1. R2 D T4 N2 Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pNO
Human colon AdenoCa (SRCC750) [CT17] Cl pT3 pNl
Human colon AdenoCa (SRCC751 ) [CT1] MO, Rl B pT3 NO
Human colon AdenoCa (SRCC752) [CT4] B pT3 M0
Human colon AdenoCa (SRCC753) [CT5] G2 Cl pT3 pNO Human colon AdenoCa (SRCC754) [CT6] pMO, RO B pT3 pNO
Human colon AdenoCa (SRCC755) [CT7] Gl A pT2 pNO
Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2
Human colon AdenoCa (SRCC757) [CT11] B T3 NO
Human colon AdenoCa (SRCC758) [CT18] MO. RO B pT3 pNO
DNA Preparation:
DNA was prepared from cultured cell lines, primary tumors, and 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 108 per tip and pelleted by centrifuging at
1000 rpm for 5 minutes at 4°C, followed by washing again with 1/2 volume of PBS and 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. Quiagen protease #19155 was diluted into 6.25 ml cold ddH20 to a final concentration of 20 mg/ml and equilibrated at 4°C. 10 ml of G2 Buffer was prepared by diluting Quiagen RNAse A stock (100 mg/ml) to a final concentration of 200 μg/ml.
Buffer Cl (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 centrifuging in a Beckman swinging bucket rotor at 2500 rpm at 4°C for 15 minutes. The supernatant was discarded and the nuclei were suspended with a vortex into 2 ml Buffer Cl (at 4°C) and 6 ml ddH,0, followed by a second 4°C centrifugation at 2500 rpm for 15 minutes. The nuclei were then resuspended into the residual buffer using 200 μl per tip. G2 buffer (10 ml) was added to the suspended nuclei while gentle vortexing was applied. Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds. Quiagen protease (200 μl, prepared as indicated above) was added and incubated at 50"C for 60 minutes. The incubation and centrifugation were repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4°C).
Solid human tumor sample preparation and lysis:
Tumor samples were weighed and placed into 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 into 6.25 ml cold ddH20 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 in 2L ddH20, followed by G2 buffer (50 ml). If tissue was still present on the generator tip, the apparatus was disassembled and cleaned. Quiagen protease (prepared as indicated above, 1.0 ml) was added, followed by vortexing and incubation at 50°C for 3 hours. The incubation and centrifugation were repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4°C).
Human blood preparation and lysis:
Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip. Quiagen protease was freshly prepared by dilution into 6.25 ml cold ddH20 to a final concentration of 20 mg/ml and stored at 4°C. G2 buffer was prepared by diluting RNAse A to a final concentration of 200 μg/ml from 100 mg/ml stock. The blood ( 10 ml) was placed into a 50 ml conical tube and 10 ml C 1 buffer and 30 ml ddH20 (both previously equilibrated to 4°C) were added, and the components mixed by inverting and held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm, 4°C for 15 minutes and the supernatant discarded. With a vortex, the nuclei were suspended into 2 ml Cl buffer (4°C) and
6 ml ddH20 (4°C). Vortexing was repeated until the pellet was white. The nuclei were then suspended into the residual buffer using a 200 μl tip. G2 buffer (10 ml) was added to the suspended nuclei while gently vortexing, followed by vigorous vortexing for 30 seconds. Quiagen protease was added (200 μl) and incubated at 50°C for
60 minutes. The incubation and centrifugation were repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4°C).
Purification of cleared lysates:
(1) Isolation of genomic DNA:
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 silanized, 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 rpm 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 rpm for 10 minutes at 4°C. The pellet location was marked and the supernatant discarded. The tubes were then placed on their side in a drying rack and dried 10 minutes at 37°C, taking care not to overdry the samples. After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) and placed at 50°C for 1-2 hours. Samples were held overnight at 4°C as dissolution continued. The DNA solution was then transferred to 1.5 ml tubes with a 26 gauge needle on a tuberculin syringe. The transfer was repeated 5x in order to shear the DNA. Samples were then placed at 50°C for 1 -2 hours.
(2) Ouantitation of genomic DNA and preparation for gene amplification assay: The DNA levels in each tube were quantified by standard A2( A280 spectrophotometry on a 1 :20 dilution
(5 μl DNA + 95 μl ddH20) using the 0.1 ml quartz cuvettes in the Beckman DU640 spectrophotometer. A26(/A28(l ratios were in the range of 1.8-1.9. Each DNA sample was then diluted further to approximately 200 ng/ml in TE (pH 8.5). If the original material was highly concentrated (about 700 ng/μl), the material was placed at 50°C for several hours until resuspended. Fluorometric 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 Hoechstdye working solution (#H33258, lOμl, 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 μl, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 +/- 10 units. Each sample was then read at least in 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 fluorometricly determined concentration was then used to dilute each sample to 10 ng/μl in ddH20. 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 Taqman™ 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 in 1.0 ml aliquots at -80"C. Aliquots 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 PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 compounds of the invention were screened in the following primary tumors and the resulting ΔCt values are reported in Table 5. Table 5 ΔCt values in lung and colon primary tumor and cell line models
en
I 10
Table 5 Continued ΔCt values in lung and colon primary tumor and cell line models
Figure imgf000119_0001
I
Figure imgf000119_0002
Table 5 Continued ΔCt values in lung and colon primary tumor and cell line models
:__ 5
00
Figure imgf000120_0001
Table 5 Continued ΔCt values in lung and colon primary tumor and cell line models
I
Figure imgf000121_0001
Table 5 Continued ΔCt values in lung and colon primary tumor and cell line models
10
15
Figure imgf000122_0002
Figure imgf000122_0001
Figure imgf000123_0001
PRO201 , PR0327 and PROl 265:
PRO201 (DNA30676-1223), PR0327 (DNA381 13-1230) and PR01265 (DNA60764-1533) were also reexamined along with selected tumors from the above initial screen with framework mapping. Figure 29 and Table 6 indicate the chromosomal mapping of the framework markers that were used in the present example. The framework markers are located approximately every 20 megabases and were used to control aneuploidy.
PRO201 (DNA30676-1223), PR0327 (DNA381 13-1230) and PR01265 (DNA60764-1533) were also reexamined with epicenter mapping. The markers indicated in Tables 7A, 7B, and 7C are located in close proximity (in the genome) to DNA30676, DNA381 13 and DNA60764, respectively, and are used to assess the relative amplification in the immediate vicinity of Chromosome 19 wherein the respective molecule is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-35441 is the marker found to be the closest to the location on Chromosome 19 where DNA30676 maps, and SHGC-33698 is closest to DNA60764.
Table 6 Framework Markers Along Chromosome 19
Figure imgf000124_0001
Table 7A Epicenter Markers Along Chromosome 19 Used For DNA30676
Figure imgf000124_0002
Table 7B Epicenter Markers Along Chromosome 19 Used For DNA381 13
Figure imgf000125_0001
Table 7C Epicenter Markers Along Chromosome 19 Used for DNA60764
Figure imgf000125_0002
The ΔCt values of the above described framework markers along Chromosome 19 relative to PRO201, PR0327 and PRO 1265 are indicated for selected tumors in Table 8 A, 8B and 8C, respectively. Table 8A Amplification of Framework Markers Relative to DNA30676 (ΔCt )
Figure imgf000126_0001
Table 8B Amplification of Framework Markers Relative to DNA381 13 (ΔCt )
Figure imgf000127_0001
Table 8C Amplification of Framework Markers Relative to DNA60764 (ΔCt)
Figure imgf000128_0001
Tables 9A, 9B and 9C indicate the ΔCt values for results of epicenter mapping relative to DNA30676, DNA381 13, and DNA60764, respectively, indicating the relative amplification in the region more immediate to the actual location of DNA30676, DNA38113, and DNA60764 along Chromosome 19.
Table 9A Amplification of Epicenter Markers Relative to DNA30676 (ΔCt)
Figure imgf000129_0001
Table 9A Continued Amplification of Epicenter Markers Relative to DNA30676 (ΔCt)
Figure imgf000130_0001
Table 9B Amplification of Epicenter Markers Relative to DNA381 13 (ΔCt)
Figure imgf000130_0002
Table 9B Continued Amplification of Epicenter Markers Relative to DNA381 13 (ΔCt)
Figure imgf000131_0001
Table 9C indicates the ΔCt values for the results of epicenter mapping relative to DNA60764, indicating relative amplification in the region more immediate to the actual location of DNA60764 along Chromosome 19. DNA34353, DNA40620 and DNA54002 are other independently identified molecules which have been observed to map to the same region of Chromosome 19 as DNA60764. Table 9C Amplification of Epicenter Markers Relative to DNA60764 (ΔCt )
Figure imgf000132_0001
PRQ292:
PR0292 (DNA35617) was also examined with framework mapping. Figure 30 and Table 10 indicate the chromosomal mapping of the framework markers that were used in this analysis. The framework markers are located approximately every 20 megabases and were used to control aneuploidy.
Table 10 Framework Markers Used on Chromosome 1 1 for DNA35617
Figure imgf000133_0001
The ΔCt values of the above described framework markers along Chromosome 1 1 relative for PR0292 are indicated for selected tumors in Table 1 1.
Table 11 Amplification of Framework Markers Relative to DNA35617
Figure imgf000134_0001
PRQ343 and PRQ882:
PRO 343 (DNA43318-1217) and PR0882 (DNA58125) were also reexamined with both framework and epicenter mapping. Figure 31 and Table 12 indicate the chromosomal mapping of the framework markers that were used in this analysis. The framework markers are located approximately every 20 megabases and were used to control aneuploidy. Tables 13A and 13B indicate the epicenter markers used for the mapping of DNA43318 and DNA58125. The markers shown in Tables 13A and 13B are located in close proximity (in the genome) to DNAs DNA43318 and DNA58125, respectively, and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein the respective molecules map. The distance between individual markers is measured in centirays (cR), which is equivalent to 20 kilobases. The markers AFMa061yb5 and SHGC-36123 are located the closest to the location on Chromosome 16 where DNA43318 and DNA58125, respectively, map.
Table 12 Framework Markers Used on Chromosome 16 for DNA43318 and DNA58125
Figure imgf000135_0001
Table 13A Epicenter Markers Along Chromosome 16 Used for DNA43318
Figure imgf000135_0002
Table 13B Epicenter Markers Along Chromosome 16 Used for DNA58125
Figure imgf000136_0001
The ΔCt values of the framework markers of Table 12 along Chromosome 16 relative for PR0343 and PR0882 are indicated for selected tumors in Table 14.
Table 14 Amplification of Framework Markers Relative to DNA43318 and DNA58125
Figure imgf000137_0001
Table 14 Continued Amplification of Framework Markers Relative to DNA43318 and DNA58125
Figure imgf000138_0001
Tables 15A and 15B indicate the ΔCt values from epicenter mapping relative to DNA43318 and DNA58125, respectively, indicating the relative amplification in the region more immediate to the actual location of the respective molecules along Chromosome 16.
Table 15A Amplification of Epicenter Markers Relative to DNA43318 (ΔCt )
Figure imgf000138_0002
Table 15 A Continued Amplification of Epicenter Markers Relative to DNA43318 (ΔCt )
Figure imgf000139_0001
Table 15B Amplification of Epicenter Markers Relative to DNA58125 (ΔCt )
Figure imgf000140_0001
Table 15B Continued Amplification of Epicenter Markers Relative to DNA58125 (ΔCt )
Figure imgf000141_0001
PRO1017:
PRO1017 (DNA56112-1379) was also reexamined with framework mapping. Figure 32 and Table 16 indicate the chromosomal mapping of the framework markers that were used in this analysis. The framework markers are located approximately every 20 megabases and were used to control aneuploidy.
PRO1017 (DNA56112-1379) was also reexamined with epicenter mapping. Table 17 indicates the epicenter markers which are located in close proximity to DNA56112 which were employed to assess the relative amplification in the immediate vicinity of Chromosome 7 wherein DNA561 12 is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a marker found to be the closest to the location on Chromosome 7 where DNA561 12 maps.
Table 16 Framework Markers Used on Chromosome 7 for DNA56112
Figure imgf000141_0002
Table 17 Epicenter Markers Along Chromosome 7 Used for DNA561 12
Figure imgf000142_0001
Table 18 indicates the ΔCt values for the results of epicenter mapping relative to DNA561 12, indicating the relative amplification in the region more immediate to the actual location of DNA561 12 along Chromosome 7.
Table 18 Amplification of Epicenter Markers Relative to DNA56112 (ΔCt )
Figure imgf000142_0002
Table 18 Continued Amplification of Epicenter Markers Relative to DNA561 12 (ΔCt )
Figure imgf000143_0001
The ΔCt values of the above described framework markers along Chromosome 7 relative to DNA561 12 indicated for selected tumors in Table 19. Table 19 Amplification of Framework Markers Relative to DNA561 12
Figure imgf000144_0001
PR0715 and PRQ853:
PR0715 (DNA52722- 1229) and PR0853 (DNA48227- 1350) were also reexamined with both framework and epicenter mapping. Figures 33A and 33B and Table 20 indicate the chromosomal localizations of the framework markers that were used for the procedure. The framework markers are located approximately every 20 bases and were used to control aneuploidy. Tables 21 A and 21B indicate the epicenter mapping markers that were used in the procedure. The epicenter markers were located in close proximity to DNA52722 and DNA48226, respectively, and are used to determine the relative DNA amplification in the immediate vicinity of DNA52722 and DNA48226. The distance between individual markers is measured in centirays, which is a radiation breakage unit approximately equal to a 1% chance of a breakage between two markers. One cR is very roughly equivalent to about 20 kilobases. In both Tables 21 A and 21B, "BAC" means bacterial artificial chromosome. The ends of a BAC clone which contained the gene of interest were sequenced. TaqMan™ primers and probes were made from this sequence, which are indicated in the respective tables. BAC clones are typically 100 to 150 Kb, so these primers and probes can be used as nearby markers to probe DNA for tumors. In Figure 33A, the marker SHGC-31370 is the marker found to be the closest to the location on chromosome 17 where DNA52722 maps. In Figure 33B, the marker SHGC-37126 is the marker found to be the closes to the location on chromosome 17 where DNA48227 maps.
Table 20 Framework Markers Used Along Chromosome 17 for DNA52722 and DNA48227
Figure imgf000145_0001
Table 21 A Epicenter Markers Used on Chromosome 17 in Vicinity of DNA52722
Figure imgf000145_0002
Table 21 B Epicenter Markers Used on Chromosome 17 in Vicinity of DNA48227
Figure imgf000146_0001
Table 22 indicates the ΔCt values of the above described framework markers along Chromosome 17 relative to DNA52722 and DNA48227 for selected tumors. While not shown, the similar ΔCt values for the framework markers in the analysis of DNA48227 were reported.
Table 22 Amplification of Framework Markers Relative to DNA52722
Figure imgf000147_0001
Table 23 indicates the ΔCt values for the indicated epicenter markers, indicating the relative amplification along Chromosome 17 in the immediate vicinity of DNA52722.
Table 23 Amplification of Epicenter Markers Relative to DNA52722
Figure imgf000148_0001
Table 23 Continued Amplification of Epicenter Markers Relative to DNA52722
Tables 24A and 24B indicate the ΔCt values for the indicated epicenter markers, indicating the relative amplification of selected lung and colon tumors, respectively, along Chromosome 17 in the immediate vicinity of DNA48227.
Table 24A Amplification of Epicenter Markers in the Vicinity of DNA48227 on Chromosome 17 in Selected Lung Tumors
Figure imgf000149_0002
Table 24A Continued Amplification of Epicenter Markers in the Vicinity of DNA48227 on Chromosome 17 in Selected Lung Tumors
Figure imgf000150_0001
Table 24B Amplification of Epicenter Markers In Vicinity of DNA48227 on Chromosome 17 in Selected Colon Tumors
Figure imgf000150_0002
Table 24B Continued Amplification of Epicenter Markers In Vicinity of DNA48227 on Chromosome 17 in Selected Colon Tumors
Figure imgf000151_0001
PRQ357:
PR0357 (DNA44804-1248) was reexamined with selected tumors from the above initial screen with framework mapping. Figure 34 and Table 25 indicate the chromosomal mapping of the framework markers that were used in the present example. The framework markers are located approximately every 20 megabases and were used to control aneuploidy. PR0357 (DNA44804-1248) was also examined with epicenter mapping. The markers indicated in Table
26 are located in close proximity (in the genome) to DNA44804 and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein DNA44804 is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between the two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-6154 is the marker found to be the closest to the location on Chromosome 16 where DNA44804 maps.
Table 25 Framework markers for DNA44804
Figure imgf000151_0002
Table 26 Epicenter Markers for DNA44804 Along Chromosome 16
Figure imgf000152_0001
The ΔCt values of the above described framework markers along Chromosome 16 relative to DNA44804 described in Table 27.
Table 27 Amplification of Framework Markers Relative to DNA44804 (ΔCt )
Figure imgf000152_0002
Table 27 Continued Amplification of Framework Markers Relative to DNA44804 (ΔCt )
Figure imgf000153_0001
Table 28 indicates the ΔCt values for the results of epicenter mapping relative to DNA44804, indicating the relative amplification in the region more immediate to the actual location of DNA44804 along Chromosome 16.
Table 28 Amplification of Epicenter Markers Relative to DNA44804
Figure imgf000153_0002
Table 28 Continued Amplification of Epicenter Markers Relative to DNA44804
Figure imgf000154_0001
DISCUSSION AND CONCLUSION: PRO201 (DNA30676-1223):
The ΔCt values for DNA30676-1223 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA30676-1223 encoding PRO201 occurred: (1 ) in primary lung tumors: LTla, LT3, LT6, LT7, LT9, LT10, LT1 1 , LT13, LT15, LT16, LT17, LT18, LT19, and LT21 ; (2) in primary colon tumors: CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CT17, CTl, CT4, CT5, CT6, CT7, CT9, CTl 1, and CT18; (3) lung tumor cell lines: Calu-1 , Calu-6, H157, H441, SKMES-1, H522, and H810; and (4) colon tumor cell lines Colo320 and Colo205.
Amplification has been confirmed by framework mapping for DNA30676-1223: (1 ) in primary lung tumors: LT3, LT15, LT16, LT17, and LT18; and (2) in primary colon tumors: CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, and CT17. Epicenter mapping for DNA30676-1223 resulted in confirmation of significant amplification: (1) in primary lung tumors: LT3, LT13, LT15, LT16, and LT18; and (2) in primary colon tumors:
CTl, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CTl 1, CT12, CT14, CT15, CTl 6, CT17, and CT18.
In contrast, the amplification of the closest known framework markers (with one exception, i.e., S50) (Table 8 A) or epicenter markers (Table 9 A) does not occur to a greater extent than that of DNA30676- 1223. This strongly suggests that DNA30676-1223 is the gene responsible for the amplification of the particular region on Chromosome 19.
Because amplification of DNA30676-1223 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA30676-1223 (PRO201 ) would be expected to have utility in cancer therapy.
PRQ292 (DNA35617):
The ΔCt values for DNA35617 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA35617 encoding PR0292 occurred : (1 ) in primary lung tumors: LT1 , LTla, LT1 1 , LT12, LT13, LT15, LT17, LT19 and LT21 ; (2) in primary colon tumors: CT2, CT8, CT10 and CT14; in lung tumor cell lines: H441 and H810; and (4) in colon tumor cell lines: SW620, Colo320, HT29, and LS174T. Amplification has been confirmed by framework mapping for DNA35617: (1 ) in primary lung tumors:
LT12, LT13, LT15, and LT16; and (2) in primary colon tumors: CT2, CT8, CT10 and CTl 4. In contrast, the amplification of the closest known framework markers (Table 1 1 ) does not occur to a greater extent than that of DNA35617. This strongly suggests that DNA35617 is the gene responsible for the amplification of the particular region on Chromosome 1 1. Because amplification of DNA35617 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA65617 (PR0292) would be expected to have utility in cancer therapy.
PRQ327 (DNA381 13-1230):
The ΔCt values for DNA38113-1230 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA38113-1230 encoding PR0327 occurred: (1) in primary lung tumors: LTl a, LT3, LT6, LT10, LT11, LT12, LT13, LT15, LT16, LT17, and LT19; (2) in primary colon tumors: CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CT17, CTl , CT4, CT5, CT6, CT9, CTl 1 , and CTl 8; (3) in lung tumor cell lines: H157, H441 , H460, and SKMES-1 ; and (4) in colon tumor cell lines: SW620, Colo320, HCC2998, and KM 12.
Amplification has been confirmed by framework mapping for DNA38113-1230 (Table 8B): (1 ) in primary lung tumor LT10; and (2) in primary colon tumors: CT2, CT3, CT8, CT10, CT12, CT14, and CT16. Epicenter mapping for DNA38113- 1230 resulted in confirmation of significant amplification (Table 9B): ( 1 ) in primary lung tumors: LT12, LT13, LT15, LT16, and LT17; and (2) in primary colon tumors: CTl, CT2, CT3, CT4, CT5, CT6, CT8, CT9, CT10, CTl 1 , CTl 2, CT14, CTl 6, and CTl 8.
With the exception of S41 , amplification of the closest markers to DNA381 13- 1230 does not occur to a greater extent than that of DNA381 13-1230 itself. This supports the notion that DNA38113 is the gene which is driving the amplification of this particular region of Chromosome 19. However, the amplification of marker S41
(which does not map closely to DNA381 13) could be an independent amplification event or even an error in the ordering of the markers.
Because amplification of DNA381 13-1230 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA381 13-1230 (PR0327) would be expected to have utility in cancer therapy.
PRO 1265 (DNA60764-1533):
The ΔCt values for DNA60764-1533 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA60764- 1533 encoding PROl 265 occurred: in primary lung tumors: LT3, LT12, LT13, LT15, LT16, and LT17.
Amplification has been confirmed by framework mapping for DNA60764- 1533 (Table 8C) in primary lung tumor LT16. Epicenter mapping for DNA60764-1533 also resulted in confirmation of significant amplification (Table 9C): ( 1 ) in primary lung tumors: LT12, LT13, LT15, and LT16; and (2) in primary colon tumors: CTl , CT4,
CT5, CT7, and CTl 1. In contrast, the amplification of the closest known framework markers, epicenter markers and comparison sequences does not occur to a greater extent than that of DNA60764-1533. This strongly suggests that DNA60764-1533 is the gene responsible for the amplification of the particular region on Chromosome 19.
Because amplification of DNA60764-1533 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA60764-1533 (PR01265) would be expected to have utility in cancer therapy.
PRQ344 (DNA40592-1242):
The ΔCt values for DNA40592-1242 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA40592-1242 encoding PR0344 occurred: (1 ) in primary lung tumors: LT1 1 , LT12, LT13, LT15, LT16, LT17, LT19 and LT21 ; and (2) in primary colon tumors:
CT2, CTl 4, CTl 5, CTl , CT4, CT5, and CT11.
Because amplification of DNA40592- 1242 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA40592-1242 (PR0344) would be expected to have utility in cancer therapy.
PRQ343 (DNA43318-1217):
The ΔCt values for DNA43318-1217 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA43318-1217 encoding PR0343 occurred: (1) in primary lung tumors: LT1 1 , LT12, LT13, LT15, LT16, LT17, LT18 and LT19; and (2) in primary colon tumors: CT2, CT3, CT8, CT10, CTl 2, CTl 4, CTl 5, CTl 6, CTl 7, CT4, CT5, CT7, and CTl 1.
Amplification has been confirmed by framework mapping for DNA43318-1217 (Table 14): ( 1 ) in primary lung tumors: LT12, LT13, LT15, LT16, and LT18; and (2) in primary colon tumors: CT2, CTl, CT5, CT8, CT10, CT14, CT15 and CT16.. Epicenter mapping for DNA43318-1217 also resulted in confirmation of significant amplification (Table 15A): (1 ) in primary lung tumors: LT12, LT13, LT15, and LT16; and (2) in primary colon tumors: CT4, CT5, CT6, CTl 1 , and CT2. In contrast, the amplification of the closest known framework markers, and epicenter markers (with one exception, i.e., Pl 07) does not occur to a greater extent than that of DNA43318- 1217. This strongly suggests that DNA43318-1217 is the gene responsible for the amplification of the particular region on Chromosome 16. Because amplification of DNA43318-1217 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA43318-1217 (PR0343) would be expected to have utility in cancer therapy.
PRQ347 (DNA44176-1244):
The ΔCt values for DNA44176-1244 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA44176- 1244 encoding PR0347 occurred: (1 ) in primary lung tumors: LT1 , LTla, LT3, LT6, LT9, LT10, LT1 1 , LT12, LT13, LT15, LT17, LT19, and LT21 ; and (2) in primary colon tumors: CTl , CT2, CT3, CT5, CT8, CTl 1 , CTl 4, CTl 5, and CTl 6. Because amplification of DNA44176- 1244 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g. , antibodies) directed against the protein encoded by DNA44176- 1244 (PR0347) would be expected to have utility in cancer therapy.
PRQ357 (DNA44804-1248):
The ΔCt values for DNA44804-1248 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA44804-1248 encoding PR0357 occurred: (1) in primary lung tumors: LT1 a, LT3, LT6, LT9, LT10, LT11 , LT12, LT13, LT15, LT16, LT17, LT18, LT19, and LT21 ; and (2) in primary colon tumors: CT2, CT8, CT10, CTl 4, CTl 5, CTl 6, CTl, CT4, CT5, CT6, CT7, and CTl 1. Amplification has been confirmed by framework mapping for DN A44804- 1248 (Table 27) in primary lung tumors: LT3, LT10, LT1 1 , LT12, LT13, LT15, LT17, LT19 and LT21. Epicenter mapping for DNA44804- 1248 also resulted in confirmation of significant amplification (Table 28) in primary lung tumors: LT1 1, LT12, LT13,
LT15, LT17 and LT19. In contrast, the amplification of the closest known framework markers and epicenter markers does not occur to a greater extent than that of DNA44804-1248. This strongly suggests that DNA44804-
1248 is the gene responsible for the amplification of the particular region on Chromosome 16.
Because amplification of DNA44804-1248 occurs in various lung tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g. , antibodies) directed against the protein encoded by DNA44804-1248 (PR0357) would be expected to have utility in cancer therapy.
PRQ715 (DNA52722-1229):
The ΔCt values for DNA5271 1-1229 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA5271 1-1229 encoding PR0715 occurred: (1 ) in primary lung tumors: LT1 , LTl a, LT9, LT10, LT1 1 , LT12, LT13, LT15, LT16, LT17, LT18, and LT19; and (2) in primary colon tumors: CT2, CT3, CT8, CTl 0, CTl 4, CTl 5, CTl 6; CTl 7, CTl , CT4, CT5, CT6, CT7, CTl 1 and CTl 8.
Amplification has been confirmed by framework mapping for DNA5271 1 -1229 (Table 22): ( 1 ) in primary lung tumors: LT1 1 , LT13, LT15, LT16, LT17, and LT18; and (2) in primary colon tumors: CT2, CT3, CT8, CTl 0, CTl 4, CTl 5, CTl 6, and CTl 7. Epicenter mapping for DNA5271 1 -1229 also resulted in confirmation of significant amplification (Table 23): (1 ) in primary lung tumors: LT1 1 , LT13, LT15, LT16, LT17 and LT18; and (2) in primary colon tumors: CTl , CT3, CT4, CT5, CT6, CT7, CT10, CTl 1 , CTl 4, CTl 5, CTl 6, and CTl 8. In marked contrast, the amplification of the closest known framework markers and epicenter markers does not occur to a greater extent than that of DNA5271 1 -1229. This strongly suggests that DNA52711-1229 is the gene responsible for the amplification of the particular region on Chromosome 17.
Because amplification of DNA5271 1-1229 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA5271 1-1229 (PR0715) would be expected to have utility in cancer therapy.
PRO1017 (DNA561 12-1379):
The ΔCt values for DNA561 12-1379 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA561 12-1379 encoding PRO1017 occurred: (1) in primary lung tumors: LTla, LT3, LT6, LT7, LT9, LT10, LT1 1 , LT12, LT13, LT15, LT16, LT17, LT18, LT19, and LT21 ; and in primary colon tumors: CT2, CT3, CT8, CTl 0, CTl 2, CTl 4, CTl 5, CTl 6, CTl 7, CT4, CT5, CT6, CT9, and CTl 1.
Amplification has been confirmed by framework mapping for DNA56112- 1379 (Table 19) in primary lung tumors: LT3, LT4, LT7, LT9, LT10, LT1 1 , LT12, LT13, LT15, LT16, LT18, and LT22. Epicenter mapping for DNA561 12-1379 also resulted in confirmation of significant amplification (Table 18): (1 ) in primary lung tumors: LT12, LT13, LT15, LT16, and LT18; and (2) in primary colon tumors: CT5, CT8, CT10, CT12, CT14, CT16, and CTl 7. In marked contrast, the amplification of the closest known framework markers and epicenter markers does not occur to a greater extent than that of DNA56112-1379. This strongly suggests that DNA561 12-1379 is the gene responsible for the amplification of the particular region on Chromosome 7.
Because amplification of DNA561 12-1379 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g. , antibodies) directed against the protein encoded by DNA56112-1379 (PRO 1017) would be expected to have utility in cancer therapy.
PROl 1 12 (DNA57702-1476):
The ΔCt values for DNA57702-1476 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA57702-1476 encoding PROl 1 12 occurred: (1 ) in primary lung tumors: LT10, LT11, LT12, LT13, LT15, LT17, and LT18; and (2) in primary colon tumors: CT2, CT8, CT10, CTl 2, CTl 4, CTl 5, CTl 6, CTl , CT4, CT5, CT6, and CTl 1. Because amplification of DNA57702- 1476 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA57702-1476 (PROl 1 12) would be expected to have utility in cancer therapy.
PRO509 (DNA50148):
The ΔCt values for DNA50148 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA50148 encoding PRO509 occurred: (1 ) in primary lung tumors: LT1, LTla, LT3, LT4, LT9, LT12, LT13, LT15, LT16, LT17, and LT19; and (2) in primary colon tumors: CTl 5, CTl 7, CT6, CTl 1 , CTl 8. Because amplification of DNA50148 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA50148 (PRO509) would be expected to have utility in cancer therapy.
PRQ853 (DNA48227-1350):
The ΔCt values for DNA48227-1350 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA48227-1350 encoding PR0853 occurred: (1) in primary lung tumors: LT1 1, LT12, LT13, LT15, and LT16; (2) in primary colon tumors: CT2, CT3, CT8, CT10, CTl 2, CTl 4, CTl 5, CTl 6, CTl 7, CTl , CT4, CT5, CT7, and CTl 1 ; and (3) in lung tumor cell lines: H441 , and H522. Confirmation of amplification was not confirmed in epicenter mapping for DNA48227 for primary lung tumors, but was seen in primary colon tumors (Table 24B): CTl, CT2, CT3, CT4, CT5, CT6, CT8, CT9, CT10, CTl 1 , CT12, CT14, CT15 and CT17. In contrast, the amplification of the closest known epicenter markers does not occur to a greater extent than that of DNA48227. This strongly suggests that DNA48227 is the gene responsible for the amplification of the particular region on Chromosome 17. Because amplification of DNA48227-1350 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g. , antibodies) directed against the protein encoded by DNA48227-1350 (PR0853) would be expected to have utility in cancer therapy.
PRQ882 (DNA58125):
The ΔCt values for DNA58125 in a variety of tumors are reported in Table 5. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA58125 encoding PR0882 occurred: ( 1 ) in primary lung tumors: LTla, LT3, LT6,LT9, LT10, LTl l, LT12, LT13, LT15, LT16, LT17, LT18, LT19, andLT21 ; (2) in colon tumors: CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CTl, CT4, CT5, and CTl 1 ; (3) in lung tumor cell line H441 ; and (4) in colon tumor cell lines: SW620, Colo320, HT29, SKCOl , SW403, LS174T, Colo205, HCT15, HCC2998, and KM 12. Amplification has been confirmed by framework mapping for DNA58125 (Table 14): in primary lung tumors: LT3, LT12, LT13, LT15, LT17, and LT18; and (2) in primary colon tumors: CTl , CT2, CT3, CT4, CT5, CT6, CT8, CTl 0, CTl 2, CTl 4, CTl 5 and CTl 8. Epicenter mapping for DNA58125 also resulted in confirmation of significant amplification (Table 15B): (1 ) in primary lung tumors: LT12, LT13, LT15, LT16, and LT17; and (2) in primary colon tumors: CTl , CT4, CT6, CT7, CT9, CTl 1 , CT2, CT8, CTl 0 and CTl 6. In marked contrast, the amplification of the closest known framework markers and epicenter markers does not occur to a greater extent than that of DNA58125. This strongly suggests that DNA58125 is the gene responsible for the amplification of the particular region on Chromosome 16.
Because amplification of DNA58125 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA58125 (PR0882) would be expected to have utility in cancer therapy.
EXAMPLE 18 In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision. 1 : 169-176 (1994), using PCR-generated33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37 °C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A (33-P)UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks. — P-Riboprobe synthesis 6.0 μ\ ( 125 mCi) of 3 P-UTP (Amersham BF 1002, S A<2000 Ci/mmol) were speed- vacuum dried. To each tube containing dried 33P-UTP, the following ingredients were added: 2.0 μl 5x transcription buffer l .O μl DTT (lOO mM)
2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H20) 1.0 μl UTP (50 μM) l .O μl RNAsin 1.0 μl DNA template (1 μg) 1.0 μl H2O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37 °C for one hour. A total of 1.0 μl RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes. A total of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™.
The probe was run on a TBE/urea gel. A total of 1 -3 μl of the probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer. After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70°C freezer one hour to overnight.
SP-Hybridization A. Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37 °C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each.
B. Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 °C, 15 minutes) for human embryo tissue, or 8 x proteinase K ( 100 μl in 250 ml Rnase buffer, 37 °C, 30 minutes) for formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
C. Prehybridization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H20), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC, and 9 ml SQ H20 were added, and the tissue was vortexed well and incubated at 42°C for 1 -4 hours.
D. Hybridization 1.0 x 106 cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer was added per slide. After vortexing, 50 μl 33P mix was added to 50 μl prehybridization on the slide. The slides were incubated overnight at 55 °C.
E. Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, V,=4L), followed by RNAseA treatment at 37 °C for 30 minutes (500 μl of 10 mg/ml in 250 ml
Rnase buffer = 20 μg/ml), The slides were washed 2 x10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
V 4L). F. Oligonucleotides
In situ analysis was performed on two of the DNA sequences disclosed herein. The oligonucleotides employed for these analyses are as follows:
(1 ) PRQ292 (DNA35616) (Cathepsin D) DNA35616-pl :
5 -GGA TTC TAA TAC GAC TCA CTA TAG GGC TCT TCG ACA CGG GCT CCT CCA A-3' (SEQ ID NO:96) DNA35616-p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CAG CTC GCG CAC CTC ATC CAC-3' (SEQ ID NO:97) DNA35616 (Cathepsin D), an estrogen inducible lysosomal aspartyl protease, showed widespread tissue expression. Expression has been reported to correlate with outcome in breast and lung cancer. In normal tissues: widespread expression was seen in macrophages and cells of macrophage lineage, especially osteoclasts. Expression was observed in epithelial cells of lung, liver, gall bladder, stomach (basal glands), kidney, bladder and prostate. Expression was also seen in adult cardiac myocytes, cartilage and cerebral neurones. In the fetus, expression was strongest in osteoclasts, but was also seen in thymus, splenic red pulp, fetal liver (hepatocytes and Kupffer cells), bronchial epithelium, choroid plexus, neurones and spinal ganglia.
Additional notable expression was observed in diseased tissues: DNA35617 was widely expressed in macrophages at sites of injury; in tumor tissues, expression was seen at varying levels in malignant epithelium in all of the lung cancers. In nine out of fifteen tumors, expression was higher in the malignant epithelium than it was in the benign epithelium - a finding that is consistent with amplification. Expression was always higher in tumor associated macrophages, than it was in the malignant epithelium and in one case expression was striking in multinucleated giant cells.
(2) PRQ327 (DNA381 13-1230) (Prolactin Rceptor Homlog) DNA381 13-pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CCC CCT GAG CTC TCC CGT GTA-3' (SEQ ID NO:98) DNA381 13-p2:
5'-CTATGAAATTAACCCTCACTAAAGGGAAGGCTCGCCACTGGTCGTAGA-3'(SEQIDNO:99)
High expression was observed in developing mouse and human fetal lung, while normal adult lung, including bronchial epithelium was negative. Expression was also seen in human fetal trachea, including with high probability, smooth muscle cells. Expression was also observed in non-trophoblastic cells in the human placenta. These data are consistent with a potential role in bronchial development.
In addition, DNA381 13 was identified as being amplified in a panel of lung cancers. Accordingly, expression was examined in a series of lung cancers: eight squamous carcinomas and eight adenocarcinomas were examined. Based on observing strong expression on the radiographic film, three tumors were examined after a two week exposure, all other sections were examined after a 4 week exposure. DNA381 13 was highly expressed in three out of the eight adenocarcinomas. Moderate, more focal expression, was seen in four other adenocarcinomas. None of the squamous carcinomas showed significant expression over the malignant epithelium, although low level expression was detected .
Expression was not restricted to malignant epithelium, additional sites of expression included: benign bronchiolar epithelium; expression in stromal spindle shaped cells; expression in smooth muscle of arteries (in one specimen the expression was in the outer third of the wall); and in bronchial and in small nerves. Expression of DNA381 13 was consistent with the amplification data (shown above). Expression was especially prominent in three tumors studied. Based on the expression data, this may be a therapeutic target for lung adenocarcinomas.
The expression pattern in fetal lung suggests a possible role in lung growth and repair.
(3) PRQ1265 (DNA60764-1533) (Fig-1 Homolog) DNA60764-pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CGC GCT GTC CTG CTG TCA CCA-3' (SEQ ID NO: 100)
DNA60764-p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GTT CCC CTC CCC GAG AAG ATA-3' (SEQ ID NO: 101 )
Fifteen of the sixteen lung tumors examined were suitable for analysis (eight adeno and seven squamous carcinomas). Most of the tumors showed some expression of DNA60764. Expression was largely confined to monnuclear cells adjacent to the infiltrating tumor. In one squamous carcinoma, expression was seen by the malignant epithelium.
Expression was also seen over cells in fetal thymic medulla of uncertain histogenesis. Expression was seen over mononuclear cells in damaged renal interstitium and in interstitial cells in a renal cell carcinoma. Expression was also seen over cells in a germinal center, consistent with the fact that most Fig-1 positive cells are probably inflammatory in origin.
(4) PRQ343 (DNA43318-1217) (Human Prostasin Homolog) DNA43318-P1 :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCG GCG AGG ACA GCA CTG ACA G-3' (SEQ ID NO:102)
DNA43318-p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCG GGC CCC CAG AGG TAG AGG-3' (SEQ ID
NO:103)
Expression was observed in lung carcinomas as well as in benign normal fetal and adult tissues. Expression was observed in five out of eight adenocarcinomas, and three out of seven squamous carcinomas. Expression was seen over malignant epithelium. Expression was accentuated in cells adjacent to areas of necrosis suggesting that this gene may be upregulated by hypoxia and/or that it may be associated with cell death or cell shedding. Expression was also seen in two sarcomas and in a renal cell carcinoma.
In benign tissues, expression was seen over developing esophageal and gastric epithelium, with expression higher in superficial cells. No specific expression was seen in adult human gastric epithelium, but expression was seen in the basal glands of chimp stomach. Low level expression was observed in fetal and adult bronchial epithelium as well as in bronchial cartilage. Fetal limb showed expression adjacent to sites of bone formation. Expression was also seen in placenta. Stromal cells in the wall of rhesus monkey penis showed expression and regional expression was seen over neurones in monkey cerebrum.
(5) PRQ357 (DNA44804-1248) (ALS Homolog): DNA44804-pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TGC CCG CAA CCC CTT CAA CTG-3' (SEQ ID NO: 104) DNA44804-p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCG CAG CTG GGT GAC CGT GTA-3'(SEQ ID NO: 105) Low to moderate expression was seen at sites of bone formation in fetal tissues and in the malignant cells of an osteosarcoma. Expression was also observed at low level in the placenta and umbilical cord.
(6) PRQ715 (DNA52722-1229) (TNF Homolog) DNA52722-pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CGC CCC GCC ACC TCC T-3' (SEQ ID NO: 106)
DNA52722-p2: 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CTC GAG ACA CCA CCT GAC CCA-3' (SEQ ID NO: 107)
DNA52722-p3:
5'-GGATTCTAATACGACTCACTATAGGGCCCAAGGAAGGCAGGAGACTCT-3'(SEQIDNO:108)
DNA52722-p4:
5'-CTA TGA AATTAA CCC TCA CTA AAG GGA CTA GGG GGT GGG AAT GAA AAG-3'(SEQ ID NO: 109) Generalized high expression level was observed in many tissues: the highest signals were seen over placenta, osteoblasts, injured renal tubules, injured liver, colorectal liver metastasis and gall bladder. Tested samples had acetominophen induced liver injury and hepatic cirrhosis.
Tissues also examined included eight adenocarcinomas and eight squamous carcinomas of the lung. Strong expression was seen over macrophages in all tumors examined. In one case, there was weak to moderate expression over benign bronchial epithelium. Expression was also observed in a non-tumor containing lung sample with moderate inflammatory changes.
(7) PROl 017 (DNA56112-1379) (Sulfotransferase Homolog) DNA56112-pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCA GCA GGC GGA GCG GAG GAG-3'(SEQ ID NO:110)
DNA56112-p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CAC GGC GAA CTT GCG GTA GAA-3' (SEQ ID NO: 1 1 1 ) A positive signal was seen in a multi-tumor block: expression was seen in squamous carcinoma, sarcoma, and hepatocellular carcinoma. In lung cancers: two out of eight adenocarcinomas and two out of seven squamous carcinomas showed a positive signal over the malignant epithelium. A positive signal was also seen over cortical and hippocampal neurones in adult rhesus monkey brain. A possible signal was seen in fetal small intestinal epithelium.
(8) PRQ853 (DNA48227-1350) (Reductase Homolog): DNA48227-pl : 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CCA ACA GCG GCA TCG GAA AGA-3'(SEQ ID NO: 1 12) DNA48227-p2:
5'-CTA TGA AATTAA CCC TCA CTA AAG GGA GGA GCA CCA GCC AAG CCA ATG-3'(SEQ ID NO: 1 13) Elevated expression was observed in the mucosa of the chimp stomach.
EXAMPLE 19 Use of PRO201 , PRQ292, PRQ327, PRQ1265, PRQ344. PRQ343. PRQ347. PRQ357, PRQ715, PRO1017.
PRQ1 1 12. PRO509. PRQ853 or PRQ882 as a hybridization probe The following method describes use of a nucleotide sequence encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide as a hybridization probe. DNA comprising the coding sequence of a full-length or mature "PRO201 ", "PR0292", "PR0327",
"PR01265", "PR0344", "PR0343", "PR0347", "PR0357", "PR0715", "PRO1017", "PROl 1 12", "PRO509", "PR0853" or "PR0882" polypeptide as disclosed herein and/or fragments thereof may be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882) 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 PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PROl 112-, PRO509-, PR0853- or PR0882-derived 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 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can then be identified using standard techniques known in the art.
EXAMPLE 20
Expression of PRO201. PRQ292. PRQ327. PROl 265. PRQ344. PRQ343. PRQ347. PRQ357. PRQ715. PRO1017, PROl 112, PRO509, PRQ853 or PRQ882 Polypeptides in E. coli. This example illustrates preparation of an unglycosylated form of PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 by recombinant expression in E. coli. The DNA sequence encoding the PRO polypeptide of interest 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. coli; 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 trp promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 coding region, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli 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 in 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, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PR0327 was successfully expressed in E. coli in a poly-His tagged form using the following procedure. The DNA encoding PR0327 was initially amplified using selected PCR primers. The primers contained restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants were first grown in LB containing 50 mg/ml carbenicillin at 30°C with shaking until an O.D. of 3-5 at 600 nm was reached. Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2S04, 0.71 g sodium citrate«2H20, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36g Sheffield hycase SF in 500 ml water, as well as 1 10 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30CC with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture was centrifuged to pellet the cells. Cell pellets were frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate were added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract was loaded onto a 5 ml Qiagen Ni 2+-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The proteins were eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4CC. Protein concentration was estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
The protein was refolded by diluting sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes were chosen so that the final protein concentration was between 50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C for 12-36 hours. The refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded protein was chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1 % TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A28() absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PR0327 protein were pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
EXAMPLE 21
Expression of PRO201. PRQ292. PRQ327, PROl 265. PRQ344, PRQ343, PRQ347. PRQ357, PRQ715,
PRO1017. PROl 112. PRO509, PRQ853 or PRQ882 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PRO201, PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 by recombinant expression in mammalian cells.
The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, thePRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357,PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 DNA using ligation methods such as described in Sambrook et al, supra. The resulting vector is called pRK5-PRO201 , pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK5-PRO1017, pRK5- PROl 1 12, pRK5-PRO509, pRK5-PR0853 or pRK5-PR0882.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRO201 , pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK5- PRO1017, pRK5-PR01 1 12, pRK5-PRO509, pRK5-PR0853 or pRK5-PR0882 DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya etal, CeH, 3J.:543 (1982)] and dissolved in 500 μl of 1 mM Tris- HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac etal, Proc. Natl. Acad. Sci., 12:7575 (1981 ). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO201, pRK5-PR0292, pRK5- PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK5-PRO1017, pRK5-PR01112, pRK5-PRO509, pRK5-PR0853 or pRK5-PR0882 DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PRO715, PRO1017, PRO1 112, PRO509, PRO853 orPRO882can be expressed inCHOcells. ThepRK5-PRO201 , pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5- PR0357, pRK5-PR0715, pRK5-PRO 1017, pRK5-PRO 1 1 12, pRK5-PRO509, pRK5-PR0853 or pRK5-PR0882 vector can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methionine. After determining the presence of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method.
Epitope-tagged PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 may also be expressed in host CHO cells. The PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector. The poly-His tagged PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 insert can then be subcloned into a S V40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the S V40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography. Expression in CHO and/or COS cells may also be accomplished by a transient expression procedure.
PRO 1265, PRO 1017 and PRO509 were expressed in CHO cells by a stable expression procedure, whereas PR0292, PR0715 and PRO509 were expressed in CHO cells by a transient procedure. Stable expression in CHO cells was performed using the following procedure. The proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g., extracellular domains) of the respective proteins were fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or in a poly-His tagged form.
Following PCR amplification, the respective DNAs were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used for expression in CHO cells is as described in Lucas et al, Nucl. Acids Res., 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells were grown as described in Lucas etal, supra. Approximately 3 x IO"7 cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA were thawed by placement into a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mis of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 ml of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells were then aliquoted into a 100 ml spinner containing 90 ml of selective media. After 1 -2 days, the cells were transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37°C. After another 2-3 days, 250 ml, 500 ml and 2000 ml spinners were seeded with 3 x 105 cells/ml. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in US Patent No. 5, 122,469, issued June 16, 1992 was actually used. 3L production spinner was seeded at 1.2 x IO6 cells/ml. On day 0, the cell number and pH were determined. On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33°C, and 30 ml of 500 g/L glucose and 0.6 ml of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) added. Throughout the production, the pH was adjusted as necessary to keep at around 7.2. After 10 days, or until viability dropped below 70%, the cell culture was harvested by centrifugation and filtered through a 0.22 μm filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins were purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4"C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs were purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. PR0292, PR0327, PR0344, PR0347, PR0357, and PR0853 were also produced by transient expression in COS cells.
EXAMPLE 22
Expression of PRO201. PRQ292. PRQ327. PRQ1265. PRQ344. PRQ343. PRQ347. PRQ357. PRQ715.
PRO1017, PROl 1 12, PRO509. PRQ853 or PRQ882 in Yeast The following method describes recombinant expression of PRO201 , PR0292, PR0327, PROl 265,
PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO20I , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 from the ADH2/GAPDH promoter. DNA encoding PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882. For secretion, DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PRO715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882.
Yeast cells, such as yeast strain ABI 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
Recombinant PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 may further be purified using selected column chromatography resins.
EXAMPLE 23 Expression of PRO201. PRQ292. PRQ327, PRQ1265. PRQ344, PRQ343. PRQ347, PRQ357. PRQ715. PRO1017. PRO! 1 12. PRO509. PRQ853 or PRQ882 in Baculovirus-infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected insect cells. The sequence coding for PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1112, PRO509, PR0853 or PR0882 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 orPR0882 or the desired portion of the coding sequence of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA
(Pharmingen) into Spodopterafrugiperda ("Sf9") cells (ATCC CRL 171 1 ) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
Expressed poly-His tagged PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert er a/.. Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1 % NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 M phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 M phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882, respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
While expression is actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g., 8 L) preparations. The proteins are expressed as an IgG construct (immunoadhesin), in which the protein extracellular region is fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly- His tagged forms.
Following PCR amplification, the respective coding sequences are subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVLl 393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28 °C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni +-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis are repeated, as necessary, until expression of the spinner culture is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows.
The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
PR0327, PR0344 and PRO509 were expressed in Baculovirus -infected Sf9 insect cells by the above procedure.
Alternatively, a modified baculovirus procedure may be used incorporating high 5 cells. In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly- His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl - 1 (Novagen). The pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably- transformed insect cells. The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for iel -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIEl-1 and pIEl-2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. For example, derivatives of pIE 1 - 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of ) the desired sequence. Preferably, the vector construct is sequenced for confirmation.
High 5 cells are grown to a confluency of 50% under the conditions of 27 °C, no C02, NO pen/strep. For each 150 mm plate, 30 μg of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media: Ex -Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100 μl ofCellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) is mixed with 1 ml of Ex-Cell medium. The two solutions are combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex- Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on high 5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The conditioned media from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows. The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the sequence is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
PR0327, PROl 265, PR0344 and PR0882 were successfully expressed by the above modified baculovirus procedure incorporating high 5 cells. EXAMPLE 24 Preparation of Antibodies that Bind PRO201. PRQ292, PRQ327, PRQ1265. PRQ344, PRQ343. PRQ347, PRQ357, PRQ715. PROl 017. PROl 1 12. PRO509. PRQ853 or PRQ882 This example illustrates preparation of monoclonal antibodies which can specifically bind PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882.
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO201, PR0292, PR0327, PR01265,
PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 fusion proteins containing PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715,
PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 and cells expressing recombinant PRO201 , PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 orPR0882 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 -100 micrograms.
Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton,
MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO201, anti-PR0292, anti-PR0327, anti-
PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PROl 017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. l , available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO201, PR0292, PR0327,
PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882.
Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or
PR0882 is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti- PR0347, anti-PR0357, anti-PR0715, anti-PRO 1017, anti-PRO 1 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
Deposit of Material:
The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC):
Material ATCC Deposit No.: Deposit Date
DNA30676-1223 209567 12/23/97
DNA381 13-1230 209530 12/10/97
DNA60764-1533 203452 1 1/10/98
DNA40592-1242 209492 1 1/21/97
DNA43318-1217 209481 1 1/21/97
DNA44176- 1244 209532 12/10/97
DNA44804-1248 209527 12/10/97
DNA52722-1229 209570 1/7/98
DNA561 12-1379 209883 5/20/98
DNA57702-1476 209951 6/9/98
DNA48227-1350 209812 4/28/98
These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and the ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. § 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. § 1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated antibody that binds to a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
2. The antibody of Claim 1 which specifically binds to said polypeptide.
3. The antibody of Claim 1 which induces the death of a cell that expresses said polypeptide.
4. The antibody of Claim 3, wherein said cell is a cancer cell that overexpresses said polypeptide as compared to a normal cell of the same tissue type.
5. The antibody of Claim 1 which is a monoclonal antibody.
6. The antibody of Claim 5 which comprises a non-human complementarity determining region (CDR) or a human framework region (FR).
7. The antibody of Claim 1 which is labeled.
8. The antibody of Claim 1 which is an antibody fragment or a single-chain antibody.
9. A composition of matter which comprises an antibody of Claim 1 in admixture with a pharmaceutically acceptable carrier.
10. The composition of matter of Claim 9 which comprises a therapeutically effective amount of said antibody.
11. The composition of matter of Claim 9 which further comprises a cytotoxic or a chemotherapeutic agent.
12. An isolated nucleic acid molecule that encodes the antibody of Claim 1.
13. A vector comprising the nucleic acid molecule of Claim 12.
14. A host cell comprising the vector of Claim 13.
15. A method for producing an antibody that binds to a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, said method comprising culturing the host cell of Claim 14 under conditions sufficient to allow expression of said antibody and recovering said antibody from the cell culture.
16. An antagonist of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
17. The antagonist of Claim 16, wherein said antagonist inhibits tumor cell growth.
18. An isolated nucleic acid molecule that hybridizes to a nucleic acid sequence that encodes a PRO201 ,
PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, or the complement thereof.
19. The isolated nucleic acid molecule of Claim 18, wherein said hybridization is under stringent hybridization and wash conditions.
20. A method for determining the presence of a PRO201, PR0292, PR0327, PR01265, PR0344,
PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in a sample suspected of containing said polypeptide, said method comprising exposing the sample to an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PROl 265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody and determining binding of said antibody to said polypeptide in said sample.
21. The method of Claim 20, wherein said sample comprises a cell suspected of containing a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide.
22. The method of Claim 21 , wherein said cell is a cancer cell.
23. A method of diagnosing tumor in a mammal, said method comprising detecting the level of expression of a gene encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained.
24. A method of diagnosing tumor in a mammal, said method comprising (a) contacting an anti- PRO201 , anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between said antibody and aPRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.
25. The method of Claim 24, wherein said antibody is detectably labeled.
26. The method of Claim 24, wherein said test sample of tissue cells is obtained from an individual suspected of having neoplastic cell growth or proliferation.
27. A cancer diagnostic kit comprising an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti- PRO509, anti-PR0853 or anti-PR0882 antibody and a carrier in suitable packaging.
28. The kit of Claim 27 which further comprises instructions for using said antibody to detect the presence of a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROIOI 7, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in a sample suspected of containing the same.
29. A method for inhibiting the growth of tumor cells, said method comprising exposing tumor cells that express a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide to an effective amount of an agent that inhibits a biological activity of said polypeptide, wherein growth of said tumor cells is thereby inhibited.
30. The method of Claim 29, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type.
31. The method of Claim 29, wherein said agent is an anti-PRO201 , anti-PR0292, anti-PR0327, anti- PRO1265, anti-PRO344, anti-PRO343, anti-PRO347, anti-PRO357, anti-PRO715, anti-PRO1017, anti-PRO1 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
32. The method of Claim 31 , wherein said anti-PRO201, anti-PR0292, anti-PR0327, anti-PRO 1265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti- PRO509, anti-PR0853 or anti-PR0882 antibody induces cell death.
33. The method of Claim 29, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent.
34. A method for inhibiting the growth of tumor cells, said method comprising exposing tumor cells that express a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to an effective amount of an agent that inhibits the expression of said polypeptide, wherein growth of said tumor cells is thereby inhibited.
35. The method of Claim 34, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type.
36. The method of Claim 34, wherein said agent is an antisense oligonucleotide that hybridizes to a nucleic acid which encodes the PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or the complement thereof.
37. The method of Claim 36, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent.
38. 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 inhibiting the growth of tumor cells and wherein the label on the container indicates that the composition is effective for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide in said tumor cells as compared to in normal cells of the same tissue type.
39. The article of manufacture of Claim 38, wherein said active agent inhibits a biological activity of and/or the expression of said PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
40. The article of manufacture of Claim 39, wherein said active agent is an anti-PRO201 , anti-PR0292, anti-PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti- PRO1017, anti-PROl 112, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
41. The article of manufacture of Claim 39, wherein said active agent is an antisense oligonucleotide.
42. A method of identifying a compound that inhibits a biological or immunological activity of a
PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide, said method comprising contacting a candidate compound with said polypeptide under conditions and for a time sufficient to allow the two components to interact and determining whether a biological or immunological activity of said polypeptide is inhibited.
43. The method of Claim 42, wherein said candidate compound is an anti-PRO201 , anti-PR0292, anti- PR0327, anti-PR01265, anti-PR0344, anti-PR0343, anti-PR0347, anti-PR0357, anti-PR0715, anti-PRO1017, anti-PROl 1 12, anti-PRO509, anti-PR0853 or anti-PR0882 antibody.
44. The method of Claim 42, wherein said candidate compound or said PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide is immobilized on a solid support.
45. The method of Claim 44, wherein the non-immobilized component is detectably labeled.
46. A method of identifying a compound that inhibits an activity of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, said method comprising the steps of (a) contacting cells and a candidate compound to be screened in the presence of said polypeptide under conditions suitable for the induction of a cellular response normally induced by said polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist, wherein the lack of induction of said cellular response is indicative of said compound being an effective antagonist.
47. A method for identifying a compound that inhibits the expression of a PRO201 , PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in cells that express said polypeptide, wherein said method comprises contacting said cells with a candidate compound and determining whether expression of said polypeptide is inhibited.
48. The method of Claim 47, wherein said candidate compound is an antisense oligonucleotide.
49. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO:13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), and Figure 28 (SEQ ID NO:53).
50. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ), Figure 3 (SEQ ID NO:5), Figure 5 (SEQ ID NO:7), Figure 7 (SEQ ID NO: 12), Figure 9 (SEQ ID NO: 14), Figure 1 1 (SEQ ID NO:22), Figure 13 (SEQ ID NO:27), Figure 15 (SEQ ID NO:32), Figure 17 (SEQ ID NO:39), Figure 19 (SEQ ID NO:41 ), Figure 21 (SEQ ID NO:43), Figure 23 (SEQ ID NO:45), Figure 25 (SEQ ID NO:47), and Figure 27 (SEQ ID NO:52).
51. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO:l ), Figure 3 (SEQ ID NO:5), Figure 5 (SEQ ID NO:7), Figure 7 (SEQ ID NO: 12), Figure 9 (SEQ ID NO: 14), Figure 1 1 (SEQ ID NO:22), Figure 13 (SEQ ID NO:27), Figure 15 (SEQ ID NO:32), Figure 17 (SEQ ID NO:39), Figure 19 (SEQ ID NO:41 ), Figure 21 (SEQ ID NO:43), Figure 23 (SEQ ID NO:45), Figure 25 (SEQ ID NO:47), and Figure 27 (SEQ ID NO:52).
52. Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number 209567, 209530, 203452, 209492, 209481 , 209532, 209527, 209570, 209883, 209951 or 209812.
53. A vector comprising the nucleic acid of any one of Claims 49 to 52.
54. The vector of Claim 53 operably linked to control sequences recognized by a host cell transformed with the vector.
55. A host cell comprising the vector of Claim 53.
56. The host cell of Claim 55, wherein said cell is a CHO cell.
57. The host cell of Claim 55, wherein said cell is an E. coli.
58. The host cell of Claim 55, wherein said cell is a yeast cell.
59. The host cell of Claim 55, wherein said cell is a Baculovirus-infected insect cell.
60. A process for producing a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide comprising culturing the host cell of Claim 55 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
61. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), and Figure 28 (SEQ ID NO:53).
62. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), and Figure 28 (SEQ ID NO:53).
63. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA deposited under ATCC accession number 209567, 209530, 203452, 209492, 209481 , 209532, 209527, 209570, 209883, 209951 or 209812.
64. A chimeric molecule comprising a polypeptide according to any one of Claims 61 to 63 fused to a heterologous amino acid sequence.
65. The chimeric molecule of Claim 64, wherein said heterologous amino acid sequence is an epitope tag sequence.
66. The chimeric molecule of Claim 64, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
67. An antibody which specifically binds to a polypeptide according to any one of Claims 61 to 63.
68. The antibody of Claim 67, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
69. Isolated nucleic acid having at least 80% nucleic acid sequence identity to: (a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4
(SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), lacking its associated signal peptide; (b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), lacking its associated signal peptide.
70. An isolated polypeptide having at least 80% amino acid sequence identity to: (a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:6), Figure 6 (SEQ ID
NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), lacking its associated signal peptide; (b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID
NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), with its associated signal peptide; or (c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID
NO:6), Figure 6 (SEQ ID NO:8), Figure 8 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 15), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:28), Figure 16 (SEQ ID NO:33), Figure 18 (SEQ ID NO:40), Figure 20 (SEQ ID NO:42), Figure 22 (SEQ ID NO:44), Figure 24 (SEQ ID NO:46), Figure 26 (SEQ ID NO:48), or Figure 28 (SEQ ID NO:53), lacking its associated signal peptide.
PCT/US1999/030095 1994-09-08 1999-12-16 Compositions and methods for the treatment of tumor WO2000037640A2 (en)

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MXPA01006345A MXPA01006345A (en) 1998-12-22 1999-12-16 Compositions and methods for the treatment of tumor.
KR1020017007877A KR20010102960A (en) 1998-12-22 1999-12-16 Compositions and Methods for The Treatment of Tumor
AU21928/00A AU2192800A (en) 1998-12-22 1999-12-16 Compositions and methods for the treatment of tumor
IL14321299A IL143212A0 (en) 1998-12-22 1999-12-16 Compositions and methods for the treatment of tumor
JP2000589694A JP2003524390A (en) 1998-12-22 1999-12-16 Compositions and methods for treating tumors
EP99966374A EP1141289A2 (en) 1998-12-22 1999-12-16 Compositions and method for the treatement of tumors
CA002353775A CA2353775A1 (en) 1998-12-22 1999-12-16 Compositions and methods for the treatment of tumor
CA002361840A CA2361840A1 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/004342 WO2000078961A1 (en) 1999-06-23 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU28837/00A AU2883700A (en) 1999-06-23 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00907314A EP1263948A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2000603377A JP2004513602A (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
KR1020017011399A KR20030002292A (en) 1999-03-08 2000-02-18 Secreted and Transmembrane Polypeptides and Nucleic Acids Encoding the Same
AU28839/00A AU2883900A (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/004414 WO2001004311A1 (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/005841 WO2000053758A2 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
AU35144/00A AU3514400A (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
KR1020017011406A KR20010103046A (en) 1999-03-08 2000-03-02 Compositions and Methods for the Treatment of Immune Related Diseases
CA002362427A CA2362427A1 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
EP00913764A EP1220905A2 (en) 1999-03-08 2000-03-02 Composition and methods for the treatment of immune related diseases
JP2000603379A JP2004516227A (en) 1999-03-08 2000-03-02 Compositions and methods for treating immune-related diseases
CA002383254A CA2383254A1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00916675A EP1210418B1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/008439 WO2000073454A1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU37743/00A AU3774300A (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001500766A JP2004522402A (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AT00916675T ATE478145T1 (en) 1999-06-02 2000-03-30 SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS CODING THEREFOR
EP00939307A EP1212417B1 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
PT00939307T PT1212417E (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
CA002376116A CA2376116A1 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US2000/013705 WO2000073445A2 (en) 1999-06-02 2000-05-17 Interleukin-1-receptor associated kinase-3 (irak3)
ES00939307T ES2307515T3 (en) 1999-06-02 2000-05-17 ACTIVATION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION.
JP2001500757A JP4297317B2 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
DK00939307T DK1212417T3 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and vascularization
AU54412/00A AU5441200A (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
AT00939307T ATE393825T1 (en) 1999-06-02 2000-05-17 STIMULATION OR INHIBITION OF ANGIOGENESIS AND CARDIAC VASCULARIZATION
CA002373915A CA2373915A1 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
JP2001500672A JP2003524406A (en) 1999-06-02 2000-05-30 Compositions and methods for inhibiting tumor cell growth
AT00941164T ATE357518T1 (en) 1999-06-02 2000-05-30 METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL GROWTH
EP07005022A EP1870464A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07004855A EP1820860A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
DK00941164T DK1185648T3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07005021A EP1873244A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/014941 WO2000073348A2 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
ES00941164T ES2287020T3 (en) 1999-06-02 2000-05-30 PROCEDURE AND COMPOSITIONS TO INHIBIT THE GROWTH OF NEOPLASSIC CELLS.
EP07005023A EP1867719A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP00941164A EP1185648B1 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
DE60041266T DE60041266D1 (en) 1999-08-31 2000-08-23 COMPOSITION AND METHOD FOR THE TREATMENT OF IMMUNOMATED DISEASES
AT00959474T ATE419348T1 (en) 1999-08-31 2000-08-23 COMPOSITION AND METHOD FOR TREATING IMMUNE-RELATED DISEASES
CA002384055A CA2384055A1 (en) 1999-08-31 2000-08-23 Compositions and methods for the treatment of immune related diseases
JP2001520865A JP3988821B2 (en) 1999-08-31 2000-08-23 Compositions and methods for treating immune related diseases
ES00959474T ES2317847T3 (en) 1999-08-31 2000-08-23 COMPOSITIONS AND PROCEDURES FOR THE TREATMENT OF IMMUNOLOGICAL TYPE DISEASES.
EP00959474A EP1208201B9 (en) 1999-08-31 2000-08-23 Compositions and methods for the treatment of immune related diseases
PCT/US2000/023522 WO2001016319A2 (en) 1999-08-31 2000-08-23 Compositions and methods for the treatment of immune related diseases
AU70793/00A AU7079300A (en) 1999-08-31 2000-08-23 Compositions and methods for the treatment of immune related diseases
CA002494705A CA2494705A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000581A EP1666494A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU20554/01A AU2055401A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002391455A CA2391455A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002492070A CA2492070A1 (en) 1999-12-01 2000-12-01 Lung tumor marker pro4329 polypeptides and nucleic acids encoding the same
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
CA002491433A CA2491433A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05025102A EP1672070A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000583A EP1686134A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds and nucleic acids encoding the same
EP06000588A EP1690873A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA2709291A CA2709291A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000586A EP1688497A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA002491610A CA2491610A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000589A EP1661997A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491258A CA2491258A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding same
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002496312A CA2496312A1 (en) 1999-12-01 2000-12-01 Colon tumour marker pro4799 polypeptides and nucleic acids encoding the same
CA002490909A CA2490909A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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US09/905,291 US20020160374A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,877 US20030186358A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,859 US20030036060A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,088 US20030073077A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,992 US20030135025A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,125 US6664376B2 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,938 US20030211569A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,766 US20030152999A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,056 US20030054441A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,348 US20030064923A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,449 US6965011B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,820 US20030036094A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/904,920 US6806352B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,381 US6818746B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,532 US7151160B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,553 US20030059828A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,462 US6878807B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,838 US20030148370A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,485 US20030064367A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,760 US20030096340A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,722 US6946262B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,777 US20030148371A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,838 US7070979B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,815 US7094567B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,618 US6828146B2 (en) 1997-09-17 2001-07-16 Nucleic acid encoding PRO229 polypeptides
US09/906,742 US20030023054A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,700 US6723535B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,728 US20030190611A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,841 US7033825B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,093 US20030017498A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,824 US20020197671A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,925 US20030054352A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,942 US7087738B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,575 US20030073079A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,979 US20030082542A1 (en) 1994-09-08 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,613 US20030027145A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,652 US20030104469A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,088 US20020146709A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
US09/908,576 US20040005553A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,204 US20030036061A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,413 US20020156004A1 (en) 1998-09-16 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,403 US20020165143A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/943,664 US20040091972A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/943,762 US20020142958A1 (en) 1998-09-16 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,449 US20020102647A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/943,780 US20030096742A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/943,851 US20020150976A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,396 US20020132981A1 (en) 1997-12-03 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,457 US6734288B2 (en) 1997-12-03 2001-08-30 Antibodies against a secreted polypeptide that stimulates release of proteoglycans from cartilage
US09/944,432 US20020142419A1 (en) 1998-09-16 2001-08-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/945,587 US6936254B2 (en) 1997-12-03 2001-08-31 Method of inducing fetal hemoglobin synthesis
US09/944,896 US7189566B2 (en) 1997-12-03 2001-08-31 PRO347 nucleic acids
US09/944,884 US7018837B2 (en) 1997-12-03 2001-08-31 Nucleic acids encoding secreted polypeptides that stimulate release of proteoglycans from cartilage
US09/944,944 US6929947B2 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/945,584 US6908993B2 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,929 US7550573B2 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,852 US20030083479A1 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,862 US20020115145A1 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,654 US20020142959A1 (en) 1998-09-16 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,907 US20020198147A1 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/945,015 US20020132768A1 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/946,374 US20030073129A1 (en) 1998-09-01 2001-09-04 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,193 US20030073624A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,608 US20030045462A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,403 US20030050240A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,423 US20030069178A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,585 US20030049633A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,802 US20030199674A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,728 US20030186368A1 (en) 1998-05-13 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,036 US20050227342A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,834 US20030064407A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,830 US20030077700A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,967 US20030148373A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,829 US20030195344A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,928 US20030215905A1 (en) 1998-10-07 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,711 US20030032023A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,687 US20020198149A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,427 US20030073809A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,440 US20030060407A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,436 US20020198148A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,726 US20030054359A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,562 US20030027985A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,667 US20030022187A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,521 US20030083461A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,643 US20030228656A1 (en) 1998-06-10 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,073 US20020127576A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,438 US20030027754A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/997,585 US20030119055A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,683 US20030059783A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US20030134284A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US20030087305A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US7244816B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,573 US20030049682A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,156 US20030044806A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US20030054404A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,614 US20030124531A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US20030027163A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/997,542 US20030068647A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,559 US20030054403A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US7309775B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,628 US20030059782A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,428 US20030027162A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,440 US20030059833A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,857 US20030064375A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US7309761B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,150 US20030194760A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,734 US7491529B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,862 US20030130182A1 (en) 1997-11-05 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,279 US7083978B2 (en) 1997-06-16 2001-11-19 Nucleic acid encoding PRO1111 polypeptides
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,721 US20020142961A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,728 US7029873B2 (en) 1997-06-16 2001-11-20 Nucleic acids to PRO1387 polypeptides
US09/989,725 US20030139329A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/001,054 US20020192209A1 (en) 1997-09-17 2001-11-30 Methods and compositions for inhibiting neoplastic cell growth
US10/006,768 US6936697B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,041 US6951921B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,172 US7081514B2 (en) 1998-09-01 2001-12-06 PRO1347 polypeptides
US10/007,236 US7034123B2 (en) 1998-09-01 2001-12-06 Anti-PRO1347 antibodies
US10/006,485 US7026448B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,746 US7026449B2 (en) 1999-01-05 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,818 US20030054406A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,117 US7071304B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,130 US7098312B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,063 US20030114652A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/007,194 US7041805B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,116 US20030082626A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,856 US7538086B2 (en) 1998-09-01 2001-12-06 PRO1303 polypeptides
US10/012,754 US7375184B2 (en) 1998-09-01 2001-12-07 PRO1382 polypeptides
US10/011,795 US7012131B2 (en) 1998-09-01 2001-12-07 Pro1410 polypeptides
US10/011,671 US20030096954A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,149 US7038019B2 (en) 1998-09-01 2001-12-07 Antibodies to PRO1410
US10/012,755 US20030096955A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,752 US7026455B2 (en) 1998-09-01 2001-12-07 Anti-pro 1343 antibodies
US10/011,692 US20030109672A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,121 US7022817B2 (en) 1998-09-01 2001-12-07 PRO1325 polypeptides
US10/012,137 US20030187189A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,753 US7488796B2 (en) 1998-09-01 2001-12-07 PRO1269 polypeptides
US10/012,101 US20030187239A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,064 US6953841B2 (en) 1999-01-05 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,754 US20030187191A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,237 US20030191281A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,231 US6924355B2 (en) 1998-09-01 2001-12-07 PRO1343 polypeptides
US10/011,833 US6951920B2 (en) 1998-09-01 2001-12-07 PRO1340 polypeptides
US10/013,907 US20030064925A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,911 US20030187193A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,915 US20030204053A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,910 US7057018B2 (en) 1999-01-05 2001-12-10 Pro 1474 polypeptides
US10/013,909 US20030186318A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,906 US20030191282A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,913 US20030083462A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,822 US20030130491A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,430 US20030092883A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US20030073130A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,393 US6951737B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,389 US6936436B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,480 US7074912B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US7189530B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,653 US20030187195A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,519 US7033785B2 (en) 1998-09-01 2001-12-11 PRO1347 nucleic acids
US10/015,499 US20030065142A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,671 US6946263B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,715 US7033786B2 (en) 1998-09-01 2001-12-12 Pro1340 nucleic acids
US10/015,391 US20030120053A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,385 US20030195347A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,387 US20030135034A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,390 US20030216562A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,388 US20030191299A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,386 US7022498B2 (en) 1998-09-01 2001-12-12 Pro 1325 nucleic acids
US10/017,306 US20030170718A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,867 US20030180792A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,407 US20030125535A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,527 US20030082628A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,063 US20030119097A1 (en) 1999-01-05 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,610 US20030113795A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,187 US20030096744A1 (en) 1997-10-17 2002-01-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/062,923 US20030191283A1 (en) 1998-04-23 2002-01-31 Nsp molecules
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,061 US20030082761A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,062 US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,049 US20030022239A1 (en) 1997-06-18 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,235 US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,816 US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,823 US20030199062A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/137,865 US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2002341509A JP4017507B2 (en) 1999-06-02 2002-09-25 Medicament containing polypeptide
US10/429,667 US20030207401A1 (en) 1997-12-03 2003-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/735,014 US7442772B2 (en) 1997-12-03 2003-12-12 Antibodies to PRO361 polypeptide
US10/771,187 US7355002B2 (en) 1997-11-12 2004-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/805,667 US20050037458A1 (en) 1999-03-10 2004-07-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/943,353 US20050059115A1 (en) 1997-12-03 2004-09-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/970,823 US7307152B2 (en) 1997-10-17 2004-10-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/025,607 US20050181478A1 (en) 1998-09-01 2004-12-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
JP2005229454A JP4145314B2 (en) 1999-06-02 2005-08-08 Compositions and methods for tumor cell growth inhibition
JP2005264293A JP2006068016A (en) 1999-12-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/296,155 US20060127983A1 (en) 1997-12-03 2005-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
JP2006225771A JP4074645B2 (en) 1999-06-02 2006-08-22 Compositions and methods for tumor cell growth inhibition
JP2006225772A JP2007029098A (en) 1999-06-02 2006-08-22 Method and composition for inhibiting neoplastic cell growth
JP2006225770A JP4072181B2 (en) 1999-06-02 2006-08-22 Compositions and methods for tumor cell growth inhibition
JP2006225879A JP2007037552A (en) 1999-08-31 2006-08-22 Composition and method for treatment of immune-associated disorder
US11/518,609 US20070077623A1 (en) 1997-09-17 2006-09-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/796,725 US20090197301A1 (en) 1998-09-01 2007-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007172879A JP5512073B2 (en) 1999-06-02 2007-06-29 Medicament containing polypeptide
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007325484A JP2008148699A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
US12/079,178 US20090170158A1 (en) 1997-12-03 2008-03-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2008145176A JP2009019032A (en) 1999-06-02 2008-06-02 Promotion or inhibition of angiogenesis and cardiovascularization
JP2011138276A JP2011236224A (en) 1999-06-02 2011-06-22 Medicine containing polypeptide

Applications Claiming Priority (18)

Application Number Priority Date Filing Date Title
US11329698P 1998-12-22 1998-12-22
US60/113,296 1998-12-22
PCT/US1999/005028 WO1999046281A2 (en) 1998-03-10 1999-03-08 Novel polypeptides and nucleic acids encoding the same
USPCT/US99/05028 1999-03-08
USPCT/US99/12252 1999-06-02
PCT/US1999/012252 WO1999063088A2 (en) 1998-06-02 1999-06-02 Membrane-bound proteins and nucleic acids encoding the same
USPCT/US99/20111 1999-09-01
PCT/US1999/020111 WO2000012708A2 (en) 1998-09-01 1999-09-01 Further pro polypeptides and sequences thereof
PCT/US1999/021090 WO2000015796A2 (en) 1998-09-16 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US99/21090 1999-09-15
PCT/US1999/028409 WO2000032778A2 (en) 1998-12-01 1999-11-30 Methods and compositions for inhibiting neoplastic cell growth
PCT/US1999/028313 WO2000032221A2 (en) 1998-12-01 1999-11-30 Promotion or inhibition of angiogenesis and cardiovascularization
USPCT/US99/28313 1999-11-30
USPCT/US99/28409 1999-11-30
PCT/US1999/028301 WO2000032776A2 (en) 1998-12-01 1999-12-01 Secreted amd transmembrane polypeptides and nucleic acids encoding the same
USPCT/US99/28301 1999-12-01
PCT/US1999/028565 WO2000037638A2 (en) 1998-12-22 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
USPCT/US99/28565 1999-12-02

Related Parent Applications (10)

Application Number Title Priority Date Filing Date
PCT/US1998/019330 Continuation-In-Part WO1999014328A2 (en) 1994-09-08 1998-09-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US21851798A Continuation 1996-11-06 1998-12-22
US21851798A Continuation-In-Part 1996-11-06 1998-12-22
US38013799A Continuation-In-Part 1997-06-18 1999-06-02
PCT/US1999/012252 Continuation-In-Part WO1999063088A2 (en) 1996-11-06 1999-06-02 Membrane-bound proteins and nucleic acids encoding the same
PCT/US1999/028301 Continuation WO2000032776A2 (en) 1994-09-08 1999-12-01 Secreted amd transmembrane polypeptides and nucleic acids encoding the same
PCT/US1999/028565 Continuation-In-Part WO2000037638A2 (en) 1994-09-08 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
PCT/US1999/028551 Continuation-In-Part WO2000053750A1 (en) 1997-03-31 1999-12-02 Compositions and methods for the treatment of tumors
PCT/US2001/017800 Continuation-In-Part WO2001093983A1 (en) 1996-11-06 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/874,503 Continuation-In-Part US20020177188A1 (en) 1997-03-31 2001-06-05 IL-17 homologous polypeptides and therapeutic uses thereof

Related Child Applications (18)

Application Number Title Priority Date Filing Date
PCT/US1999/030911 Continuation-In-Part WO2000075316A1 (en) 1994-09-08 1999-12-20 Methods and compositions for inhibiting neoplastic cell growth
PCT/US1999/030999 Continuation-In-Part WO2001005836A1 (en) 1994-09-08 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
PCT/US1999/031274 Continuation-In-Part WO2000053752A2 (en) 1997-03-31 1999-12-30 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US1999/031243 Continuation-In-Part WO2000053751A1 (en) 1997-03-31 1999-12-30 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/000219 Continuation-In-Part WO2000053753A2 (en) 1994-09-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US2000/003565 Continuation WO2001053486A1 (en) 1994-09-08 2000-02-11 Compositions and methods for the treatment of tumor
PCT/US2000/003565 Continuation-In-Part WO2001053486A1 (en) 1994-09-08 2000-02-11 Compositions and methods for the treatment of tumor
PCT/US2000/004414 Continuation-In-Part WO2001004311A1 (en) 1994-09-08 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/008439 Continuation-In-Part WO2000073454A1 (en) 1994-09-08 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/866,028 Continuation-In-Part US6642360B2 (en) 1996-11-06 2001-05-25 Secreted polypeptides that stimulate release of proteoglycans from cartilage
US87203501A Continuation-In-Part 1996-11-06 2001-06-01
US88263601A Continuation 1996-11-06 2001-06-14
US88263601A Continuation-In-Part 1996-11-06 2001-06-14
US09/903,749 Continuation US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/941,992 Continuation US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/944,862 Continuation US20020115145A1 (en) 1997-12-03 2001-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 Continuation US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same

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CA (1) CA2353775A1 (en)
IL (1) IL143212A0 (en)
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US6541224B2 (en) 1996-03-14 2003-04-01 Human Genome Sciences, Inc. Tumor necrosis factor delta polypeptides
US7217788B2 (en) 1996-03-14 2007-05-15 Human Genome Sciences, Inc. Human tumor necrosis factor delta polypeptides
US6482612B1 (en) 1997-08-26 2002-11-19 Zymogenetics, Inc. Adipocyte-specific protein homologs
US7381809B2 (en) 1998-09-09 2008-06-03 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
WO2001016319A3 (en) * 1999-08-31 2001-10-04 Genentech Inc Compositions and methods for the treatment of immune related diseases
US7576182B1 (en) 1999-08-31 2009-08-18 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
WO2001016319A2 (en) * 1999-08-31 2001-03-08 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
US7282562B2 (en) 1999-08-31 2007-10-16 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
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US7189820B2 (en) 2001-05-24 2007-03-13 Human Genome Sciences, Inc. Antibodies against tumor necrosis factor delta (APRIL)
JP2006512903A (en) * 2002-09-11 2006-04-20 ジェネンテック・インコーポレーテッド Novel compositions and methods for the treatment of immune related diseases
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US7947436B2 (en) 2004-12-13 2011-05-24 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in the process of bone remodeling
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ZA200103885B (en) 2002-05-14
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CA2353775A1 (en) 2000-06-29
EP1141289A2 (en) 2001-10-10
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